DEVICE FOR THE LYSIS OF MICROORGAMSMS PRESENT IN AN
ENVIRONMENTAL OR CLINICAL SAMPLE AND THE EXTRACTION OF
NUCLEIC ACIDS FROM SAID MICROORGANISMS FOR ANALYSIS
The technical field of the present mvention is that of biological analysis More particularly, the present mvention relates to a device for the lysis of microorgamsms present m an environmental sample, such as an air sample, or a clinical sample
For several years, a resurgence m nosocomial infections m hospitals has been observed These infections are explained by the contamination of hospitalized, and therefore by definition lmmunodepressed, individuals with pathogenic microorgamsms present m the hospital environmental sphere, which have not been destroyed despite the always great care given to the disinfection of instruments and surfaces and to air treatment With regard to these mcreasmgly common cases of environmental microbiological contamination, the development of devices and methods for improving and facilitating environmental controls has come to represent major stakes for healthcare professionals
In addition to the problem of nosocomial infections, the controlling of environmental conditions has also over the past several years become a recurrent concern m the industrial environment, m particular the food-processing industries or the pharmaceutical or cosmetics industries In the food-processing industries, the disastrous consequences to consumer health that the contamination of products, or even of startmg matenals, with a pathogenic microorganism can have are known Indeed, food poisoning due to bacteria such as those of the Listeria or Salmonella genus is today a common occurrence The controlling of air quality is also a key process in the quality approach of the pharmaceutical or cosmetics industries
Moreover, these controls must meet an mcreasmgly high level of requirement owing to mcreasmgly strict regulations
Among the tools available to health professionals or manufacturers for canymg out environmental controls, aerobiocollectors are solutions of choice for detectmg
microorganisms m the air These devices are placed at appropriate sites on the premises where it is desired to measure the aerobiocontamination They are generally constituted of an air collector coupled to a culture medium The air collected by the air collector comes mto contact with the culture medium, the microorganisms possibly contamed m the collected air becoming deposited on the culture medium The culture medium is then recovered and placed m an mcubator m order to promote growth of the microorganisms It is thus possible to detect and identify said microorganisms by means of conventional microbiology techniques
These devices nevertheless have a major drawback which is linked to the technology used This drawback is the tune required to obtain the result of the analysis This is because the use of conventional microbiology, m particular bacteriology, techniques means that mcubation times necessary for cell growth, or even tunes for phases of re-inoculation on specific culture media so as to enable identification, have to be adhered to It follows that the time required to obtain a result is relatively long, mdeed even too long, when the mtention is to detect and identify a pathogenic organism responsible for a nosocomial infection or for food poisoning
Another drawback of this type of device is that the use of culture media, while making it possible to discriminate between bacterial genera and species, does not generally make it possible to discriminate between the strains of the same bacterial species Now, it is known that the pathogenicity of a microorganism can vary significantly according to the strain under consideration
There are, moreover, devices for recovering particles present m the air, m particular microorganisms Document GB-2 254 024 thus describes a device for collecting the particles contamed m the air, the prmciple of which is based on the cyclone effect While such a device is found to be suitable for collecting the particles contamed m the air, including the microorganisms, it is m no way studied for treating the sample thus obtained, m particular for extracting the genetic material intended to be used for the analysis
More generally, the most relevant techniques m terms of identifying microorganisms
and/or of rapidity of providing results, whether with respect to cluneal or environmental samples, are without any doubt the molecular diagnostics techniques These techniques, based on analyzmg the genetic matenal of microorganisms, and m particular certam specific sequences of interest, make it possible to obtain a very precise identification of the microorganisms m a record time, smce they make it possible to do away with the culture steps
Nevertheless, the use of such techniques presents a certam number of limits, among which the most important is the potential limited amount of microorganisms present m the air and therefore recoverable for carrying out the analysis In fact, it is known that environmental samples, but also some clinical samples, have relatively low amounts of microorganisms It follows that the amount of genetic matenal obtained from this starting matenal is small The effectiveness of the technique used to extract the nucleic acids, m terms of yield, then becomes an essential parameter
Moreover, most of the existing techniques for the lysis of microorganisms are long, requiring the involvement of qualified staff to carry out the manual steps
Document WO-A-2005/038025 desenbes a method for the extraction of nucleic acids from microorganisms taken in particular from the air This method consists in performing three different lysis methods, namely chemical lysis, thermal shock lysis and mechanical lysis While such a method without doubt makes it possible to optimize the nucleic acid extraction yield and therefore to mcrease the amount of genetic matenal available for analysis, it nevertheless remains the case that this yield is still dependent on the amount of microorganisms recovered However, there is nothmg desenbed m this document for optimizing the recovery of said microorganisms
Document US-5,707,861 desenbes a device for disintegrating living cells of the microorganism type This device makes it possible to lyze the cells by using not only glass beads but also the effect of vibration due to the gap that exists between the tubes containing the microorganisms and the holes of the holder carrying said tubes Thus, such a device makes it possible to optimize the cell lysis and therefore to optimize the extraction of the
genetic material Such a device and the method used by the latter present the same limits as those mentioned above, namely that they remain dependent on the amount of microorganisms recovered Moreover, they have the additional drawback of havmg to subsequently carry out a nucleic acid concentration step m order to isolate said nucleic acids from the cell debris Finally, they require manual recovery of the nucleic acids, at the end of the concentration step
These problems also arise with the device descnbed m document US-5,567,050
Systems that are more integrated have also been descnbed Thus, document WO-A-2004/018704 descnbes a device and a method using the PCR (Polymerase Cham Reaction) amplification technique for collecting microorganisms m the air and identifying them This system is especially suitable for combatmg attempted biological contamination attacks m mail sorting centers This system is made up of an air collection device placed along the mail transport circuit, a device for filtering/separating the particles by means of a cyclone effect, a device for concentrating/recovenng the particles in a liquid sample, and a device for transferring a fraction of the sample to a GeneXpert PCR analysis cartndge from the company Cepheid The cartndge is then transferred manually to an independent automated biological analysis device m order to identify the microorganism(s) collected from the air
While this system makes it possible to solve many of the technical problems linked to the devices and methods descnbed above, it nevertheless has major drawbacks The first of these drawbacks is that the system for treating the sample (collection, separation, concentration/recovery) pnor to transfer to the analytical cartndge is relatively complex and cumbersome A second drawback is that the microorganisms collected are recovered in a liquid sample of which only one fraction is analyzed This means that the nsk of not recovering all the microorganisms and therefore all the nucleic acids is very high, greatly limiting the relevance of the analysis Moreover, despite its complexity, this system requires the manual transfer of the cartndge to the GeneXpert automated analysis device
Thus, a first objective of the present mvention is to provide a device and a method for universal lysis, which are effective both for environmental samples and for cluneal
amples, for a large diversity of microorganisms, whether they are bacteria, viruses or else fungi, possibly m the vegetative state or m the form of spores
Another objective of the present mvention is to provide a device capable of effectively lyzing said microorganisms contained m an environmental sample such as the air, or m a clinical sample, m order to extract the nucleic acids therefrom and to recover said nucleic acids for analysis, m an integrated manner
Another objective of the present mvention is to provide a device capable of collecting all the microorganisms contained in an air sample
Another objective of the present mvention is to provide a device having a simple design
Another objective of the present mvention is to provide an excessively compact device
Another objective of the present mvention is to provide a closed device in which the various steps stated above take place without any risk of outside contamination
Another objective of the present mvention is to provide a device m which said steps take place without transfer of the sample by the operator, thereby preventing contamination thereof
Finally, another objective of the present mvention is to provide a device capable of providing target nucleic acids m a buffer that can be directly used m molecular diagnostics steps comprising, for example, amplification and detection steps, without additional pretreatment steps such as centnfugation or filtration
These objectives, among others, are achieved by virtue of the present mvention which relates firstly to a cartridge which can be positioned inside an air collection means and receive a means for recovering nucleic acids, said cartridge being substantially cylindrical and comprising a microorganism retaining zone, said retaining zone comprising microorganism lysis means
Advantageously, the microorganism retainmg zone comprises a matenal capable of retainmg the microorganisms, of keeping the lysis means m place and of dissolving m the presence of a liquid The matenal is preferably a gelled matenal.
