Claims:WE CLAIM:
1. An assay for diagnosing sepsis or septicaemia in a biological sample through detection of plurality of pathogens causing sepsis or septicaemia comprising:
extracting DNA from said biological sample;
amplifying said DNA through three optimised multiplex Polymerase Chain Reactions (PCRs) comprising a plurality of primers specific to a plurality of target genes of said plurality of pathogens and said plurality of primers depicted by SEQ ID NO 1 to SEQ ID NO 36; said three optimised multiplex PCR mixtures comprise a first PCR mixture having primers depicted by SEQ ID NO 1 to SEQ ID NO 14, a second PCR mixture having primers depicted by SEQ ID NO 15 to SEQ ID NO 26, and a third PCR mixture having primers depicted by SEQ ID NO 27 to SEQ ID NO 36.

2. The assay as claimed in claim 1, wherein said pathogens are bacterial and/or fungal.

3. The assay as claimed in claim 2, wherein said bacterial pathogens are detected as gram-positive or gram-negative.

4. The assay as claimed in claim 3, wherein said gram-positive bacterial pathogens comprising Staphylococcus aureus, Group-B Streptococcus, Streptococcus pneumonia, Streptococcus pyogenes, and Enterococcus species and said gram-negative bacteria comprising Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Enterobacter aerogenes, Proteus mirabilis, Neisseria meningitidis, Hemophilus influenzae-B, Acinetobacter baumanii, Leptospira species, Pseudomonas aeruginosa, Bacteroides fragilis.

5. The assay as claimed in claim 1, wherein said fungal pathogens comprising Candida albicans and Aspergillus species.

6. The assay as claimed in claim 1, wherein said plurality of target genes are selected from Dnase B, EF-Tu gene, ompP6, crgA gene, scpB gene, Nuc gene, Enterotoxin gene, 5.8s rRNA, InvA gene, 18s rRNA, Stxholotoxin A & B subunit, oprL gene, PBP 2B gene, Oxa gene, carbapenemase (blaKPC-2) gene, BlaSHV12-TR gene, ureA and ureF genes.

