Abstract
The present invention relates to a ready to use ambient stable diagnostic kit with extendedself life, containing dry powder primarily, with minimum aqueous stable components likethe DNA probe. The kit has an added advantage of detecting microorganisms directlyfrom clinical as well as biopsy or autopsy samples in situ, without requiring the timeconsuming and cumbersome strain or gene purifications. The DNA probe is generatedand detected by Quantum dots or other visible colors.Being a simple and uniform procedure, the kit and the method can be used by Semiskilled workers like Technicians, dispensing the need for large and differentinfrastructure and expertise for identification of different infections, even under fieldconditions.
Specification
FIELD OF INVENTION:
The present invention relates to DNA diagnostic kit. The present invention in particular relates to
DNA diagnostic kit for identification of different microorganisms as well as geneti~ diseases.
BACKGROUND AND PRIOR ART:
Identification of microorganisms is traditionally made by the combination of microbiological,
biochemical, physical, in vivo animal experimentation, cell culture and other methods. This
necessitates different infrastructure, biological and specialized expertise for identification of
different microorganisms causing infection and diseases like cancer. These methods are
expensive but are unavoidable need of human, animal and plants. The constituents are short lived
and highly perishable and require cryo-preservation. All these methods involve purification of
strains of microorganisms by repeated culture and subculture which is time consuming,
cumbersome and expensive. In spite of all the above limitations, these methods, quite often lead
to false positive or false negative detections, because of reasons like gene expression, presence of
dead and debilitated micrQ2rg@llisms, insensitivities, interference growth,, cross reactivity etc.
Molecular methods of detections DNA probe is a· well known molecular method with varied
applications like identification of infection in man, animals and plants, identification of genetic
·diseases like cancer, identification of recombinant clone, DNA fingerprinting etc. It is also being
used extensively in forensic sciences
Publication No. KR20120038430 provides a probe for detecting pathogenic microorganism
causing diseases infected by sexual contact is provided to ensure high specificity and sensitivity.
Publication No. KR20110137642 provides a DNA cllip containing a probe which
complementarity binds to 44 types of HPV nucleic acids is provided to accurately diagnose
complex infection ofHPV and to predict..cervical cancer
..,
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Publication No. CNtOlSIS213 discloses a gene chip for detecting human papiUomavirus (HPV),
comprising a solid carrier and a human papiHomavirus detecting probes fixed on the solid
earner.
Publication No. KR100968360 describes a method for diagnosing breast cancer caused by
variation of the number of Her-2 gene replication is provided to ~onfirm amplif~:attion by
variation of the number ofHer-2 gene and to diagnose breast cancer.
Publication No. CN101240335 provides a gene chip for detecting common pathogen in dairy,
which comprises a solid-phase vector and an oligonucleotide probe fixed on the solid-phase
vector. It also provides a gene chip for detecting common pathogen in dairy, which comprises a
solid-phase vector and an oligonucleotide probe fixed on the solid-phase vector, wherein the
oligonucleotide probe includes DNA fragment
Pubiicatio.Q.Nff. CN 10140783 7 describes a gene chip used for detecting blood pathogen and a kit
used for detection. The gene .chip includes ·a solid phase vector and an oligonucleOtide probe
fixed on the solid phase vector, wherein the oligonucleotide probe mainly comprises DNA
segments selected from the 16S rRNA gene sequence.
Publication No. KR20080011257 provides a kit comprising a gene probe capable of detecting a
gene of 16S rRNA commonly included in a ~aterborne pathogen is provided to~ able to deteCt
the waterborne pathogen contaminated by a plurality of samples and identify the. detected
pathogen individually, thereby being widely and effectively utiiized for preventing waterbofi1e .
infectious diseases.
Publication No. KR20 100006282 describes a multiplex kit and chip for 11etermining pathogen
analysis and antibiotics resistance are provided to accurately ronfirm .pathogen 'Qf respiratory
iRfection and reduce antibiotics resistance rate.
-4-
Becton Drive,Franklin Lakes, USA provides BD Affirm™ VPIU DNA probe that offers a
dependable, rapid means for the differential detection and identification of the causative agents
for vaginitis: Candida species, Gardnerella vaginalis and Trichomonas vagina/is .. The test
features an easy..:to-read visible color reaction that is more accurate than current microscQpic
methods for detecting the causative agents of vaginitis.
PathVysion provides a HER-2 DNA probe which is designed to detect amplification of the HER-
2/neu gene via fluorescence in situ hybridization (FISH) in formalin-fixed, paraffin-embedded
human breast cancer tissue specimens.
MetaSystems offers a wide range of high quality DNA probes routinely applied in genetics and
allows nearly unlimited and targeted visualization of genomic DNA using either metaphase
spreads, interphase nuclei, tissue sections, or living cells:
- "'Life Technologies Corporation provides MicroSEQ® detection kit for simply, reliably and
rapidly detecting Listeria spp. in fOod and environmental sampfes. The assay uses the
polymerase chain reaction (PCR) to amplify a unique microorganism-specific DNA target
sequence and a TaqMan® probe to detect the amplified sequence. This system comes with a
PrepSEQ™ Nucleic Acid Extraction Kit.