The gelled matenal may advantageously be a microorganism culture medium
Accordmg to one vanant of the device, the latter compnses a means for connection to an analyzing device
Preferably, the lysis means are constituted of beads Even more preferably, the diameter of the beads is between 200 and 600 una
The invention also relates to a device for collecting microorganisms contained m the air,
said device compnsing
an air collection means, compnsmg an upper element compnsing an air inlet duct and a lower element compnsing an air outlet duct, it being possible for said upper and lower elements to be interlocked with one another such that a current of air can be created inside said air collection means, a substantially cylindncal cartridge compnsmg a microorganism retaining zone, said retainmg zone compnsmg microorganism lysis means, said cartridge bemg positioned inside said air collection means
The air collection means is capable of being connected to an an- recycling circuit
The mvention also relates to a device for microorganism lysis, with the aim of isolating the
nucleic acids from said microorganisms, said device compnsmg
a cartridge accordmg to the mvention, said cartridge compnsmg microorganisms placed in the microorganism retainmg zone, a substantially cylindncal means for recovering nucleic acids, that can be fitted mto the cartridge, said recovering means cooperating with the microorganism lysis means in order to lyze said microorganisms and enable release of the nucleic acids
Advantageously, the means for recovermg nucleic acids compnses a means for drawmg up/delivering liquid
Notably, the means for recovermg nucleic acids also compnses a liquid storage zone
Accordmg to one preferred embodiment, the internal diameter of the cartndge is greater than the external diameter of the means for recovermg nucleic acids, such that, when the means for recovermg nucleic acids is fitted mto the cartndge, the distance separatmg the internal wall of the cartndge from the external wall of the means for recovermg nucleic acids is sufficiently large to allow the lysis means to sit in this interstitial space and sufficiently small for the lysis means to be m contact with one or other of said walls
The mvention also relates to a method for concentrating microorganisms contained m the air, said method compnsing the steps consisting m
a) placing a cartndge mside the air collection means, such that the retaining zone, inside said cartndge, is m communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropnate means,
c) concentrating the microorganisms contained m the air m the retaining zone of the cartndge
Accordmg to one particular embodiment, it also compnses a step d) consisting m growing the microorganisms m the retaining zone
Advantageously, the microorganisms are retained on the lysis means present in the retaining zone
The mvention also relates to a method for the lysis of microorganisms contained m the air, said method compnsing the steps consisting m
a) placmg a cartridge inside the air collection means, such that the retaining
zone, mside said cartndge, is m communication with the air inlet duct of the
air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorgamsms contamed m the air, m the retaimng zone of the cartridge,
d) removing the cartridge from the air collection means,
e) placing the means for recovering nucleic acids m the cartridge,
f) introducing a liquid of mterest mto the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartridge being placed m suspension, and
g) mechanically lyzing the microorgamsms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorgamsms are retained
The mvention also relates to a method for the lysis of microorgamsms contamed m the air, said method comprising the steps consisting in
a) placing a cartridge mside the an- collection means, such that the retaimng zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorgamsms contamed in the air, m the retaimng zone of the cartridge,
d) removing the cartridge from the air collection means,
e) fitting the means for recovering nucleic acids mto the cartridge,
f) causing the delivery of a liquid of mterest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/dehvenng means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located m the microorganism retaimng zone of the cartridge bemg placed in suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids, and
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorgamsms are retained
Another subject of the mvention concerns a method for the extraction of nucleic acids from microorgamsms contamed m the air, said method comprising the steps consisting m
a) placing a cartridge inside the air collection means, such that the retaimng zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentratmg the microorgamsms contamed in the air, m the retaimng zone of the cartridge,
d) removmg the cartridge from the air collection means,
e) placing the means for recovering nucleic acids in the cartridge,
f) introducing a liquid of mterest mto the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartridge being placed m suspension, and
g) mechanically lyzing the microorgamsms by rotating the means for recovering nucleic acids, mside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorgamsms are retained, and
h) drawing up the liquid of mterest containing the nucleic acids of said microorganisms, released during the lysis
The mvention also relates to a method for the extraction of nucleic acids from microorgamsms contamed m the air, said method comprising the steps consisting m
a) placing a cartridge inside the air collection means, such that the retaimng zone, inside said cartridge, is m communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentratmg the microorgamsms contamed in the air, m the retaimng zone of the cartridge,
d) removing the cartridge from the air collection means,
e) fitting the means for recovering nucleic acids into the cartndge,
f) causing the delivery of a liquid of mterest previously placed m the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivenng means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartndge being placed m suspension, said lysis means coming to sit between the vertical internal wall of the cartndge and the vertical external wall of the means for recovering nucleic acids,
g) mechanically lyzmg the microorgamsms by rotating the means for recovering nucleic acids, inside the cartndge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, thus releasing the nucleic acids from said microorgamsms, and
h) causing the liquid of mterest m the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid of mterest thus drawn up containing the nucleic acids of said microorgamsms, released during the lysis
These extraction methods preferentially compnse an additional step d') consistmg m growing the concentrated microorgamsms in the retaining zone of the cartndge
This growing is obtained by mcubation of the cartndge m an incubator for a penod of time ranging from 2 to 24 hours
Another subject of the mvention concerns a method for the lysis of microorgamsms contained m the air, said method compnsmg the steps consistmg m
a) obtaining a cartndge m which microorganisms are placed in the vicinity of the retaining zone,
b) fitting the means for recovering nucleic acids into the cartridge,
c) causing the delivery of a liquid of mterest previously placed m the storage zone of the means for recovering nucleic acids, said delivery bemg obtained by
means of the drawing up/delivenng means of the means for recovermg nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovermg nucleic acids and the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartridge being placed m suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovermg nucleic acids, and d) mechamcally lyzmg the microorganisms by rotatmg the means for recovermg nucleic acids, inside the cartridge, said means for recovermg nucleic acids rotatmg the lysis means on which the microorganisms are retained
Another subject of the mvention concerns a method for the lysis of microorganisms, said method comprising the steps consisting in
a) obtaining a cartridge m which microorganisms are concentrated m the retaining zone,
b) placing the means for recovering nucleic acids in the cartridge,
c) introducing a liquid of interest mto the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartridge being placed m suspension, and
d) mechamcally lyzing the microorganisms by rotatmg the means for recovermg nucleic acids, inside the cartridge, said means for recovermg nucleic acids rotatmg the lysis means on which the microorganisms are retained
Another subject of the mvention concerns a method for the extraction of nucleic acids from microorganisms, said method comprising the steps consisting m
a) obtaining a cartridge in which microorganisms are concentrated m the retaining zone,
b) fining the means for recovermg nucleic acids mto the cartridge,
c) causing the delivery of a liquid of interest previously placed m the storage zone of the means for recovermg nucleic acids, said delivery being obtained by means of the drawing up/dehvenng means of the means for recovermg nucleic acids, the liquid thus delivered filling the interstitial space located between the
means for recovermg nucleic acids and the cartridge, which leads to the lysis means located m the microorganism retaining zone of the cartridge bemg placed m suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovermg nucleic acids,
d) mechamcally lyzmg the microorgamsms by rotatmg the means for recovermg nucleic acids, inside the cartridge, said means for recovermg nucleic acids rotatmg the lysis means on which the microorgamsms are retained, thus releasmg the nucleic acids from said microorgamsms, and
e) causing the liquid of mterest m the storage zone of the means for recovermg nucleic acids to be drawn up, said drawing up bemg obtained by means of the drawing up/dehvenng means of the means for recovermg nucleic acids, the liquid of mterest thus drawn up containing the nucleic acids of said microorgamsms, released during the lysis
Another subject of the mvention concerns a method for the extraction of nucleic acids from microorgamsms, said method comprising the steps consisting m
a) obtaining a cartridge m which microorgamsms are concentrated m the retaining zone,
b) placmg the means for recovermg nucleic acids m the cartridge,
c) introducing a liquid of interest mto the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge bemg placed in suspension, and
d) mechamcally lyzmg the microorgamsms by rotating the means for recovermg nucleic acids, inside the cartridge, said means for recovering nucleic acids rotatmg the lysis means on which the microorgamsms are retained, and
e) drawing up the liquid of mterest containing the nucleic acids of said microorgamsms, released during the lysis.
The mvention also concerns a method for the lysis of microorgamsms, said method compnsmg the steps consisting m
a) introducing a liquid sample containing said microorgamsms mto a cartridge
according to the invention, m the vicinity of the retaining zone, such that said liquid sample leads to the lysis means located m said microorganism retaining zone of the cartridge being placed m suspension,
b) placing the means for recovering nucleic acids m the cartridge,
c) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained
The term "liquid sample" is mtended to mean any liquid sample that may contam microorganisms It may be a liquid sample of human or animal ongm This sample may be, for example, a urine sample, a whole blood sample, a plasma sample or a sample of any other bodily fluid The liquid sample may be of food origin, such as a drink It may also be of environmental ongm, such as water Moreover, the liquid sample may also be a "transfer" liquid, in which possible microorganisms contained on a surface-sampling device, of the swab type, such as those sold by the company Copan, under the name flockedSWABS, have been resuspended by agitation of said swab m said transfer liquid
In addition, the invention concerns a method for the extraction of nucleic acids from microorganisms, said method compnsmg the steps consisting m
a) introducing a liquid sample containing said microorganisms mto a cartridge according to the mvention in the vicinity of the retaining zone, such that said liquid sample leads to the lysis means located m said microorganism retaining zone of the cartridge being placed m suspension,
b) placmg the means for recovenng nucleic acids m the cartridge,
c) mechanically lyzing the microorganisms by rotating the means for recovenng nucleic acids, inside the cartridge, said means for recovenng nucleic acids rotating the lysis means on which the microorganisms are retained,
d) drawing up the liquid of mterest containing the nucleic acids of said microorganisms, released during the lysis
Another subject of the mvention concerns, in addition, a method for identifying one or more microorganisms, compnsmg the steps consisting m
a) isolating the nucleic acids from the microorganisms contained m said sample by means of the device according to the mvention,
b) identifying the microorganism(s) thus isolated