, Description:FIELD OF THE INVENTION
The present embodiment relates to molecular techniques for detection and diagnosis of septicaemia or sepsis, and more particularly to oligonucleotides for detection of bacterial and fungal pathogens responsible for sepsis or septicaemia, through a multiplex PCR assay.
BACKGROUND OF INVENTION
Septicaemia is systemic illness of a body due to invasion or infection of the bloodstream by virulent bacteria and fungi, often originating from a local site of infection. Septicaemia is a form of Blood Stream Infection (BSI) and is at times called as BSI too. Septicaemia is also known as blood poisoning if the presence of the microbial toxins is observed in the blood stream. Other known name of septicaemia is bacteraemia. If septicaemia is left untreated, it often progresses to a life-threatening condition called Sepsis, in which a body's response to infection injures its own tissues and organs.
Septicaemia is caused often by an infection in one or the other part of body. Many types of microbes including bacteria and fungi can cause septicaemia. The most common infections that lead to septicaemia are urinary tract infections, lung infections, such as pneumonia, kidney infections and infections in the abdominal area.
BSIs show high rates of morbidity and mortality throughout the world. These conditions, particularly sepsis, typically, but not always, result from an interaction between a pathogenic microorganism and the host's defense system that triggers an excessive and dysregulated inflammatory response in the host. Early detection of the disease condition allows for a more effective therapeutic treatment with a correspondingly more favourable clinical outcome. Inappropriate antimicrobial treatment is a major concern, reason for which is, lack of coverage of the underlying pathogen and antimicrobial resistance of the causative pathogen in infections by emerging multi drug resistant microorganisms. Thus, an incomplete understanding of the disease pathogenesis, in turn, contributes to the difficulty in finding useful diagnostic biomarkers. Early and reliable diagnosis is imperative, however, because of the remarkably rapid progression of sepsis into a life-threatening condition.
Early and accurate detection of the pathogenic microorganisms that are clinically significant to sepsis has proven difficult. Causative microorganisms are typically detected by culturing a subject's blood, sputum, urine, wound secretion, in-dwelling line catheter surfaces, etc. Causative microorganisms, however, may reside only in certain body microenvironments such that the sample that is cultured may not contain the contaminating microorganisms. Detection may be complicated further by low numbers of microorganisms at the site of infection. Low numbers of pathogens in blood present a particular problem for diagnosing sepsis by culturing blood. In one study, for example, positive culture results were obtained in only 17% of subjects presenting clinical manifestations of sepsis. Diagnosis can be further complicated by contamination of samples by non-pathogenic microorganisms. For example, a study found that only 12.4% of detected microorganisms were clinically significant in a study of 707 subjects with septicaemia.
Yet, blood culture methods remain a gold standard in diagnosis and detection of blood stream pathogens although they fall short in accurate and timely diagnosis because of risk of contamination, long time taken, less sensitivity and specificity. These methods have evolved towards fully automated systems, thereby removing the risk of contamination and associated errors, but still the overall time taken is far too long for clinicians to take immediate treatment decisions.
In order to overcome the limitations of blood or biological fluid culture methods, several molecular detection techniques have been implemented in the recent years for detection of pathogen DNA and aim at improvisation in terms of time taken for detection, cost involved, sensitivity, specificity and number of pathogens to be detected in a single step. The sensitivity of such assays depends on yield of DNA obtained, which is often challenged by factors like laboratory cross contamination, low pathogen load etc. The isolation and detection of fungal DNA is even more difficult owing to thick fungal cell walls and low pathogen load.
Fluorescent in situ hybridization (FISH) is among the most studied commercial techniques suitable for detection of pathogens in positive blood cultures. FISH may identify more than 95% bacteria and yeasts commonly found in blood within 2.5- 3 hours approximately. Despite being specific, these methods pose the limitation that certain bacteria may be identified at the genus level only if no species-specific probes are available.
AdvanDx, USA uses peptide nucleic acid probes targeting 16s rRNA for direct identification of S. aureus from positive blood cultures within 3 hours. However, these hybridisation-based methods require microorganisms to be grown in blood cultures followed by single colony growth on solid media. This results in a delay in time and also introduces a bias based on culture methods employed. WO 2007083852 discloses a microarray-based method for detection of sepsis causing bacteria through genus specific as well as species specific probes and the method is also capable of distinguishing between gram-positive and gram-negative bacteria.
Recently, several broad range PCR amplification-based methods have been implemented that recognize conserved sequences of bacterial/fungal genes. However, these methods pose limitations, as further genus/ species-specific identification procedures are required to decide upon exact mode of treatment to be taken.