However, the kits available in prior arts have remained as a high-:tech .and an expensive affair
dealing with highly degradable and perishable components requiring. adequate. expertise and
laboratory facilities.
The present invention provides -stable, inexpensive DNA diagnostic kit and a technofogy fm-tbe
identification of microorganisms as well as genetic diseases such as but not iimited to caooer
-even under field conditions.
OBJECTS OF THE INVENTION:
-5-
The .principal object of the present invention is to provide, ready to use diagnostic kit
provided in the form of dry powder with minimum aqueous stable -components like the DNA
probe.
Another object of the present invention is to provide a diagnostic kit which detects
microorganisms directly from clinical samples without the need for time consuming and
cumbersome strain purifications
Yet another object of the present invention is to provide a diagnostic kit which is simple,
inexpensive and uniform for all microorganisms and genetic diseases such as but not limited to
cancer.
Still another object of the present invention is to provide a diagnostic kit which can be used
by semi skilled persons
Yet another object of the present invention is to provide dia&nostic kit having extended shelf
life under ambient conditions.
Still another object of the present invention is to provide a diagnostic kit which dispenses the
necessity of a large and different infrastructure and different expertise for identification of
different infections and diseases.
At the outset of the· description that follows, it. is to be unde~stood that the ensuing
description only ·illustrates a particular form of this invention. Howev~r, such a particular .
form is only an exemplary embodiment and is not intended to be -taken restrictively to
imply any limitation on the scope of the present invention~
SUMMARY OF THE INVENTION:
The present invention provides a technology and a diagnostic kit in the form of dry powder with
minimum aqueous stable components like the immortalized DNA probe in a ready to use state.
The kit is simple, inexpensive and unif-orm for all micr-oorganisms and genetic drneases -such as
but not limited to cancer. The kit has an extended (may be infinite) shelf life under ambient
renditions. The·disclosed kit i-s ~able -of detecting almost every infection and ~ic disea-ses
-6- c such as but not limited to cancer simply by changing the DNA probe sequence. Being uniform, it
can be used by semi skilled workers {ike the technician, in Reference Laboratories or even under
Field Conditions.
The present invention dispenses the need of a large and different infrastructuce and different
expertise for identification of different infections and diseases in human, animal and plants.
In a preferred embodiment of the present invention, the ambient stable, inexpensive DNA
diagnostic kit is provided with an immortalized aqueous stable and ready-to-use DNA probe and
it follows only a single, simple and uniform method for identification.
In another embodiment of the present invention, the DNA kit with an immortalized aqueous
stable and ready-to-use DNA probe can be used for the identification of microorganisms as well
as genetic and metabolic diseases in human, animal a~d plants, such as but not limited to cancer
and under field conditions.
In ·yet another embodiment of the present invention, the DNA kit is provided with an
immortalized aqueous stable and ready-to-use DNA probe on cellulose acetate (MIS Whatman
541) membrane which is not fragile unlike the commonly used nitrocellulose and also does nQt
require baking at 80°C under vacuum for one hour or UV cross-linking as required for different
membranes to retain the DNA on the membrane.
· In still another embodiment of the prese~t invention, the DNA kit is ·provided with ready-touse
immortalized DNA probe on cellulose acetate membrane.
In still another embodiment of the present invention, the DNA kit dispense the need fQf
vacuum baking or UV cross-linking also dispenses pre-hybridization before DNA-DNA
hybridization of test sample and also enables rapid detection and better sensitivity and specificity
<>f detection than other routinely used membranes andmetOOds.
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, In yet another embodiment of the present invention, the present DNA kit is provided with an
immortalized aqueous stable and ready-to-use DNA probe that has been ~abcled or bound with
Quantum Dots (QDs) or nanoparticles by the functional muiety on the particle.
In still another embodiment of the present invention, the immortal DNA kit -can use -chemical
labeling and detection of DNA-DNA hybridization in an ambient-stable manner.
In yet another embodiment of the present invention, the kit dispenses the need of sterilization
and pH adjustment
In yet another embodiment of the present invention, the DNA kit with an immortalized
aqueous stable and ready-to-use DNA probe detect the sample by Auto-Fluorescence of the
!?article that can be viewed under UV lamp/torch or by naked eye, preferably in dark.
In still another embodiment of the present invention, the present DNA kit can- use fluorescent dye &/or enhancers to enhance the signal enabling easy detection of DNA-DNA hybridization on
white membrane.
Yet another embodiment of the present invention is that the quantum dots or nanoparticles
can be prevented from joining up by silver, silica etc. coating .
.. In still another embodiment of the present invention, the DNA kit r reUs briefly
-contacted from a agar culture plate (by Dot blot or Colony hybridizatk>n) on the membrane and
tysed.
Atso tbe double stranded DNA is denatured to single stranded .furm and 'ihe "Single stranded
DNA then again is bound t.o the membrane easily by a rapid and single step<>f exposure