According to one advantageous variant of the method of identification according to the mvention, said method also comprises an intermediate step consisting m purifying the nucleic acids This purification step makes it possible to separate the nucleic acids from the other cell constituents released m the lysis step This step generally makes it possible to concentrate the nucleic acids, and can be adapted for the purification of DNA or of RNA By way of example, it is possible to use magnetic particles optionally coated with oligonucleotides, by adsorption or covalence (m this respect, see patents US 4,672,040 and US 5,750,338), and thus to purify the nucleic acids which are bound to these magnetic particles, by means of a washing step This nucleic acid purification step is particularly advantageous if the mtention is to subsequently amplify said nucleic acids. One particularly advantageous embodiment of these magnetic particles is described in patent applications WO-A-97/45202 and WO-A-99/35500 Another advantageous example of a method for purifying nucleic acids is the use of silica, either m the form of a column, or m the form of inert particles (Boom R et al, J Clm Microbiol, 1990, n° 28(3), p 495-503) or magnetic
particles (Merck MagPrep^ Silica, Promega MagneSil^M Paramagnetic particles) Other very widely used methods are based on ion exchange resins m a column or m a paramagnetic particulate format (Whatman DEAE-Magarose) (Levison PR et al, J Chromatography, 1998, p 337-344) Another method, which is very relevant but not exclusive for the mvention, is that of adsorption onto a metal oxide support (the company
Xtrana Xtra-Bind™ matrix)
In particular, the identification step comprises the substeps consisting in
a) specifically amplifying the isolated nucleic acids,
b) detecting the nucleic acids thus amplified
According to one preferential variant of the method of identification, the identification step is earned out m an identification device in fluid communication with the cartridge of the
device accordmg to the invention
Thus, the isolated nucleic acids are transferred from the means for recovering nucleic acids, of the device accordmg to the invention, to the identification device
The transfer of the nucleic acids is advantageously obtained by delivery of the liquid of interest containing the nucleic acids, said liquid of interest bemg contained m the storage zone of the means for recovering nucleic acids, by means of the drawing up/dehvenng means of the means for recovering nucleic acids
The microorganisms are taken from the group comprising bacteria, viruses, yeasts, molds and parasites
The samples from which the microorganisms are isolated are of environmental origin Thus, they may be an air sample or a liquid sample, such as water, or surface samples The samples may also be of cluneal origin, 1 e any sample of human or animal origin, capable of bemg the subject of an analysis for searching for and identifying a microorganism, optionally a pathogenic microorganism
The presence of the target nucleic acids is demonstrated by the visualization of hybridization reactions The term "hybridization reaction" is mtended to mean any reaction between a capture nucleic acid and a target nucleic acid which has been isolated or generated by means of a step of transcnption, of reverse transcnption or of amplification of NASBA (Nucleic Acid Sequence Based Amplification) or PCR (Polymerase Cham Reaction) type
The term "nucleic acid" is intended to mean oligonucleotides, deoxynbonucleic acids and nbonucleic acids, and also denvatives thereof The term "oligonucleotide" denotes a senes of at least two natural or modified nucleotides (deoxynbonucleotides or nbonucleotides, or both) capable of hybndizmg, under suitable hybndization conditions, with an at least partially complementary oligonucleotide The term "modified nucleotide" is intended to mean, for example, a nucleotide compnsing a modified base and/or compnsmg a
modification at the level of the mtemucleotide bond and/or at the level of the backbone By way of example of a modified base, mention may be made of mosme, methyl-5-deoxycytidine, dimethylamino-5-deoxyuridine, diammo-2,6-purme and bromo-5-deoxyundme
In order to illustrate a modified intemucleotide bond, mention may be made of phosphorothioate, N-alkylphosphoramidate, alkylphosphonate and alkylphosphodiester bonds
Alpha-ohgonucleotides such as those described m FR-A-2 607 507, LNAs such as phosphorothioate-LNA and 2'-thio-LNA described m Bioorganic & Medicinal Chemistry Letters, Volume 8, Issue 16, 18 August 1998, pages 2219-2222, and PNAs which are the subject of the article by M Egholm et al, J Am Chem Soc (1992), 114, 1895-1897, are examples of oligonucleotides made up of nucleotides of which the backbone is modified
The hybndization reactions can be visualized by any detection means such as direct or indirect means
In the case of direct detection, I e without gomg through labelmg, the hybndization reactions are observed by plasmon resonance or by cyclic voltametry on an electrode bearing a conductive polymer
In the case of indirect detection, l e by means of labelmg, the labelmg can be earned out either directly on the target nucleic acids, or by means of a prelabeled bmdmg partner specific for said nucleic acids
The expression "bmdmg partner specific for the target nucleic acids" is mtended to mean any partner capable of binding with the target nucleic acid, and examples that will be given are nucleic acids, oligonucleotides or polynucleotides and enzyme substrates
The term "labelmg" is mtended to mean the attachment of a label capable of directly or indirectly generating a detectable signal A nonlimmng list of these labels consists of
enzymes which produce a signal that can be detected, for example, by electrochemistry, colonmetry, fluorescence, luminescence, or enzymes such as horseradish peroxydase (HRP), alkaline phosphatase (ALP), a-galactosidase or glucose-6-phosphate dehydrogenase, enzyme inhibitors, enzyme cofactors, particles such as gold particles, magnetic latices, liposomes, chromophores such as luminescent or coloring compounds, radioactive molecules such as P, S or I, fluorescent molecules such as fluorescein, rhodamine, Alexa®, umbelliferone, luminol or phycocyamns In the case of fluorescence, it may involve the fluorescent product of an enzyme-substrate reaction, a fluorophore-quencher combination, fluorescence quenching, or any other system based on fluorescence properties
Indirect systems may also be used, for example involving another pan-, the ligand/antiligand parr Ligand/antiligand pairs are well known to those skilled m the art, and mention may, for example, be made of the following pairs biotin/streptavidin, sugar/lectin, polynucleotide/sequence complementary to the polynucleotide In this case, it is the hgand which carries the binding agent The antihgand may be detectable directly via the labels descnbed in the precedmg paragraph, or may itself be detectable via a ligand/antiligand
These indirect detection systems may, under certain conditions, result m amplification of the signal This signal amplification technique is well known to those skilled in the art, and reference may be made to pnor patent applications FR-A-2 781 802 or WO-A-95/08000 by the applicant or to the article J Histochem Cytochem 45 481-491,1997
The prelabeling of the target nucleic acids may be earned out by direct or indirect incorporation of label by means of a polymerase or by means of a kinase, randomly or specifically, at the ends, or by incorporation "within" the molecule.
The labeling of bmdmg partners specific for target analytes is widely known to those skilled m the art and is descnbed, for example, by Greg T Hermanson m Bioconjugate Techniques, 1996, Academic Press Inc, 525B Street, San Diego, CA92101 USA
Dependmg on the type of labeling of the conjugate used, for instance using an enzyme, those skilled in the art will add reagents for visualizing the labeling This step corresponds to the revealing It is preceded by the use of a washing buffer which makes it possible to remove the fractions of analytes or of elements not involved m the reaction, or weakly or nonspecifically bound, m order to limit the background noise
The objectives and advantages of the device accordmg to the present invention will be understood more clearly m the light of the following example, which is in no way limiting, with reference to the drawing, m which
Figure 1 represents an exploded view m longitudinal section of the air collection means, accordmg to a first embodiment of the mvention
Figure 2 represents a view m longitudinal section of the air collection means m which a cartndge has been placed, during the step of collecting the microorganisms, according to a first embodiment of the mvention
Figure 3 represents a view in longitudinal section of the cartndge and of the means for recovering nucleic acids at an initial stage of the process for fitting said means inside the cartndge, accordmg to a first embodiment of the mvention
Figure 4 represents a view m longitudinal section of the cartndge and of the means for recovering nucleic acids at an advanced stage of the process for fitting said means inside the cartndge, accordmg to a first embodiment of the mvention
Figure 5 represents a view m longitudinal section of the cartndge and of the means for recovering nucleic acids at the final stage of the process for fitting said means inside the cartndge, accordmg to a first embodiment of the mvention
Figure 6 represents a view m longitudinal section of the means for recovering nucleic acids, fitted mto the cartndge, during the step of mechanical lysis of the microorganisms, accordmg to a first embodiment of the invention
Figure 7 represents a view m longitudinal section of the means for recovering nucleic acids, fitted mto the cartndge, during the step of drawing the liquid of mterest containing the nucleic acids up mto the storage zone of the means for recovermg nucleic acids, accordmg to a first embodiment of the mvention
Figure 8 represents a view m longitudinal section of the means for recovering nucleic acids, fitted mto the cartndge, when all the liquid of mterest containing the nucleic acids has been drawn up mto the storage zone of the means for recovermg nucleic acids, accordmg to a first embodiment of the invention
Figure 9 represents a view m longitudinal section of the means for recovering nucleic acids, fitted into the cartndge, during the presentation of the means for identifying microorganisms, according to a first embodiment of the mvention
Figure 10 represents a view m longitudinal section of the means for recovenng nucleic acids, fitted into the cartndge, once the means for identifying microorganisms is m fluid communication with the cartndge, according to a first embodiment of the mvention
Figure 11 represents a view in longitudinal section of the means for recovenng nucleic acids - cartndge - means for identifying microorganisms assembly, m the initial phase of transfer of the liquid of mterest containing the nucleic acids, into the means for identifying microorganisms, according to a first embodiment of the invention
Figure 12 represents a view in longitudinal section of the means for recovenng nucleic acids - cartndge - means for identifying microorganisms assembly, after transfer of the liquid of mterest containing the nucleic acids, mto the means for identifying microorganisms, according to a first embodiment of the mvention
Figure 13 represents a view m longitudinal section of the air collection means in which a cartndge has been placed, during the step of collecting the microorganisms, accordmg to a second embodiment
Figure 14 represents a view in longitudinal section of the cartndge, during the step of manual distribution of the liquid of interest, according to the second embodiment
Figure 15 represents a view m longitudinal section of the means for recovering nucleic acids, placed m the cartndge, during the step of mechanical lysis of the microorganisms, according to the second embodiment
Figure 16 represents a view in longitudinal section of the means for recovering nucleic acids, placed in the cartndge, during the step of drawing up the liquid of interest containing the nucleic acids, accordmg to the second embodiment
Figure 17 represents a functional scheme of a test bench mtended to test the capacity for capture of bactena contained m aerosols, with the device accordmg to the mvention
Figure 18 is a graph showing the collection efficiency of the device accordmg to the mvention as a function of the size of the aerosol particles produced
Figure 19 is a graph comparing the bactenal lysis efficiency with the device accordmg to the mvention and alternative methods of the pnor art
Figure 20 is a graph showing the real tune detection, using the Real Tune NASBA technology, of the bactena contained m vanous suspensions, after lysis of these bactena and recovery of the nucleic acids by means of the device accordmg to the invention
Figure 21 is a graph showing the real time detection, using the Real Tune NASBA technology, of the bactena contained m vanous whole blood samples, after lysis of these bactena and recovery of the nucleic acids by means of the device accordmg to the mvention
According to a first embodiment, the first element constituting the device accordmg to the invention is an air collection means 10 This means is composed of an upper element 12 and of a lower element 14 The upper element 12 has a generally cylindncal shape The
lower end of this cylinder is free, whereas the upper end is partially closed off, by means of a horizontal wall 13 In its center, this wall 13 has an orifice extending inside the upper element 12, by means of a duct 18, the base of which is substantially corneal m shape This part in fact constitutes the duct for air inlet mto the air collection means 10 Accordmg to one particular embodiment, this air inlet duct may be connected to a pipe of an air recyclmg circuit by any appropriate means