US20120088696 A1 discloses a cost-effective disposable micro-electrochemical multiplex real-time PCR platform that can be used to rapidly amplify, examine, and quantify target nucleotides in real-time, and can be used in sepsis diagnosis. Although, the reference discloses a high throughput and technically sound platform for detection of several pathogen specific biomarker sequences, the challenge lies in choice of biomarkers, primers and protocol design for simultaneous detection of differently sized amplicons and as such in for detection of septicaemia or sepsis much remains unavailable or undeveloped.
WO2014189398A1 discloses a multiplex PCR based method for simultaneous detection of bacteria and fungi in a given sample. This method differentiates fungi into mould fungi & yeast fungi while bacteria are discriminated as gram-negative and gram-positive bacteria. This necessitates further genus and species-specific identification by methods like hybridization and sequencing.
Hyplex Blood Screen (BAG, Germany) is a multiplex PCR assay that identifies 10 bacterial species from positive blood cultures using an ELISA-like format. Bacteria identified by this method include Streptococcus spp, Staphylococcus spp, Enterococcus spp, Enterobacter, Pseudomonas & Kleibsiella spp. The detection time taken by this method is 3-4 hours approximately and the reported sensitivity and specificity ranges from 96.6%-100% & 92.5%-100% respectively.
StaphPlex system (Qiagen USA) is yet another multiplex PCR based assay that amplifies and detects 18 Staphylococcus spp genes as well as few drug resistance marker genes simultaneously in a single reaction. The assay gives results demonstrating up to 100% sensitivity within 5 hours. However, the assay provides no detection for fungal pathogens responsible for sepsis or septicaemia.
All methods described above require microorganisms to be grown in blood cultures followed by single colony growth on solid media and hence are dependent on positive blood culture which brings in some uncertainty and are time- consuming as discussed previously.
SepsiTest (Molzym, Germany) is broad range PCR based detection and sequence identification system for organisms causing sepsis. The assay detects & amplifies conserved bacterial & fungal 16S and 18S rRNA genes in a sample, following which sequence analysis is performed to identify more than 300 bacteria and fungi within a span of 8-12 hours. However, SepsiTest first requires Human DNA to be isolated or pathogenic DNA to be enriched before the detection is carried out. This is the reason for the increased sensitivity of the test. As may be seen, this adds an additional step of removal of human or sample DNA. VYOO® (SIRS-Lab GmbH, Jena) is another multiplex PCR-based assay that combines a bead-based mechanical lysis protocol, followed by separation and relative enrichment of microbial DNA from background human DNA, and a multiplex PCR protocol. It enables detection of 34 bacterial, 7 fungal and 5 most common antibiotic resistance genes in a given sample. Since, the assay firstly removes human DNA and enriches pathogen DNA by affinity chromatography, followed by gel based extraction. It results in 10 fold improvement in sensitivity. The average turn-around time is 8 hours. The results obtained by Vyoo kit appear satisfactory, but they are complex and time consuming as they involve DNA enrichment, which also adds to the overall cost of the assay.
Despite having a number of efficient DNA-based detection methods, a need exists for development of more cost effective, reproducible and universal detection assays/methods that can reliably detect all possible pathogen types with improved specificity and sensitivity, sufficiently early to allow effective intervention and prevention, without any complicated processes such as enrichment of pathogenic DNA or removal of human DNA.
Accordingly, there is a dire need to expand the scope of the methods and assays to detect sepsis or septicaemia to include a variety of bacterial species as well as fungal pathogens on the panel that can be detected simultaneously, directly via a biological fluid e.g. blood, to give fast and reliable results in patients suffering from septicaemia or sepsis.
SUMMARY OF THE INVENTION
As mentioned in the foregoing, there is a need for development of a comprehensive method or assay for detection of sepsis or septicaemia, the embodiment herein present a method and assay for detection of sepsis/septicaemia with optimised multiple PCR mixtures.
In an aspect, an assay for diagnosing sepsis or septicaemia in a biological sample through detection of number of pathogens causing sepsis or septicaemia is provided. The assay includes: extracting DNA from the biological sample; amplifying the DNA through a number of multiplex Polymerase Chain Reactions (PCRs) by preparing three optimised multiplex PCR mixtures including number of primers specific to a number of target genes of the number of pathogens and depicted by SEQ ID NO 1 to SEQ ID NO 36. The three optimized multiplex PCR mixtures are made so that their amplicons don’t overlap to produce false results or indicate false diagnosis, and thereby enabling accurate diagnosis. The pathogens detected by the assay are bacterial and fungal pathogens. The bacterial pathogens are detected as either gram-positive or gram-negative. The gram-positive bacterial pathogens, as detected by the assay, are Staphylococcus aureus, Group-B Streptococcus, Streptococcus pneumonia, Streptococcus pyogenes, and Enterococcus species and the gram-negative bacteria includes Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Enterobacter aerogenes, Proteus mirabilis, Neisseria meningitidis, Hemophilus influenzae-B, Acinetobacter baumanii, Leptospira species, Pseudomonas aeruginosa, Bacteroides fragilis. And the fungal pathogens include Candida albicans and Aspergillus species.
In another aspect, three multiplex PCR mixtures are prepared; wherein a first PCR mixture includes primers depicted by SEQ ID NO 1 to SEQ ID NO 14, a second PCR mixture includes primers depicted by SEQ ID NO 15 to SEQ ID NO 26, and a third PCR mixture includes primers depicted by SEQ ID NO 27 to SEQ ID NO 36.