The air collection means 10 may, for example, have an external diameter of between 10 and 40 mm, preferably 20 mm The internal diameter of the air inlet duct is, for example, 6 mm
This air collection means 10 may advantageously be made of a material that can be sterilized, m particular by autoclavmg It may thus be made of a metal, such as aluminum or steel It may also be made of a polymer, such as poly(methyl methacrylate) (PMMA)
The lower end of the vertical circular wall 20 of the upper element 12 comprises, on its external face, a shoulder 201 This recess is made over the entire circumference of the wall 20 It is mtended to facilitate the interlocking of the upper element 12 and the lower element 14
The lower element 14 is also generally cylindrical m shape The upper end of this cylmder is free, whereas the lower end is partially closed off, by a horizontal wall 21 comprising, m its center, a substantially cylindrical an- outlet duct 22, the base of said duct being integral with the horizontal wall The lower end of the duct 22 is free This duct is m communication with the inside of the lower element 14, such that, when the upper element 12 and the lower element 14 of the air collection means 10 are mterlocked, the duct 22 plays the role of an outlet duct for the air which was allowed mto the air collection means 10, by means of the air inlet duct 18
The upper end of the vertical circular wall 24 of the lower element 14 comprises, on its internal face, a shoulder 241 This shoulder is made over the entire circumference of the wall 24 It is also mtended to facilitate the interlocking of the upper element 12 and the
lower element 14, owing to the fact that the ends of the walls 20 and 24 have a cross section of complementary shape, facilitating the fitting together It is important first of all for this interlocking to be reversible Once fitted together, the elements 12 and 14 should be able to disengage from one another
An alternative means of interlocking the elements 12 and 14 may be interlocking by screwing one element onto the other To this end, the end of the wall of one of the elements 12 or 14 may carry a male thread and the end of the wall of the second element a corresponding female thread
The important thing is that the collection means is hermetically closed, in order to prevent any parasitic entry of air
Accordmg to one particular mode of use of the air collection means, the lower end of the air outlet duct 22 may be connected to an air suction pump (not represented) or any other equivalent pumpmg means This pump makes it possible to suck the ambient air mto the air collection means, when it is desired, for example, to perform an analysis of the ambient air ui a given environment, such as a hospital room or a room for the production of pharmaceutical products or food-processing products To this end, it may be preferable to have an autonomously operating pumpmg means
Figure 2 represents the air collection means 10, during operation thereof, combmed with a cartridge 30 As can be seen m this figure, the cartridge 30 is arranged inside the air collection means 10 To do this, the element 12 and 14 constituting the air collection means 10 are separated The cartridge 30 is placed to bear m the lower element 14 of the air collection means 10 The upper elements 12 is then repositioned on the lower element 14 and these two means are interlocked The assembly constituted of the air collection means 10 and the cartridge 30 is then either connected to a recycling circuit, or to a pumpmg device, in order to bring about circulation of the air inside the air collection means 10, as described above
The cartridge 30 represented m figure 2, m longitudinal section, has the general shape of a
cylmder with a substantially circular transverse section The upper end of the cylinder is free, whereas the lower end is constituted of a wall, having, in its center, an orifice, there being, along the extension of said orifice, a duct 32 extending inside the cartridge 30 It is noted that the internal cross section of the duct tends to become smaller as one approaches the upper end of the duct It is noted, moreover, that the lower end of the duct is closed off by a membrane 34, which plays the role of a septum The matenal constituting the membrane 34 is suitable for being burst by simple pressure with a pointed object This process is descnbed below in relation to figures 9 and 10 This matenal is , for example, a polyethylene terephthalate (PET) or a polycarbonate
As can be seen m figure 2, the internal face of the lower wall 35 of the cartridge 30 is on a slant, the lowest point of the slope bemg m contact with the duct 32 and the highest point m contact with the vertical wall 36 of the cartndge 30 This wall 35 serves as a support for the lysis means 38 and constitutes the microorganism retaining zone These lysis means are m this case constituted by beads of identical size Accordmg to one preferred embodiment, these beads are made of glass They could nevertheless be constituted of any other equivalent matenal, such as iron These beads have a diameter advantageously of between 200 and 800 micrometers (urn)
Accordmg to one advantageous vanant, the beads may be of different sizes It may thus be particularly suitable to use a mixture of beads of diameter between 200 and 300 um with beads of which the diameter is between 400 and 600 urn
The beads are kept m place m the form of one or more superimposed layers by means of a gelled matenal, deposited m the form of a layer, m which the layer(s) of beads is (are) embedded The gelled matenal should satisfy several constraints The first is that it should be inert, so as not to influence the processes which are earned out inside the cartndge The second is that it should have the capacity to dissolve m a liquid, m order to release the particles that it is trapping for the implementation of the microorganism lysis Such a matenal may, for example, be agarose
Alternatively, the gelled matenal may advantageously be a culture medium for the
microorgamsms In fact, agar culture media have been conventionally used m the field of m vitro diagnostics for a very long time The use of such a culture medium has several advantages The main advantage is that it enables a growth phase for the microorgamsms before lysis of the latter is earned out. Even though the device according to the mvention seeks to meet a need linked to the rapid detection of pathogenic microorganisms, it nevertheless remains that a growth phase of a few minutes to a few hours would enable multiplication of the microorgamsms, which has the du-ect effect of havmg a greater amount of nucleic acids A second advantage of the use of culture media is that the latter can be selective for one or more given species It follows that the use of such media can enable selective growth and therefore selective detection of certain pathogenic microorgamsms to the detriment of other microorgamsms that are of no interest, and that can possibly interfere in the analysis Thus, it can be envisioned to have several specialized cartridges, each being suitable for the detection of a specific species of microorganism
The dimensions of the cartridge 30 may, for example, be from 8 to 16 mm as regards the internal diameter, preferably 12 mm The total thickness of the layers of beads is typically between 1 and 2 mm, which corresponds to an amount of glass beads of between 0 4 and 1 gram
The cartridge 30 is advantageously made using an injection-molding technique The material used is, for example, polypropylene, polystyrene, polycarbonate or PMMA
When the circulation of air m the air collection means is initiated, the path followed by said air is represented by the arrows in figure 2 Thus, it is noted that the air enters the air collection means 10 via the air inlet duct 18 Since the duct 32 of the cartridge 30 is partially inserted mto the air inlet duct 18, the air flow m the latter becomes converted, at the top of the duct 32, mto a peripheral flow This is explained by the fact that the upper end of the duct 32 is substantially corneal m shape Moreover, the presence of the membrane 34 at the base of the duct 32 prevents the air from penetrating mto the duct 32, since the latter is very rapidly m a state of mcreased pressure Since the interstitial space between the wall of the inlet duct 18 and the wall of the duct 32 narrows abruptly by virtue of the corneal shape of the end of the duct 32, there is an acceleration of the peripheral air
flow It follows that the latter breaks on the upper layer of beads 38, leading, at this site, to the retention, by impaction, of the microorganisms transported m the air flow, at the surface of the gelled material or of the culture medium Such a process is thus therefore relatively similar to that which occurs m aerobiocollectors
The air, for its part, continues its route, drawn by the pumping means It rises back up along the internal face of the vertical wall 36 of the cartndge 30, at the level of which it loses speed owing to widening of the circulation path It redescends along the external face of this same wall and again becomes converted mto a centralized flow at the entry of the air outlet duct 22, which escapes from the an- collection means 10 via said air outlet duct
The flow rate of air collected m the air collection means 10 may, for example, be between 20 and 100 liters/minute (1/min) It is advantageously 501/min
Once the microorganism retaining/concentrating step inside the cartndge has been earned out, the latter is removed from the air collection means by unfastening the upper element 12 and lower element 14 of said means, either by dislocation or by unscrewing
At this stage, there is the opportunity of mcubatmg the cartridge m an mcubator at 37°C, if it is desired to culture the microorganisms concentrated m said cartndge As already explained above, this mcubation generally lasts a few tens of minutes to a few hours, so as not to extend the analysis tune too greatly Nevertheless, if the obtaining of a result of analysis is not urgent, it may be envisioned to mcubate the cartndge for a longer penod of time more suitable to the constraints of bacterial growth, namely approximately twenty-four hours
Similarly, it may also be envisioned to store the cartndge in an environment suitable for this storage, such as a cold room, if it is desired to defer the analysis
According to one vanant of the mvention, the cartndge 30 may be provided directly placed in the air collection means 10 This type of presentation has the advantage of avoidmg the user having to place the cartndge m the air collection means and therefore thus reducmg
the risks of contamination during this step It is particularly advantageous, m this situation, for the air collection means 10 to be made of the same material as the cartndge 30, namely of polypropylene, polycarbonate or PMMA Manufacture by injection-molding is then particularly suitable
When the analysis is earned out, the cartndge 30 is interlocked with the means for recovering nucleic acids 40, as represented m figure 3 Said means is inserted mto the cartndge via the free end of the latter, according to a vertical translational movement represented by the arrow A
The means for recovering nucleic acids 40, represented m longitudinal section m figure 3, compnses a body 42 of generally cylmdncal shape having a circular cross section The body 42 has, on the upper part of its external wall, a shoulder 43, the function of which is to ensure that the cartridge 30 - means for recovering nucleic acids 40 assembly is leaktight, when said means for recovering nucleic acids is completely inserted m the cartndge 30, by bearing against the upper end of the vertical wall 36 of the cartndge 30 The external wall of the body 42 has, moreover, several successive recesses in its top part The recess 44 located under the shoulder 43 itself plays the role of a shoulder srnce it bears against the shoulder 45 made at the upper end of the vertical wall 36, when the means 40 is completely inserted mto the cartndge 30, as represented in figure 5 The second recess 46 positioned under the recess 44 has, for its part, the function of enablmg the diameter of the body 42 to be reduced, such that there remains an interstitial space between the external surface of the vertical wall of the body 42 and the internal surface of the vertical wall 36 of the cartndge 30 The role of this space will be explained below Moreover, all these shoulders and recesses make it possible to reinforce the leaktightness of the assembly constituted of the cartndge 30 and the means for recovering nucleic acids 40
At its base, the body 42 has, in the central position, a cavity 47 which is substantially cylmdncal in shape with a circular cross section, the upper end of which is substantially corneal in shape Similarly, it also compnses a cavity 48 m its upper part, m the central position This cavity 48, which is substantially cylmdncal m shape with a circular cross section, the lower end of which is substantially corneal in shape, constitutes a storage zone
m which a liquid of interest 50 is placed This liquid makes it possible to resuspend the glass beads 38, but also to recover the nucleic acids, once the microorganisms have been lyzed It is therefore important for this liquid to be inert with respect, on the one hand, to the microorganisms and, on the other hand, to the nucleic acids, but also with respect to biological protocols Thus, this liquid of interest is typically a buffer suitable for the implementation of biological protocols The volume of liquid of mterest contained m the cavity 47 may be between, for example, 0 1 and 0 4 milliliter (ml).
In order to enable the liquid of mterest to play its role, the cavity 48 cooperates with a drawing up/dehvenng means constituted of a plunger 52 of which the shape is complementary to the lower part of the cavity 48 and an arm 54 m the vertical position, which is integral with the plunger 52, and which extends outside the cavity 48 The end of the arm opposite that attached to the plunger 52 may have a head of particular shape capable of engaging a means for actuatmg (not represented) the drawing up/dehvenng means translationally accordmg to a substantially vertical movement This actuatmg means is preferably an automated means
The cavity 48 is in fluid communication with the cavity 47 by means of a channel 56, connecting the lower end of the cavity 48 to the upper end of the cavity 47 In figure 3, the channel 56 is closed off by means of a membrane 58 positioned at the upper end of the cavity 47, preventmg emptying of the cavity 47 This membrane is also made of a material that can be perforated In particular, this membrane may be perforated by means of the upper end of the duct 32 of the cartridge 30
As shown in figure 4, the external shapes of the means for recovering nucleic acids 40 are perfectly complementary to the internal shapes of the cartridge 30 It follows that the means 40 sits perfectly inside the cartridge 30, the duct 32 playmg the role of a guide by inserting itself into the cavity 47 The means 40 is positioned until the lower wall 60 of the means 40 is bearing against the beads 38
At this stage, there is a pressure movement on the body 42 of the means for recovering nucleic acids and simultaneously on the drawing up/dehvenng means m accordance with
the arrow B represented m figure 5 This translational movement of the drawing up/dehvermg means leads to the delivery of the liquid of mterest 50 mto the cavity 47 via the channel 56, and more particularly mto the interstitial space between the internal wall of the cavity 47 and the external wall of the duct 32 This delivery is possible since the pressure exerted by the liquid on the membrane 58 leads to the perforation or the detachment of said membrane The liquid of mterest 50 then travels to the retainmg zone of the cartridge where the glass beads 38 are located, and fills this zone, creating the suspending of the gelled material trapping the beads, such that the latter are entrained m the flow of liquid of interest, created by the drawing up/dehvermg means, to the interstitial space 62, between the means for recovering nucleic acids 40 and the cartridge 30 It should be noted that this interstitial space preferably has a width between 600 and 800 urn This width is directly linked to the diameter of the beads 38 used. Specifically, the beads should be able to readily circulate m this space, but should also be able to be rotated by the means for recovering nucleic acids 40 To this end, the external vertical wall 64 of the body 42 preferably has a rough surface, which facilitates the rotating of the beads
Once all the beads are positioned along the external vertical wall 64 of the body 42, the means for recovering nucleic acids can be completely inserted mto the cartridge, thus generating the leaktightness of the assembly, by virtue m particular of the shoulders 43 and 44, which respectively bear against the end of the wall of the cartridge 30 and against the shoulder 45, as represented in figure 5
Of course, the microorganisms retained both on the gelled material and on the beads are transferred mto the interstitial space 62 m the same way as the beads 3, by the flow of the liquid of interest
With regard to the transfer of the liquid of mterest, it appears that the presence of the membrane 34 at the base of the duct 32 prevents the air contained m the duct 32 from being expelled when the liquid of mterest is pushed mto the channel 56 It follows that the pressure generated m the duct 32 by the trapped air prevents the liquid of mterest from entering the duct, said liquid of mterest therefore infiltrating the interstitial space between the internal wall of the cavity 47 and the external wall of said duct 32
Once the beads 38 are distributed along the external vertical wall 64 of the body 42 and the means for recovering nucleic acids 40 - cartridge 30 assembly is interlocked, the lysis step per se is initiated This step is represented m figure 6 As can be seen m this figure, the means for recovering nucleic acids 40 is rotated m the direction represented by the arrow C For this, the body 42 of the means for recovering nucleic acids 40 is interlocked, by any suitable mechanical coupling means, with an automated device Such a system makes it possible in particular to precisely adjust the speed of rotation
It can nevertheless be envisioned to rotate the means for recovering nucleic acids manually For this, gnppmg means (not represented) may be specifically provided on the upper part of the body 42 These gnppmg means can then be seized with one hand, while the cartridge 30 is held with the other hand
By way of example, the speed of rotation values may be between 300 and 2000 rpm, preferably 1000 rpm
The rotation time is, for its part, generally between 1 and 2 minutes
It is quite obvious that the choice of these two parameters depends on the type of microorganisms that it is intended to lyze Thus, m order to lyze yeasts of the Saccharomyces cerevisiae type, the optimal lysis conditions consist of a speed of rotation equal to 1000 rpm, for 2 minutes
During this step, the beads 38 are rotated around the axis of symmetry by friction against the body 42 of the means for recovering nucleic acids This double rotation leads to the mechanical lysis of the microorganisms which are trapped between the beads 38 and the body 42 This results in release of the nucleic acids into the liquid of interest
Once the lysis step has been completed and the nucleic acids released, the liquid of interest is drawn up mto the cavity 48 by means of the drawing up/dehvenng means, as represented m figure 7 In detail, the drawing up/dehvenng means is moved by vertical translation according to the arrow D, by any suitable automatic or manual means of traction on the arm
54 This translational movement > leads to the drawmg up of the liquid of mterest 50 mto the cavity 48 Durmg this drawmg up, the liquid of mterest follows the reverse path of that which it follows durmg its delivery prior to the lysis step In particular, it progresses mto the interstitial spaces between the means for recovering nucleic acids 40 and the cartridge 30, goes back up the channel 56 and ends up m the cavity 48 As can be seen m figures 7 and 8, the hqmd of mterest which fills the cavity 48 is loaded with target nucleic acids 70 of the microorganisms
Since the means for recovering nucleic acids 40 is entirely inserted mto the cartridge 30, the interstitial space located between the lower wall 60 of said means 40 and the bottom of the cartridge, playmg the role of microorganism retaining zone, freed of the beads, is not sufficient to allow the beads 38 to travel It follows that the beads 38 remain positioned along the vertical wall 64 of the body 42
Similarly, the interstitial space between the internal wall of the cavity 47 and the external wall of the duct 32 is particularly suitable for playing the role of a filter, m particular for retaining the cell debris produced durmg the lysis step This is because, durmg the manufacture of the cartridge and of the means for recovering nucleic acids, the dimensions of said cartridge and said means for recovering nucleic acids can be adjusted in such a way that the size of this interstitial space is particularly well-controlled and optimizes the filtering effect
Once all the liquid of mterest has been drawn up mto the cavity 48, it can be envisioned to transfer said liquid mto a biological analysis device This transfer may be carried out directly into the analyzing device or mto a fluidic circuit of which the final element is the analyzing device
When the liquid of mterest is transferred directly mto the device, it is envisioned that all the nucleic acid treatment steps for identifying the microorganism(s) be earned out m the device itself Thus, it may be envisioned, for example, to carry out, m the device, the amplification of the target nucleic acids, the cleavage and the labeling of these target nucleic acids, and the detection thereof by hybridization with complementary sequences
In figure 9, a fluidic analyzing device 80 is partially represented m longitudinal section This device 80 comprises, m its upper part, a preferential zone 82 for fluidic connection to the means for recovering nucleic acids 40 - cartridge 30 assembly The preferential connection zone 82 is generally corneal m shape, more particularly complementary to the shape of the duct 32 At its top, it has an aperture 84 for gaining access to the internal fluidic circuit 86 of the device 80 As represented m this figure 9, the fluidic analyzing device 80 is connected to the means for recovering nucleic acids 40 - cartridge 30 assembly by bringing the preferential connection zone 82 up to the base of said assembly, m particular at the level of the membrane 34 closmg off the duct 32, m accordance with the arrow E Smce the end of the preferential connection zone 82 is pointed, when the latter comes mto contact with the membrane 34, it perforates said membrane, freeing the access to the duct 32 The analyzing device 80 is then inserted mto the duct 32 until it is touching, I e until the external wall of the preferential connection zone is m contact with the internal wall of the duct 32, as represented in figure 10 When the analyzing device 80 is thus connected to the means for recovering nucleic acids 40 - cartridge 30 assembly, it is noted that the end of the preferential connection zone 82 comes into contact with the lower end of the channel 56 It follows that the wall of the fluidic device 80, m the upper part of the preferential connection zone 82, cuts off the fluid communication between the channel 56 and the interstitial space between the internal wall of the cavity 47 and the external wall of the duct 32
It follows that, when the operator wishes to transfer the liquid of interest 50 loaded with target nucleic acids 70 mto the analyzing device 80, it is sufficient to exert, once again, a pressure on the arm 54 of the drawmg up/delivering means, such that said means moves by vertical translation mto the cavity 48, m accordance with the arrow B This is represented m figure 11
The movmg of the drawmg up/delivering means leads to the transfer of the liquid of interest 50 and of the nucleic acids 70 mto the channel 56 connecting the cavity 48 to the cavity 47 Smce the channel 56 is m direct fluid communication with the internal fluidic circuit 86 of the fluidic analyzing device 80 by means of the aperture 84 of the preferential
connection zone 82, the liquid of interest is then transferred directly into the analyzmg device 80, without any risk of said liquid coming mto contact with the outside environment There is also no risk of the liquid of interest escaping mto the interstitial spaces between the means for recovering nucleic acids 40 and the cartridge 30, since any fluid communication between the channel 56 and the interstitial space between the internal wall of the cavity 47 and the external wall of the duct 32 is blocked. This is represented m figure 12