The target genes to be amplified by the first PCR mixture include Dnase B, EF-Tu gene, ompP6, crgA gene, scpB gene, Nuc gene and Enterotoxin gene, primers for which are depicted by SEQ ID NO 1 to SEQ ID NO 14 and the pathogens detected by them include Streptococcus pyogenes, Enterococcus, Haemophilus influenza, Neisseria meningitides, Group B Streptococcus, Staphylococcus aureus and Bacteroides fragilis.
The target genes to be amplified by the second PCR mixture include 5.8s rRNA, InvA gene, 18s rRNA, Stxholotoxin A & B subunit and oprL gene, primers for which are depicted by SEQ ID NO 15 to SEQ ID NO 26 and the pathogens detected by them include Candida species, Salmonella, Leptospira, Aspergillus species, Escherichia coli and Pseudomonas aeruginosa.
The target genes to be amplified by the third PCR mixture include PBP 2B gene, Oxa gene, carbapenemase (blaKPC-2) gene, BlaSHV12-TR gene and urea, ureF genes, primers for which are depicted by SEQ ID NO 27 to SEQ ID NO 36 and the pathogens detected by them include Streptococcus pneumoniae, Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter aerogenesand Proteus mirabilis.
In another aspect, the assay for diagnosing sepsis or septicaemia in a biological sample through detection of number of pathogens causing sepsis or septicaemia by amplifying the DNA of select pathogens through a number of multiplex Polymerase Chain Reactions may include mixtures other than the three PCR mixtures described herein, by arranging the genes based on amplicon size.
The preceding is a simplified summary to provide an understanding of some aspects of embodiment of the present assay. This summary is neither an extensive nor exhaustive overview of the present detection assay and its various embodiment. The summary presents selected concepts of the embodiment of the present assay and method in a simplified form as an introduction to the more detailed description presented below. As will be appreciated, other embodiment of the present assay and method are possible utilizing, alone or in combination, one or more of the features set forth above or described in detail below.
BRIEF DESCRIPTION OF THE DRAWING
The above and still further features and advantages of embodiment of the present assay to detect sepsis or septicaemia will become apparent upon consideration of the following detailed description of embodiment thereof, especially when taken in conjunction with the accompanying drawings, and wherein:
FIG. 1 illustrates a flow diagram depicting a method of detection of sepsis or septicaemia, according to an embodiment herein;
FIG. 2 illustrates a table depicting list of bacterial and fungal pathogens together with their target genes as detected by the method of detection of sepsis or septicaemia of FIG. 1, according to an embodiment herein;
FIG. 3 illustrates a table depicting the primer sequences specific to each of the bacterial and fungal pathogen, depicted in FIG. 2, for use in detection of sepsis or septicaemia , according to an embodiment herein; and
FIG. 4A-4C illustrates the resuts as obtained after running the PCR amplified DNA sequences on an electrophoresis system for detection of sepsis/sepaticaemia causing pathogens, according to an embodiment herein along with details of the targets gene and their amplicon sizes .
To facilitate understanding, like reference numerals have been used, where possible, to designate like elements common to the figures.
DETAILED DESCRIPTION OF THE INVENTION
As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words “include”, “including”, and “includes” mean including but not limited to.
The phrases “at least one”, “one or more”, and “and/or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and/or C” means A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
The term “a” or “an” entity refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.
The term “PCR” refers to Polymerase Chain Reaction, which is a molecular biology technique to amplify a single copy or multiple copies of a DNA sequence to generate millions of copies of the sequence before subjecting the sequence for further detection and diagnostic assays.
The term “DNA” refers to the deoxyribonucleic acid, a nucleic acid molecule that carries the genetic information of an organism, humans, microbes, plants or otherwise, necessary for their growth, functioning, development and reproduction. The term “RNA” refers to ribonucleic acid, another nucleic molecule that is obtained from DNA via translation, before it is transcribed to produce several proteins.
The term “assay” refers to any investigative procedure done in laboratory or otherwise for qualitative and quantitative determination of a target(s) (or analyte(s)).
The term “PCR mixture” refers to a reaction mixture to be subjected to PCR. In a typical or single PCR, a PCR mixture has provision for amplifying only one DNA sequence through a single set of primer specific to the target DNA sequence; whereas, in a multiplex PCR, a PCR mixture has provision for amplifying multiple DNA sequences of interest through multiple set of primers specific to the multiple DNA sequences of interest.
As mentioned, there is a dire need for developing specialised assays and methods for detection of sepsis or septicaemia that overcome limitations of the prior art, the embodiment herein provide a comprehensive method and assay to detect presence of both bacterial and fungal pathogens responsible for sepsis or septicaemia by providing high throughput PCR mixture(s), for both single PCRs and multiplex PCR, such that the sensitivity or specificity of the detection of pathogens isn’t compromised.