Once all of the liquid of mterest 50 and of the nucleic acids 70 have been transferred mto the fluidic analyzmg device 80, said device can be disengaged from the means for recovering nucleic acids 40 - cartridge 30 assembly Smce the latter are used, they are disposed of The fluidic analyzmg device 80 is, for its part, subsequently used to carry out the identification of the microorganisms
Accordmg to one advantageous embodiment, all the steps described above m relation to figures 1 to 12, and in particular those linked to the microorganism lysis, the nucleic acid extraction and the transfer of said nucleic acids mto the fluidic analyzing device, can be automated by means of an ad hoc system
Figures 13 to 16 represent a second embodiment of the device accordmg to the mvention The device under consideration has a simpler design than the device accordmg to the first embodiment
The device accordmg to this second embodiment also comprises an air collection means 90 This means is composed of an upper element 92 and of a lower element 94 The upper element 92 is generally cylindrical m shape The lower end of this cylinder is free, whereas the upper end is partially closed off, by means of a horizontal wall 93 This wall 93 has, m its center, an onfice extending mto the upper element 92, by means of a duct 98, the base of which is substantially comcal m shape This part m fact constitutes the duct for mlet of air mto the air collection means 90 Accordmg to one particular embodiment, this an* inlet duct may be connected to a pipe of an air recyclmg circuit by any appropriate means
In its lower part, the duct 98 comprises a screen 99 The function of this screen is to allow a homogeneous distribution of the air flow inside the air collection means 90
The lower end of the vertical circular wall 100 of the upper element 92 compnses, on its external face, a shoulder 100i This recess is made over the entire circumference of the wall 100 It is mtended to facilitate the fitting together of the upper element 92 and the lower element 94
The lower element 94 is also generally cylindrical in shape The upper end of this cylinder is free, whereas the lower end is partially closed off, by means of a horizontal wall 101 compnsing, m its center, a substantially cylindncal air outlet duct 102, the base of said duct being integral with the honzontal wall The lower end of the duct 102 is free This duct is m communication with the mside of the lower element 94, such that, when the upper element 92 and the lower element 94 of the air collection means 90 are mterlocked, the duct 102 plays the role of an outlet duct for the air that was allowed into the an- collection means 90 by means of the air inlet duct 98
The upper end of the vertical circular wall 104 of the lower element 14 compnses, on its internal face, a shoulder 104i This shoulder is made over the entire circumference of the wall 104 It is also mtended to facilitate the fitting together of the upper element 92 and the lower element 94, owing to the fact that the ends of the walls 100 and 104 have a cross section of complementary shape, facilitating the fitting together It is important m the first place for this fitting together to be reversible Once fitted together, the elements 92 and 94 should be able to disengage from one another
An alternative means of fitting together the elements 92 and 94 may be fitting together by screwing one element onto the other To this end, the end of the wall of one of the elements 92 or 94 may carry a male thread and the end of the wall of the second element a corresponding female thread
The important thing is that the collection means is hermetically closed, m order to prevent any parasitic entry of air
According to one particular mode of use of the air collection means, the lower end of the air outlet duct 102 may be connected to an air suction pump (not represented) or any equivalent pumping means
A cartndge 110 is arranged inside the air collection means 90 To do this, the elements 92 and 94 constituting the air collection means 90 are separated The cartridge 110 is placed to bear m the lower element 94 of the air collection means 90 The upper element 92 is then repositioned on the lower element 94 and these two means are interlocked The assembly constituted of the air collection means 90 and the cartndge 110 is then either connected to a recycling circuit, or connected to a pumping device, in order to bring about circulation of the air rnside the an* collection means 10, as descnbed above
The cartndge 110 represented m longitudinal section has the general shape of a cylmder with a low height and a substantially circular transverse section The upper end of the cylinder is free, whereas the lower end is constituted of a wall 112 This wall 112 serves as a support for the lysis means 118 and constitutes the microorganism retaining zone These lysis means are constituted of beads, as descnbed above The beads are kept in place in the form of one or more superimposed layers by means of a gelled matenal as descnbed above, deposited m the form of a layer, m which the layer(s) of beads is (are) embedded
When the circulation of air m the an- collection means is initiated, the path followed by said air is represented by the arrows m figure 13 Thus, it is noted that the air enters the air collection means 90 via the air inlet duct 98 By virtue of the screen 99, it is distnbuted homogeneously and breaks on the upper layer of beads 118, leadmg, at this site, to the retention, by impaction, of the microorgamsms transported in the air flow, at the surface of the gelled matenal or of the culture medium
The air, for its part, continues its route, drawn by the pumping means It nses back up along the internal face of the wall of the cartndge 110 It redescends along the external face of this same wall and again becomes converted mto a centralized flow at the entry of the air outlet duct 102, which escapes from the air collection means 90 via said air outlet duct
Once the air collection has been earned out, the elements 92 and 94 of the an collection means 90 are unfastened and the cartridge 110 is recovered
The step of distribution of the liquid of interest then takes place, as represented m figure 14 During this step, the manipulator deposits, m the cartridge 110, a given volume of liquid of interest 120 using a pipette 122, partially represented m figure 14 As explained above, the liquid of mterest may, for example, be a lysis buffer The contact between the liquid of mterest and the gelled matenal trapping the beads leads to the suspendmg of said matenal and therefore the suspendmg of the beads themselves The volume of liquid of mterest deposited m the cartridge 110 may, for example, be 0 5 ml
Once this step has been earned out, the manipulator places m the cartridge 110 a means 124 for recovering nucleic acids Unlike the means 40 desenbed m the first embodiment, the means 124 compnses neither a drawing up/dehvenng means nor a cavity capable of receivmg the liquid of mterest This means 124 has a substantially cyhndncal transverse section and has, in its upper part, a preferential grippmg zone 126 The lower wall 128 of the means 124 is substantially flat and bears against the beads The lysis is then earned out by rotation of the means 124 m the direction of the arrow F around its axis of symmetry, by the manipulator The rotating of the wall 128 leads to the rotating of the beads 118 around the axis of symmetry This double rotation leads to mechanical lysis of the microorganisms which are trapped between the beads 118 and the wall 128 This results m release of the nucleic acids mto the liquid of mterest
Once the lysis step has been completed and the nucleic acids released, the liquid of mterest, loaded with said nucleic acids 70, is drawn up by the manipulator by means of the pipette 122, as represented in figure 16
Once all the liquid of mterest is m the pipette, said liquid can be transferred mto an analyzing device m order to undergo therein the vanous nucleic acid treatment steps, as desenbed above It can also be placed m a container m order to be stored or m any device mtended for carrying out steps pnor to the nucleic acid treatment steps Such a step may,
for example, be a purification step, mtended to concentrate the nucleic acids by separating them from the cellular constituents still present Such a step is particularly relevant when the device accordmg to the mvention is used in its second embodiment, smce, by virtue of the simple design of the latter, the liquid of mterest loaded with nucleic acids does not undergo any filtering, as it does m the device accordmg to the first embodiment and as explained above
Examples:
Example 1: Measurement of the efficiency of capture of Bacillus subtilis spores contained in an aerosol using the device according to the invention
A leaktight test bench was designed m order to carry out this measurement This test bench is represented m figure 17 It is constituted of a closed an circuit 200, fed with aerosols by an aerosol generator 202 This generator makes it possible to introduce, mto the an flow, a constant amount of Bacillus subtilis (CIP 52 62) spores, from a suspension of spores of known concentration Suitable aerosol generators are, for example, sold by the company TSI The air is cuculated m the circuit by means of a suction pump 204 Three THE filters 206 are present all along the circuit so as to make it possible to remove the particles, in particular the particles of dust, contained m the an A manometer 208 makes it possible to verify the pressure m the circuit and two flow meters 210 make it possible to verify the flow rate of the air m the circuit The device accordmg to the mvention is referenced 212 It is constituted of the an collection means 10, inside which a cartridge 30 is placed The collection means is connected to the an circuit as explamed above Finally two sampling sources, in the an circuit, are positioned just upstream and just downstream of the device 212 The an sampled at these levels, m the form of constant fractions, goes to a spectrometer 214 for carrying out an aerodynamic measurement of the particles, I e the Bacillus subtilis spores, contained in the fractions This spectrometer is an Aerodynamic Particle Sizer® model 3321, sold by the company TSI The transfer of an into this ancillary cncuit that goes to the spectrometer is earned out by means of valves 216, which make it possible to permit or prevent the samplings, either upstream, or downstream of the device 212 The differential analysis of the upstream and downstream fractions makes it possible
to calculate the efficiency of capture of the Bacillus subtihs spores by the device according to the invention
The flow rate of an- passing through the device according to the invention is 50 l/mm under controlled humidity and temperature conditions (25 °C, 35% relative humidity) This flow rate is obtained by means of the pump 204
The capture efficiency of the device accordmg to the mvention was studied under the following two conditions
- determination of the efficiency of capture of Bacillus subtihs spores, with a cartndge m which the beads 36 are positioned m the cartndge without gelled matenal,
- determination of the efficiency of capture of Bacillus subtihs spores, with a cartndge m which the beads 36 are positioned m the cartndge and covered with a fine film-coating of glycerol This product is sufficiently viscous to provide good adhesion of the aerosols to the collection plate and does not generally interfere with the subsequent molecular analysis
The results obtained with and without glycerol are shown in figure 18 In this figure, it is possible to correlate the efficiency of collection of the Bacillus subtihs spores as a function of the diameter of the particles of aerosols produced by the generator 202
It appears that, m the absence of gelled matenal covering the beads 36 m the cartndge, the efficiency of spore collection by the device accordmg to the mvention can range up to more than 70%
In the presence of gelled matenal covering the beads 36 m the cartndge, the collection efficiency climbs to more than 90%
The mcrease m the collection efficiency m the presence of a gelled matenal, of the glycerol type, is explained by the fact that the spores remain stuck to this matenal In the absence of gelled matenal, a slight phenomenon of spore rebound on the beads occurs, preventing them from remaining stuck to the beads
Example 2; Determination of Ivsis efficiency by comparison between the lysis method according to the invention and alternative methods
In this case, 3 methods are compared with the lysis method accordmg to the mvention
- method of mechanical lysis using the FastPrep™ instrument (type FP120, BIO 101, sold by the company Thermo Electron Corporation),
- method of enzyme lysis, using lysozyme,
- method using a chaotropic agent (guamdium isothiocyanate, GuSCN) combmed with a step of heatmg the cell suspension to 90°C, followed by three steps of freezing-thawing the cells
In this example, the cells used are Bacillus cereus m suspension After the lysis step, the recovered nucleic acids are purified using the NucliSENS® easyMAG™ extraction system sold by the applicant The purified nucleic acids are finally quantified using the SYBRGreen detection method (Molecular Probes) The precise steps of the comparative study are described below
- Step 1: preparation of the Bacillus cereus suspension
A culture of Bacillus cereus is earned out on agar culture medium m TSA (trypticase soja) plates Bacterial colomes are recovered on the agar and washed m a NucliSENS® easyMAG™ extraction buffer 3 (ref 280132), sold by the applicant Once the cells have been washed, they are diluted m 5 ml of the same extraction buffer, m order to obtain a bacterial suspension havmg an optical density of 0 5 at a wavelength of 600 nm
- Step 2a: lysis with the FastPrep® instrument
0 5 ml of the bacterial suspension is transferred to a 2 ml tube filled with a mixture of 1 g of glass beads (equal fractions of beads of various sizes 150-212 um, 420-600 um and 700-1180 um) The FastPrep® instrument is used at its maximum speed (setting at 6 0), for 60 sec Once the lysis has been completed, 100 ul of lysate are transferred mto the NucliSENS® easyMAG™ instrument so as to undergo a purification step
- Step 2b: lysis with the device according to the invention
0 3 ml of bacterial suspension is transferred mto a cartridge equivalent to the cartridge 110 described above The cartridge is then filled with 0 4 g of mixtures of glass beads (equal fractions of beads of various sizes 150-212 um, 212-300 um and 420-
600 um) The cartridge is brought into contact with a means for recovering nucleic acids The latter is rotated for 2 mm at 2000 rpm Once this lysis step has been earned out, 100 ul of lysate are transferred mto the NucliSENS® easyMAG™ instrument so as to undergo a purification step
- Step 2c: enzyme lysis
In this method of lysis with lysozyme, 100 ul of bacterial suspension are mixed with 100 ul of a solution of lysozyme (50 mg/ml) The mixture is mcubated for 30 mm at 37°C After this mcubation, the entire lysate (200 ul) is transferred mto the NucliSENS® easyMAG™ instrument so as to undergo a purification step
- Step 2d : chaotropic lysis
100 ul of bacterial suspension are mixed with 100 ul of a concentrated solution of GuSCN (5 mol/1) and the whole mixture is incubated for 30 mm After this mcubation, the entire lysate (200 ul) is transferred mto the NucliSENS® easyMAG™ instrument so as to undergo a purification step
- Step 3: purification of the lysate using the NucliSENS® easyMAG™
instrument
The purification step is earned out usmg the generic extraction protocol of the NucliSENS® easyMAG™ instrument 2 ml of NucliSENS® easyMAG™ lysis buffer (ref 280134) sold by the applicant and 50 ul of a suspension of NucliSENS® easyMAG™ magnetic silica particles (ref 280133) sold by the applicant are added to each lysate The nucleic acids of each of the lysates are eluted m 25 ul of NucliSENS® easyMAG™ extraction buffer 3
- Step 4: nucleic acid detection
In order to determine the amount of nucleic acids in each of the eluates, 10 ul of said eluates are placed m a 0 2 ml PCR tube 180 ul of SyberGreen solution are then added to the samples and the fluorescence signal is measured usmg the NucliSENS EasyQ® instrument sold by the applicant
The results of the fluorescence measurements are given in figure 19 The correspondence is the following
A = lysis with the FastPrep® instrument
B = lysis with the device according to the mvention
C = enzyme lysis
D = chaotropic lysis
By analyzing figure 19, it is noted that the lysis efficiency of the device accordmg to the mvention is similar to that of the FastPrep® mechamcal lysis method These two mechamcal methods are, moreover, sigmficantly more efficient than the method of enzyme lysis or of chemical lysis using the chaotropic agent
Example 3: Lysis of a Bacillus suspension using the device according to the invention in combination with detection by the NASBA method
0 3 ml of a suspension of Bacillus cereus diluted m the NuchSENS® easyMAG™ extraction buffer 3 is introduced mto the cartridge according to the mvention The lysis method accordmg to the mvention, as described m the precedmg example, is reproduced for 2 mm After the lysis step, 5 ul of lysate are used to carry out a detection by the real time NASBA method using primers and probes specific for Bacillus cereus, by fluorescence using the NuchSENS EasyQ® instrument The target gene used for the detection is the EF-tu gene The size of the amplification product is 115 nucleotides Various amounts of Bacillus ranging from 6 to 6 x 105 were used Figure 20 shows the real time production of fluorescence as a function of the various amounts of Bacillus cereus introduced mto the device accordmg to the mvention In this figure, the correspondence is the following
a = sample with 6 x 105 Bacilli
b = sample with 6 x 104 Bacilli
c = sample with 6 x 103 Bacilli
d = sample with 600 Bacilli
e = sample with 60 Bacilli
f = sample with 6 Bacilli
g = sample without Bacillus
From the viewpoint of figure 20, a very good correlation between the level of fluorescence detected and the number of bacteria contained m the sample is observed Furthermore, a significant difference is noted between a sample containing no bacteria and a sample containing only 6 thereof Finally, it can be deduced from the results of this analysis that the device according to the mvention offers a level of performance appropriate for the use of a nucleic acid amplification method of the NASBA type
It should be noted that the sensitivity of this analysis could be improved by concentrating the nucleic acids, before carrying out the amplification by the NASBA method Such a concentration step can be earned out using, for example, the NucliSENS® easyMAG™ instrument
Example 4: Analysis of a whole blood sample using the device according to the mvention
In this example, the device according to the mvention is used to analyze a whole blood sample to which a population of Bacillus cereus has been added The blood sample is derived from a healthy individual
A culture of Bacillus cereus is earned out on agar culture medium m TSA (trypticase soja) plates Bactenal colomes are recovered on the agar and washed with a NucliSENS® easyMAG™ extraction buffer 3 Once the cells have been washed, they are diluted in 5 ml of the same extraction buffer, in order to obtam a bactenal suspension havmg an optical density of 0 5 at a wavelength of 600 ran This stock suspension contains approximately 22 x 107 bactena/ml This stock suspension is diluted m NucliSENS® easyMAG™ extraction buffer 3, in order to obtam a suspension havmg different concentrations, one at 103 bactena/ml and one at 10 bactena/ml
The protocol subsequently used is the following for each of the dilution suspensions
1 24 ul of dilution suspension are added to 150 ul of blood and mixed,
2 125 a! of a NucliSENS® easyMAG™ extraction buffer 1 (ref 280130) are introduced mto a cartndge according to the mvention,
3 the blood sample loaded with bactena is, in turn, introduced mto the cartndge,
4 the lysis step is earned out by bringing the cartridge into contact with a means for recovering nucleic acids The latter is rotated for 2 mm at 2000 rpm,
5 100 ul of lysate are drawn up mto the cartridge,
6 purification of this lysate is earned out using the NuchSENS® easyMAG™ instrument as desenbed in step 3 of example 2, enabling 25 ul of eluate to be recovered,
7 5 ul of this eluate are used to carry out the amplification and the detection, using the Real Time NASBA technology
The results are given in figure 21 In this figure, the correspondence is the following
a = whole blood sample containmg 53 Bacilli (dilution suspension at
105 bactena/ml)
b = whole blood sample containmg 5 Bacilli (dilution suspension at
103 bactena/ml)
c = whole blood sample containmg 5 Bacilli (dilution suspension at
103 bactena/ml)
d = whole blood sample without bactena
The results given correspond to the dilution suspensions at 103 and 105 bactena/ml Moreover, the analysis for the suspension at 105 bactena/ml was duplicated
From the viewpomt of the volume of dilution suspensions introduced mto the cartndge accordmg to the mvention, the actual amounts of bactena are 5 and 530 bactena, respectively, for the suspensions at 103 and 105 bactena/ml
It is thus noted that the analysis of a sample containmg only 5 bactena with the device accordmg to the mvention produces a positive detection signal (curves b and c), which can be clearly distinguished from the result obtained with the sample containing no bactena (curve d)
It is noted, moreover, that even with small amounts of bactena, the results are
reproducible, this is shown by curves b and c
It should be noted that this very good sensitivity was merely obtained using only 5 |il of the 25 ul obtained at the end of the purification step This sensitivity can therefore be further improved by carrying out a step of concentrating the sample, after punfication
It thus emerges from these examples that the device according to the invention is an effective tool for collecting the bactena contained m a sample of air, lyzing them, and allowing the recovery of the nucleic acids from said bactena for analysis For this, the device used will be constituted, firstly, of a cartndge mtroduced mto the air collection means and, secondly, of the cartndge containmg the collected bactena, combmed with the means for recovenng nucleic acids
The device accordmg to the invention is also entirely suitable for the analysis of cluneal samples, such as whole blood samples In this case, the cartndge is used without an air collection means The liquid sample is placed in the cartndge and then the latter is combined with the means for recovenng nucleic acids in order to lyze the bactena and to recover the nucleic acids

CLAIMS
1. A cartridge which can be positioned inside an air collection means and receive a means for recovering nucleic acids, said cartridge being substantially cylindrical and comprising a microorganism retaining zone, said retaining zone comprising microorganism lysis means.