FIG. 1 illustrates a flow diagram depicting a method of or assay for detection of sepsis or septicaemia, according to an embodiment herein. The method or assays 100 includes obtaining DNA through DNA extraction 102 from a biological sample or fluid. In an embodiment, the biological sample is any biological fluid or tissues such as blood, stool, urine, plasma, a tissue etc. In a preferred embodiment, the DNA is extracted from a blood sample. In an embodiment, the DNA is extracted through established DNA isolation or extraction techniques following general procedure of lysing the cell walls to expose the DNA, subjecting the exposed DNA to surfactants to remove membrane lipids, followed by removal of proteins by adding proteases to the exposed DNA, and subsequently removal of RNA by subjecting exposed DNA to RNase. Further, the DNA may be purified using established techniques such as precipitation or column-based extractions. The PCR mixtures are so selected on basis of the size of the amplicon of each of the target genes during PCR, in order to enable efficient PCR.
The DNA extraction 102 is followed by preparation 104 of reagents. In an embodiment, the reagents to be prepared are for single as well as multiplex PCRs. In a preferred embodiment, the reagents include dNTP mixture, Taq DNA polymerase with buffer, Agarose, Nuclease-free water, Tris/Borate/EDTA (TBE) Buffer, Ethidium Bromide and DNA ladder. The preparation 104 of reagents is followed by preparation 106 of primers or set of primers for single as well as Multiplex PCR mixtures for confirmed bacterial and fungal pathogens responsible for sepsis or septicaemia. Figure 2 illustrates a list of the bacterial and fungal pathogens detected by assay or the method of Figure 1, according to an embodiment herein, together with the target gene for each of the bacterial and fungal pathogen. The bacterial pathogens are either gram-positive or gram-nevgative. The gram-positive bacteria inclue Staphylococcus aureus, Group-B Streptococcus, Streptococcus pneumonia, Streptococcus pyogenes, Enterococcus species and the gram-negative bacteria includes Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, Enterobacter aerogenes, Proteus mirabilis, Neisseria meningitidis, Hemophilus influenzae-B, Acinetobacter baumanii, Leptospira species, Pseudomonas aeruginosa, Bacteroides fragilis. The fungal pathogens include Candida albicans and Aspergillus species.
Further, the assay also detects 16s rRNA conserved region. Figure 3 illustrates the primer sequences specific to each of the bacterial and fungal pathogen responsible for sepsis or septicaemia. The various primer sequences, specific to the multiple organisms or microbes for several target genes, are referred to as SEQ ID NO 1, SEQ ID NO 2 and so on until SEQ ID NO 36 as shown in Figure 3. The target sequences are specific for detecting gram-positive as well as gram-negative bacteria.
The preparation 106 of the primers is followed by preparation 108 of several PCR mixtures. In a preferred embodiment, three PCR mixtures are prepared as shown in FIG. 3, including primers specific for target genes in different organisms. In a preferred embodiment, three PCR mixtures are prepared. The first PCR mixture includes primers depicted by SEQ ID NO 1 to SEQ ID NO 14, the second PCR mixture includes primers depicted by SEQ ID NO 15 to SEQ ID NO 26, and the third PCR mixture includes primers depicted by SEQ ID NO 27 to SEQ ID NO 36. In an embodiment, a single PCR mixture includes all the primers from SEQ ID NO 1 to SEQ ID NO 36 to detect all the pathogens in one single step. However, it is possible to have reduced sensitivity and specificity in detection of certain pathogens, hence three PCR mixtures presented here are optimised PCR mixtures to enable rapid detection with increased specificity and sensitivity. The target genes for primers are selected from Dnase B, EF-Tu gene, crgA gene, scpB gene, Nuc gene, Enterotoxin gene, 5.8s rRNA, InvA gene, 18s rRNA, Stxholotoxin A & B subunit, oprL gene, PBP 2B gene, Oxa gene, carbapenemase (blaKPC-2) gene, BlaSHV12-TR gene, ureA and ureF genes.
The three PCR mixtures including different set of primers for different set of pathogens are then subjected 110 to PCR under optimal PCR conditions specific to the three PCR mixtures.
Examples
Example 1: Preparation of reagents:
The reagents as prepared were as follows:
10mM dNTP Mixture
Taq DNA polymerase with buffer
Agarose, Nuclease-free water
0.5X TBE Buffer
10mg/ml Ethidium Bromide
50 bp or 100bp DNA ladder .
The above reagents were used in obtaining PCR mixtures and performing PCRs.
Example 2: Preparation of Primers:
Each of the primers, from SEQ ID NO 1 to SEQ ID NO 37 was dissolved in sterile 1X TE buffer to obtain a final concentration of 100 ?M. This served as stock primer solutions. The stock primer solutions were diluted in sterile nuclease-free water to obtain a final concentration of 10 ?M to obtain working primer solutions. The working primer solutions (10 µM) were freshly diluted after a period of one month.
Example 3: Preparation of PCR Mixtures:
Three PCR mixtures Mix A, Mix B and Mix C were prepared as shown below:
1. Mix-A
2X Qiagen Multiplex PCR Buffer 12.0 µl
Primer (10 ?M): SAF 0.5 µl
Primer (10 ?M): SAR 0.5 µl
Primer (10 ?M): PAF 0.5 µl
Primer (10 ?M): PAR 0.5 µl
Primer (10 ?M): Nmen F 0.5 µl
Primer (10 ?M): Nmen R 0.5 µl
Primer (10 ?M): ECF 0.5 µl
Primer (10 ?M): ECR 0.5 µl
Nuclease free water 0.8 µl _________________________________________________________________