2. The cartridge as claimed in the preceding claim, in which the microorganism retaining zone comprises a material capable of retaining the microorganisms, of keeping the lysis means in place and of dissolving in the presence of a liquid.
3. The cartridge as claimed in the preceding claim, in which the material is a gelled material.
4. The cartridge as claimed in the preceding claim, in which the gelled material is a microorganism culture medium.
5. The cartridge as claimed in one of the preceding claims, also comprising a means for connection of an analyzing device.
6. The cartridge as claimed in the preceding claim, in which the lysis means are constituted of beads.
7. The cartridge as claimed in one of the preceding claims, in which the diameter of the beads is between 200 and 600 um.
8. A device for collecting microorganisms contained in the air, said device comprising:

- an air collection means, comprising an upper element comprising an air inlet duct and a lower element comprising an air outlet duct, it being possible for said upper and lower elements to be interlocked with one another such that a current of air can be created inside said air collection means;
- a cartridge as claimed in one of claims 1 to 7, positioned inside said air collection
means.
9. The device as claimed in the preceding claim, in which the air collection means is capable of being connected to an air recycling circuit.
10. A device for microorganism lysis, with the aim of isolating the nucleic acids from said microorganisms, said device comprising:

- a cartridge as claimed in one of claims 1 to 6, said cartridge comprising microorganisms placed in the microorganism retaining zone;
- a substantially cylindrical means for recovering nucleic acids, that can be placed in the cartridge, said recovering means cooperating with the microorganism lysis means in order to lyze said microorganisms and enable release of the nucleic acids.

11. The device as claimed in the preceding claim, in which the means for recovering nucleic acids comprises a means for drawing up/delivering liquid.
12. The device as claimed in either of claims 10 and 11, in which the means for recovering nucleic acids also comprise a liquid storage zone.
13. The device as claimed in one of claims 10 to 12, in which the internal diameter of the cartridge is greater than the external diameter of the means for recovering nucleic acids, such that, when the means for recovering nucleic acids is fitted into the cartridge, the distance separating the internal wall of the cartridge from the external wall of the means for recovering nucleic acids is sufficiently large to allow the lysis means to sit in this interstitial space and sufficiently small for the lysis means to be in contact with one or other of said walls.
14. A method for concentrating microorganisms contained in the air, said method comprising the steps consisting in:
a) placing a cartridge as claimed in one of claims 1 to 7 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication
with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorganisms contained in the air in the retaining zone of the cartridge.

15. The method of concentration as claimed in the preceding claim, which also comprises a step d) consisting in growing the microorganisms in the retaining zone.
16. The method of concentration as claimed in either of claims 14 and 15, in which the microorganisms are retained on the lysis means present in the retaining zone.
17. A method for the lysis of microorganisms contained in the air, said method comprising the steps consisting in:

a) placing a cartridge as claimed in one of claims 1 to 7 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection means,
e) placing the means for recovering nucleic acids in the cartridge,
f) introducing a liquid of interest into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.
18. A method for the lysis of microorganisms contained in the air, said method
comprising the steps consisting in:
a) placing a cartridge as claimed in one of claims 1 to 7 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication
with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection means,
e) fitting the means for recovering nucleic acids into the cartridge,
f) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids, and
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.
19. A method for the extraction of nucleic acids from microorganisms contained in the air, said method comprising the steps consisting in:
a) placing a cartridge as claimed in one of claims 1 to 7 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection means,
e) placing the means for recovering nucleic acids in the cartridge,
f) introducing a liquid of interest into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, and
h) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.
0. A method for the extraction of nucleic acids from microorganisms contained in the air, said method comprising the steps consisting in:
a) placing a cartridge as claimed in one of claims 1 to 7 inside the air collection means, such that the retaining zone, inside said cartridge, is in communication with the air inlet duct of the air collection means,
b) causing air to enter said air collection means by any appropriate means,
c) concentrating the microorganisms contained in the air, in the retaining zone of the cartridge,
d) removing the cartridge from the air collection means,
e) fitting the means for recovering nucleic acids into the cartridge,
f) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids,
g) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, thus releasing the nucleic acids from said microorganisms, and
h) causing the liquid of interest in the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the
liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.
21. The method as claimed in one of claims 17 to 20, comprising an additional step d') consisting in growing the concentrated microorganisms in the retaining zone of the cartridge.
22. The method as claimed in the preceding claim, in which the growing is obtained by incubation of the cartridge in an incubator for a period of time ranging from 2 to 24 hours.
23. A method for the lysis of microorganisms, said method comprising the steps consisting in:

a) obtaining a cartridge, as claimed in one of claims 1 to 7, in which microorganisms are concentrated in the retaining zone,
b) fitting the means for recovering nucleic acids into the cartridge,
c) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids, and
d) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.
24. A method for the lysis of microorganisms, said method comprising the stepsconsisting in:
a) obtaining a cartridge, as claimed in one of claims 1 to 7, in which
microorganisms are concentrated in the retaining zone,
b) placing the means for recovering nucleic acids in the cartridge,
c) introducing a liquid of interest into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
d) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.
A method for the extraction of nucleic acids from microorganisms, said method comprising the steps consisting in:
a) obtaining a cartridge, as claimed in one of claims 1 to 7, in which microorganisms are concentrated in the retaining zone,
b) fitting the means for recovering nucleic acids into the cartridge,
c) causing the delivery of a liquid of interest previously placed in the storage zone of the means for recovering nucleic acids, said delivery being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid thus delivered filling the interstitial space located between the means for recovering nucleic acids and the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, said lysis means coming to sit between the vertical internal wall of the cartridge and the vertical external wall of the means for recovering nucleic acids,
d) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, thus releasing the nucleic acids from said microorganisms, and
e) causing the liquid of interest in the storage zone of the means for recovering nucleic acids to be drawn up, said drawing up being obtained by means of the drawing up/delivering means of the means for recovering nucleic acids, the liquid of interest thus drawn up containing the nucleic acids of said microorganisms, released during the lysis.
26. A method for the extraction of nucleic acids from microorganisms, said method
comprising the steps consisting in:
a) obtaining, as claimed in one of claims 1 to 7, a cartridge in which microorganisms are concentrated in the retaining zone,
b) placing the means for recovering nucleic acids in the cartridge,
c) introducing a liquid of interest into the cartridge, which leads to the lysis means located in the microorganism retaining zone of the cartridge being placed in suspension, and
d) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained, and
e) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.
27. A method for the lysis of microorganisms, said method comprising the steps
consisting in:
a) introducing a liquid sample containing said microorganisms into a cartridge as claimed in one of claims 1 to 7, in the vicinity of the retaining zone, such that said liquid sample leads to the lysis means located in said microorganism retaining zone of the cartridge being placed in suspension,
b) placing the means for recovering nucleic acids in the cartridge,
c) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained.
28. A method for the extraction of nucleic acids from microorganisms, said method
comprising the steps consisting in:
a) introducing a liquid sample containing said microorganisms into a cartridge as claimed in one of claims 1 to 7 in the vicinity of the retaining zone, such that said liquid sample leads to the lysis means located in said microorganism retaining zone of the cartridge being placed in suspension,
b) placing the means for recovering nucleic acids in the cartridge,
c) mechanically lyzing the microorganisms by rotating the means for recovering nucleic acids, inside the cartridge, said means for recovering nucleic acids rotating the lysis means on which the microorganisms are retained,
d) drawing up the liquid of interest containing the nucleic acids of said microorganisms, released during the lysis.
29. A method for identifying one or more microorganisms contained in a sample,
comprising the steps consisting in:
isolating the nucleic acids from the microorganisms contained in said sample by means of the device as claimed in one of claims 8 to 13, identifying the microorganism(s) thus isolated.
30. The method of identification as claimed in the preceding claim, also comprising an intermediate step consisting in purifying the nucleic acids.
31. The method of identification as claimed in either of claims 29 and 30, in which the identification step comprises the substeps consisting in:
specifically amplifying the isolated nucleic acids, detecting the nucleic acids thus amplified.
32. The method of identification as claimed in one of claims 29 to 31, in which the identification step is carried out in an identification device in fluid communication with the cartridge of the device as claimed in one of claims 10 to 13.
33. The method of identification as claimed in the preceding claim, in which the isolated nucleic acids are transferred from the means for recovering nucleic acids, of the device as claimed in one of claims 10 to 13, to the identification device.
34. The method of identification as claimed in the preceding claim, in which the transfer of the nucleic acids is obtained by delivery of the liquid of interest containing the nucleic acids, said liquid of interest being contained in the storage zone of the means
for recovering nucleic acids, by means of the drawing up/delivering means of the means for recovering nucleic acids.