2. Mix-B
2X Qiagen Multiplex PCR Buffer 6.0 µl
Primer (10 ?M): GRB Strep F 0.5 µl
Primer (10 ?M): GRB Strep R 0.5 µl
Primer (10 ?M): Salmoty 59 0.5 µl
Primer (10 ?M): Salmoty 60 0.5 µl
Nuclease free water 0.2 µl __________________________________________________________________ Total volume per reaction 8.0 µl
3. Mix-C
2X Qiagen Multiplex PCR Buffer 12.0 µl
Primer (10 ?M): P. mira F 0.3 µl
Primer (10 ?M): P. mira R 0.3 µl
Primer (10 ?M): Kleb F 0.5 µl
Primer (10 ?M): Kleb R 0.5 µl
Primer (10 ?M): Enterococcus F 0.3 µl
Primer (10 ?M): Enterococcus R 0.3 µl
Nuclease free water 1.8 µl
______________________________________________________________________
Total volume per reaction 16.0 µl

Example 4: Performing PCR and visualising results
The three PCR mixtures were subjected to PCR together with the single PCR mixtures at conditions optimal to each of the PCR mixtures.

1. Mix-A
Initial denaturation: 95?C/2 mins
Denaturation: 95?C/1 min
Annealing: 40?C/1 min 5 cycles
Extension: 72?C/45 secs
Denaturation: 95?C/45 secs
Annealing: 50?C/45 secs 35 cycles
Extension: 72?C/45 secs
Final Extension: 72?C/5 mins
Hold: 4?C/8
2. Mix-B and Mix-C

Initial denaturation: 94?C/3 mins
Denaturation: 94?C/45 secs
Annealing: 58?C/45 secs 40 cycles
Extension: 72?C/45 secs

Final Extension: 72?C/10 mins
Hold: 4?C/8
FIG. 4A-4C illustrates the resuts as obtained after running the PCR amplified DNA sequences on an electrophoresis system for detection.
The results as visualised for FIG. 4A were summarised as:
Lane No. Sample ID Organism Expected product size (bp) Result
1 100 bp DNA Ladder
2 Mix A Streptococcus pyogenes 93 bp Positive
Enterococcus 112 bp Positive
Haemophilus influenzae 156 bp Positive
Neisseria meningitidis 230 bp Positive
Group B Streptococcus 255 bp Positive
Staphylococcus aureus 279 bp Positive
Bacteroides fragilis 368 bp Positive

The results as visualised for FIG. 4B were summarised as:
Lane No. Sample ID Organism Expected product size (bp) Result
1 100 bp DNA Ladder
2 Mix B Candida species 110 bp Positive
Salmonella 244 bp Positive
Leptospira 274 bp Positive
Aspergillus species 362 bp Positive
Escherichia coli 482 bp Positive
Pseudomonas aeruginosa 504 bp Positive

The results as visualied for FIG. 4C were summarised as:
Lane No. Sample ID Organism Expected product size (bp) Result
1 100 bp DNA Ladder
2 Mix C Streptococcus pneumoniae 106 bp Positive
Acinetobacter baumannii 249 bp Positive
Klebsiella pneumoniae 260 bp Positive
Enterobacter aerogenes 927 bp Positive
Proteus mirabilis 533 bp Positive

The foregoing discussion of the present assay and method has been presented for purposes of illustration and description. It is not intended to limit the present assay or method to the form or forms disclosed herein. In the foregoing Detailed Description, for example, various features of the present assay are grouped together in one or more embodiment, configurations, or aspects for the purpose of streamlining the disclosure. The features of the embodiment, configurations, or aspects may be combined in alternate embodiment, configurations, or aspects other than those discussed above. This method of disclosure is not to be interpreted as reflecting an intention the present assay requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment, configuration, or aspect. Thus, the following claims are hereby incorporated into this detailed description, with each claim standing on its own as a separate embodiment of the present assay.
Moreover, though the description of the present assay has included description of one or more embodiment, configurations, or aspects and certain variations and modifications, other variations, combinations, and modifications are within the scope of the present assay as described, e.g., as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiment, configurations, or aspects to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter.