WO2018097799A1

WO2018097799A1 - A method for the detection of group b streptococcus

Info

Publication number: WO2018097799A1
Application number: PCT/SG2017/050579
Authority: WO
Inventors: Alexander Lezhava; Suman SARMA; Swaine CHEN; Timothy Mark Sebastian BARKHAM
Original assignee: Agency for Science Technology and Research Singapore; National University of Singapore; Tan Tock Seng Hospital
Current assignee: Agency for Science Technology and Research Singapore; National University of Singapore; Tan Tock Seng Hospital
Priority date: 2016-11-23
Filing date: 2017-11-23
Publication date: 2018-05-31
Anticipated expiration: 2019-05-23

Abstract

The invention relates to a method for the detection of a Group B Streptococcus (GBS) Sequence Type 283 (ST283) strain in a sample, wherein said GBS ST283 strain comprises a nucleic acid target region (SEQ ID NO: 39). The method comprises exposing said sample to at least one oligonucleotide that is capable of binding/hybridising to said target region and detecting the binding/hybridising for determining the presence of this ST283 strain within said sample. Also provided are oligonucleotides capable of detecting the target region, and kits for detecting GBS using the primers of the invention.

Description

A method for the detection of Group B Streptococcus Field of the Invention

The invention relates to a method for the detection of Group B Streptococcus, in particular Group B Streptococcus (GBS) bacteria strain ST283, in a sample which method comprises the binding of at least one oligonucleotide to a target region; a kit for carrying out said method; an array, comprising at least one oligonucleotide, for use in said screening method; and at least one oligonucleotide, ideally, a primer pair for use in said method.

Background of the Invention

This disclosure relates to a Molecular Diagnostics Assay for the detection of a novel Group B Streptococcus (GBS) bacteria, particularly strain ST283, linked to people who fall sick after eating raw fish. The assay is ideally, but not exclusively, a Real-Time PCR based assay and so contains an in-house designed primer set that can specifically amplify GBS ST 283 strain. Advantageously, the assay can exclusively identify GBS ST 283 strain, against other strains, and so has remarkable specificity. This is a feature that is needed to determine the presence of this strain within a sample that may contain a number of different species or strains of Streptococcus bacteria.

Additionally, the assay described herein is attractive because it is inexpensive, fast and very specific. Indeed, the assay described herein is so sensitive it allows for bacterial DNA to be directly extracted from a patient sample and used for pathogen detection, thus overcoming any need to culture a patient sample or run gel electrophoresis. This lack of a need to culture a sample or undertake any other assay, such as gel electrophoresis, means that turnaround time for clinical diagnosis is less than three hours. In fact, the assay described herein can be monitored in Real-Time mode.

The present disclosure provides a particular target region in Group B Streptococcus (GBS) bacteria, strain ST283 that is useful in a diagnostic assay. The specific target region contains approximately 144bp out of a total potential target region of about 10kb which is relatively unique to the ST283 strain. In identifying the particular target region l the inventors investigated many (>34) primers, in different combinations, and found that over 95% of their initial designs provided poor specificity and, moreover, 20% of their designs showed poor sensitivity.

Through serendipity and screening the inventors have identified oligonucleotide probes with the ability to specifically bind the selected target region. These probes thus provide the basis for a reliable and repeatable screening and/or diagnostic assay for the detection of Group B Streptococcus (GBS) bacteria, strain ST283 in a sample that may contain other species or strains of Streptococcus bacteria.

Without wishing to be technically or geographically constrained, it is thought that this assay is useful in fish/food testing in Hong Kong, Bangkok, and Singapore where the ST283 strain has been found to be causing disease in humans. The disease can be one or more of the following: bacterial meningitis, bacteremia, soft tissue infection, joint infection, endocarditis, and non-specific septicaemia. However, it has also been found to cause disease in fish farmed for human consumption in Vietnam and Bangkok and so the assay, as well as being of diagnostic value for humans, may also be of similar value in fish farms.

Statements of Invention

According to a first aspect of the invention, there is provided a method for the detection of Group B Streptococcus (GBS) bacteria, strain ST283 in a sample wherein said strain comprises a nucleic acid target region having the following sequence, or a part thereof:

I) TGGTAAGGATTGTCGATAGCTTCTCCATTAGTGTTTAACATCAAAGCA TAAGTTGCCGGTGCAGAATACCCCAGTCCGTTAAATGTAGTATAGAA GTCTACACCTACCCCAGCTGAGTTGTAGTTGCCCCTAAGACCAAGCA AA (SEQ ID NO:39);

which method comprises exposing said sample to:

a) at least one oligonucleotide that is complementary to at least a part of said target regions, or;

b) at least one oligonucleotide that is complementary to at least a part of a degenerate version of said target regions or; c) an oligonucleotide that has at least 85% sequence identity/homology with the oligonucleotides in a) or b); and

detecting the binding of said oligonucleotide to said target region and, where binding occurs, concluding that said strain is present in said sample.

Ideally, the GBS bacteria or Streptococcus is from the species agalactiae and comprises GBS bacteria or Streptococcus agalactiae of strain Sequence Type 283 (ST283). Moreover, said target region comprises bacterial polynucleotide, other than bacterial ribosomal ribonucleic acid (rRNA).

In a preferred embodiment of the invention said target region comprises:

GTAAGGATTGTCGATAGCTTCTCCATTAGTGTTTAACATCAAAGCATAAGTTGCC GGTGCAGAATACCCCAGTCCGTTAAATGTAGTATAGAAGTCTACACCTACCCCA GCTGAGTTGTAGTTGCCCCTAAGACCAAGCAAA (SEQ ID NO:40).

In yet a further preferred embodiment of the invention said target region comprises no less than about 100 base pairs (bp) and, more ideally no more than about 144 base pairs (bp) for example 142bp but the invention also extends to a target region comprising or consisting of 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 1 10, 1 1 1 , 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 , 142, 143 and 144bp.

In a further preferred embodiment of the invention said oligonucleotide is designed for use in a conventional oligonucleotide binding assay, such as but not limited to, Polymerase Chain Reaction (PCR) or Real-Time PCR, including Taq-Man assisted PCR. Accordingly, said oligonucleotide comprises a deoxyribonucleic acid (DNA) polynucleotide or a DNA oligonucleotide. Most preferably said oligonucleotide is for use in a primer pair wherein one oligonucleotide is a forward primer and the other a reverse primer and wherein the forward primer is capable of hybridizing/binding to a first strand of the target region and is capable of priming synthesis of said strand and the reverse primer is capable of hybridizing/binding to a second complementary strand of the target region of and is capable of priming synthesis of said strand. In alternative embodiments of the invention said oligonucleotide comprises a pair of oligonucleotides, or primers, used to detect the presence or absence of said target region in the sample. Ideally, there is provided at least one forward and at least one reverse primer pair.

As will be appreciated by those skilled in the art, said oligonucleotide is, advantageously, designed to have optimal properties for performing such a PCR technique, such as but not limited to, a GC content of approximately 50%, minimal self-complementarity, an optimal nucleotide length of between 15 and 50 nucleotide base pairs, or the like.

In a yet a further preferred embodiment of the invention, said oligonucleotide comprises a signaling molecule which emits a signal when said oligonucleotide exists in either a bound or an unbound state; or, alternatively, it emits a quantitative signal whose scale is representative of at least one of said states. Ideally, said signaling molecule is located immediately next to the binding nucleotides of said oligonucleotide.

Additionally or alternatively, said oligonucleotide comprises a part of a signaling system which part interacts with other parts of said system to emit a signal when either in a bound state or an unbound state whereby the binding of said oligonucleotide to said target site can be detected. As will be appreciated by those skilled in the art, there are numerous examples of such systems such as, but not limited to, conjugation of said oligonucleotide to Alkaline Phosphatase or Horseradish Peroxidase, which catalyse the cleavage of substrates to generate a signal.

In the above embodiments of the invention said signaling molecule may be a fluorescent molecule or a chemiluminescent molecule or a bioluminescent molecule or, indeed, any molecule that provides a detectable signal when said oligonucleotide exists in either a bound or unbound state. As will be appreciated by those skilled in the art, this may include but is not limited to SYBR Green, FAM (5-or 6- carboxyfluorescein), VIC, NED, Fluorescein, Digoxigenin, FITC, IRD- 700/800, CY3, CY5, CY3.5, CY5.5, Cy7, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Hydroxycumarin, Aminocoumarin, Lucifer Yellow, TruRed, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor, PET Biosearch Blue(TM), Marina Blue(R), Bothell Blue(R), CAL Fluor(R) Gold, CAL Fluor(R) Red 610, Quasar(TM) 670, LightCycler Red640(R), Quasar(TM) 705, LightCycler Red705(R), or the like.

Further, the inventors have made oligonucleotide or primer sequence modifications to include mismatches to the target genome sequence. These mismatches are designed to increase specificity. Thus, these oligonucleotides or primer sequences are not naturally occurring. Preferably, the primer sequences for use in PCR amplification are FP404-A and FP404-A-7 (5'- GGTAAGGATTGTCGATAGTTT and GTAAGGATTGTCGAGAGTTT-3') SEQ ID NO:1 and 38 and RP39 (5'- TTTGCTTGGTCTTAGGGGC-3') SEQ ID NO:2. Further, where Taqman detection is undertaken, the primer SGB-P23 (5'-CTCCATTAGTGTTTAACATCAAA-3') SEQ ID NO:3 is also used.

The use of these modified oligonucleotides is favoured because they reduce or overcome the possibly of non-specific (fluorescence) signalling if the template DNA concentration is too high or false-positive amplification occurs due to the presence of closely related but non-ST283 GBS strains.

Additionally, the use of a Taqman version of the assay also helps to overcome false signalling and so is preferred, accordingly the invention further, advantageously this employs the use of a Taqman oligonucleotide, ideally but not exclusively SGB-P23 (5'- CTCCATTAGTGTTTAACATCAAA-3') SEQ ID NO:3.

In yet a further preferred embodiment of the invention, the concentration of said oligonucleotide is typically, and without limitation, 0.2uM to 0.00002uM, including all 1 uM integers there between. In this way, the oligonucleotide are at a concentration to permit sensitive and accurate detection of said target sequences. However, as will be appreciated by those skilled in the art, the concentration of oligonucleotide will vary according to the particular oligonucleotide binding assay utilised to permit optimal binding and target detection.

In part c) above the skilled person will appreciate that homologues or derivatives of the oligonucleotides of the invention will also find use in the context of the present invention. Thus, for instance oligonucleotides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. A software program such as BLASTx can be used to identify oligonucleotides with the requisite at least 85% homology. This program will align the longest stretch of similar sequences and assign a homology value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. This type of analysis is contemplated in the present invention.

The term "homologous" as used herein refers to oligonucleotide sequences which have a sequence at least 85% homologous (or identical) to the said oligonucleotide sequence in parts a) or b). It is preferred that homologues are at least 85%, homologous to parts a) or b) and, in increasing order of preference, at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to parts a) or b).

Most ideally, said oligonucleotides are between 15 - 30 nucleotides in length and most ideally 15 - 27 nucleotides in length and even more ideally 18 - 25 nucleotides in length.

Most ideally still, said oligonucleotide is selected from the group comprising or consisting of:

SEQ ID NO :1 FP404 -A GG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO :2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO :3 SGB-P23 CTC CAT TAG TGT TTA ACA TCA AA;

SEQ ID NO :4 SGBF25 AAG TTG CCG GTG CAG A AT AC;

SEQ ID NO :5 S3F10 TAA CAG CAG TAT CGT A AT;

SEQ ID NO :6 S3F12 GGTAAGGATTGTCGATAGCT;

SEQ ID NO :7 FP40 TGG TAA GGA TTG TCG ATA GCT T;

SEQ ID NO :8 S3F12B GGTAAGGATTGTCGATAACT;

SEQ ID NO :9 S3F12D GGTAAGGATTGTCGATAGGT;

SEQ ID NO :10 S3F12E GGTAAGGATTGTCGAAAGCT;

SEQ ID NO :1 1 FP401 TGG TAA GGA TTG TCG ATA GCT;

SEQ ID NO :12 FP402 TGG TAA GGA TTG TCG ATA GGT T; SEQ ID NO 13 FP403 GT AAG GAT TGT CGA TAG CT;

SEQ ID NO 14 FP404 GG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 15 FP405 GG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 16 FP406 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 17 FP407 CTGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 18 FP408 CTGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 19 FP9 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 20 FP10 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG;

SEQ ID NO 22 FP409K GGT AAG GAT TGT CGA TAG ATT;

SEQ ID NO 23 FP410 TGGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 24 FP41 1 GGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 25 FP4021 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 26 FP415 TGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 27 FP402-A TGG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT;

SEQ ID NO 29 SGBR26 CCGTTCAAGGTTCAGGAAAA;

SEQ ID NO 30 SGBR28-2 AAG GCT AAT GGT TTC TGG GG;

SEQ ID NO 31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID NO 32 SGB_RP3 CTT TGC TTG GTC TTA GGG G;

SEQ ID NO 33 SGB RP4 GCT TTG CTT GGT CTT AGG G;

SEQ ID NO 34 SGB_RP2 TTG CTT GGT CTT AGG GGC A;

SEQ ID NO 35 RP38 TTT GCT TGG TCT TAG GGG CT;

SEQ ID NO 36 SGB-P44 AAG CAT AAG TTG CCG GTG CAG

NO:37 SGB-P56 CCG GTG CAG AAT ACC CCA GTC CGT TAA; and

SEQ ID NO:38 FP404-A-7 GTA AGG ATT GTC GAG AGT TT.

In a preferred embodiment of the invention said oligonucleotide comprises at least one pair of primers selected from the group comprising or consisting of:

SEQ ID NO:38 FP404-A-7 GTAAGGATTGTCGAGAGTTT and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO:1 FP404 -A GG TAA GGA TTG TCG ATA GTT T and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C; SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID N0:2 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID N0:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID N0:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:30 SGBR28-2 AAG G CT AATG GTTTCTG G GG ;

SEQ ID N0:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:28 SGBR28-

1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID N0:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID N0:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:30 SGBR28-

2 AAGGCTAATGGTTTCTGGGG ;

SEQ ID N0:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID N0:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID N0:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:30 SGBR28-2 AAGGCTAATGGTTTCTGGGG ;

SEQ ID N0:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID N0:31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID N0:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:32 SGB_RP3 CTTTGCTTGGTCTTAGGGG ;

SEQ ID NO:4SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:33 SGB RP4 GCTTTGCTTGGTCTTAGGG

SEQ ID N0:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID N0:31 SGBRP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID N0:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:34 SGB_RP2 TTGCTTGGTCTTAGGGGCA ;

SEQ ID N0:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:34 SGB_RP2 TTGCTTGGTCTTAGGGGCA ;

SEQ ID N0:7 FP40 TGG TAA GGA TTG TCG ATA GCT T and SEQ ID NO:35 RP38 TTT GCT TGG TCT TAG GGG CT ;

SEQ ID N0:7 FP40 TGG TAA GGA TTG TCG ATA GCT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ; SEQ ID NO:8 S3F12B GGTAAGGATTGTCGATAACT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:9 S3F12D GGTAAGGATTGTCGATAGGT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:10 S3F12E GGTAAGGATTGTCGAAAGCT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:1 1 FP401 TGG TAA GGA TTG TCG ATA GCT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:12 FP402 TGG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:13 FP403 GT AAG GAT TGT CGA TAG CT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:14 FP404 GG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:15 FP405 GG TAA GGA TTG TCG ATA GGT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:16 FP406 TGG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:17 FP407 CTGG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:18 FP408 CTGG TAA GGA TTG TCG ATA GGT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:19 FP9 ACT GCT TTT TCA TCT ACA ATG G and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:20 FP10 ACT GCT TTT TCA TCT ACA ATG G and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:22 FP409K GGT AAG GAT TGT CGA TAG ATT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:23 FP410 TGGT AAG GAT TGT CGA TAA CTT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:24 FP41 1 GGT AAG GAT TGT CGA TAA CTT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ; SEQ ID NO:25 FP4021 TGG TAA GGA TTG TCG ATA GGT T and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:26 FP415 TGG TAA GGA TTG TCG ATA GGT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ; and

SEQ ID NO:27 FP402-A TGG TAA GGA TTG TCG ATA GTT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C.

Yet more ideally still, said oligonucleotide is a Taq-man oligonucleotide selected from the group comprising:

SEQ IDNO:3 SGB-P23 CTCCATTAGTGTTTAACATCAAA ;

SEQ ID NO:36 SGB-P44 AAGCATAAGTTGCCGGTGCAGAATA ; and

SEQ ID NO:37 SGB-P56 CCGGTGCAGAATACCCCAGTCCGTTAA.

Advantageously, it has been shown that even when using a high concentration of human genomic DNA (100ng/reaction) in a GBS TaqMan Assay to assess cross reactivity, the results show that there is no non-specific amplification/cross reactivity exhibited by the assay.

In use, it will therefore be appreciated that the invention involves the identification of a target region or nucleic acid sequence in said strain of species of bacteria, which can be used to accurately and sensitively to determine the presence or absence of said bacteria in a sample. As will be appreciated by those skilled in the art, the detection of the target region or nucleic acid sequence can be achieved by oligonucleotide binding assays. The present invention therefore also concerns complementary oligonucleotide binding probes or primers for said target region or nucleic acid sequence, or probes/primers with 85% or more sequence similarity thereto, for use in assays for the detection of the disclosed target region of the invention.

Additionally, as is known to those skilled in the art, transcripts of the said target region or nucleic acid sequence can also be detected in the working of the invention, such as but not limited to, the detection of imRNA encoded by said region or sequence in the technique of RT-PCR. In a preferred aspect of the invention said sample is any matter suspected of containing said bacterial strain, such as but not limited to, an inanimate object, a food sample, earth, soil, a gut sample, faeces, urine, body fluid, food, laboratory cultures, hospital equipment, or wound dressings.

Ideally, the sample is selected from the group consisting of: blood, serum, plasma, enriched peripheral blood mononuclear cells, fecal material, urine, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, stool, skin, gastric secretions, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, peri-rectal swabs, rectal swabs, vaginal swabs, nasal aspirates, nasal wash, renal tissue, and fluid therefrom including perfusion media, pure cultures of bacterial fungal isolates, fluids and cells obtained by the perfusion of tissues of both human and non-human origin, and fluids and cells derived from culturing of human cells, including human stem cells and human cartilage or fibroblasts, pure cultures of bacterial fungal isolates, and swabs or washes of environmental surfaces, other samples derived from environmental surfaces, samples derived from fish, samples derived from raw fish or samples from food.

Most ideally said sample is taken from the gut and said method is a diagnostic method for identifying individuals, typically patients, carrying said infection. Ideally, said sample is treated to extract DNA therefrom, typically, by using conventional techniques as described herein and as known to those skilled in the art.

In a preferred embodiment the sample is from a human or a non-human species, wherein the non-human species is one selected from the group consisting of: mammal, reptile, avian, fish, or amphibian.

According to a second aspect of the invention, there is provided a kit for the detection of Group B Streptococcus (GBS) bacteria, strain ST283 in a sample wherein said strain comprises a nucleic acid target region having the following sequence:

I) TGGTAAGGATTGTCGATAGCTTCTCCATTAGTGTTTAACATCAAAGCA TAAGTTGCCGGTGCAGAATACCCCAGTCCGTTAAATGTAGTATAGAA GTCTACACCTACCCCAGCTGAGTTGTAGTTGCCCCTAAGACCAAGCA AA (SEQ IDNO:39) which kit comprises:

b) at least one oligonucleotide that is complementary to at least a part of one degenerate version of said target regions, or;

c) an oligonucleotide that has at least 85% identity/homology with oligonucleotides a) or b) and

optionally, reagents and/or instructions for practicing said detection.

In a further preferred embodiment of the second aspect of the invention said oligonucleotide is designed for use in a conventional oligonucleotide binding assay, such as but not limited to, PCR, preferably, real-Time PCR.

Most preferably said oligonucleotide comprises a primer pair selected from the group comprising or consisiting of :

SEQ ID NO:38 FP404-A-7 GTAAGGATTGTCGAGAGTTT and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C; and/or

SEQ ID NO:1 FP404 -A GG TAA GGA TTG TCG ATA GTT T and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C.

Yet more ideally still, said oligonucleotide is a Taq-man oligonucleotide selected from the group comprising or consisting of:

SEQ IDNO:3 SGB-P23 CTCCATTAGTGTTTAACATCAAA ;

SEQ ID NO:36 SGB-P44 AAGCATAAGTTGCCGGTGCAGAATA ; and

SEQ ID NO:37 SGB-P56 CCGGTGCAGAATACCCCAGTCCGTTAA.

Moreover, a plurality of different oligonucleotides, each labelled with the same or different signalling molecules, may be used to detect the presence or absence of said target region in the same sample.

In yet a further preferred embodiment of the invention, the concentration of said oligonucleotide is typically and without limitation between 0.2uM to 0.00002uM, including all 0.1 uM integers there between. In this way, the oligonucleotides are at a concentration to permit sensitive and accurate detection of said target sequences. However, as will be appreciated by those skilled in the art, the concentration of oligonucleotide will vary according to the particular oligonucleotide binding assay utilised to permit optimal binding and target detection.

In yet a further aspect of the invention there is provided an array comprising any one or more of the above oligonucleotides, including any combination thereof, including any paired combination thereof and, ideally, all of said oligonucleotides.

In yet a further aspect of the invention there is provided an oligonucleotide as described herein.

In yet still a further aspect of the invention there is provided a pair of oligonucleotides as described herein.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprises", or variations such as "comprises" or "comprising" is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The present invention will now be described by way of example only with particular reference to the following figures wherein:

Figure 1. Shows the sequence structure of the primers and probes used in one example to work the invention.

Figure 2. Shows the sequence structure of the target region, including the parts thereof to which a forward and reverse primer can bind and also the part thereof to which a probe can bind on one example used to work the invention.

Figure 3. Shows the result of a fluorescent assay using primer FP404-A SEQ ID NO:1 , unfortunately, this primer amplified two known ST-283 negative samples i.e. No 40 and 49. Sample 56 represents the positive control.

Figure 4. Shows the result of a further fluorescent assay using the modified primer FP404-A-7 SEQ ID NO:38, this primer showed positive for the control within a dilution range of 1 :10,000. It did not amplify the two known ST-283 negative samples identified with primer FP404-A SEQ ID NO:1 shown in Figure 3, indicating its enhanced specificity.

Figure 5. Shows the result of a further fluorescent assay using the modified primer FP404-A-7 SEQ ID NO:38, this primer did not amplify a range of known ST-283 negative samples although it was positive for the control.

Figure 6. Shows the result of a yet further fluorescent assay using the modified primer FP404-A-7 SEQ ID NO:38, this primer did not amplify a further range of known ST-283 negative samples although it was positive for the control and there wazs one false negative amplified No 50 SG-83. Figure 7. Shows the result of a yet further fluorescent assay that investigated those false positive samples known not to contain ST-283, however in this test no false positives were identified. The below specified reaction conditions were used in this assay.

Table 1. Shows the combination of modified primers or modified primer pairs for detecting the target region within Group B Streptococcus (GBS) bacteria, strain ST283;

Table 2 shows the combination of primer pairs that were tested during the

development of the assay; and

Table 3. Shows the Taq-Man probe sequences for use in the assay.

METHODS

Identification of the target detection region

The SG-M1 genome sequence was used as a reference for ST283 strain. 22 complete genome sequences of genotyping by sequence (GBS) (all available as of Aug 18, 2015) were downloaded from Genbank; none of these was ST283. The MUMmer package was used to identify segments of the SG-M1 genome that were not present in any of the other 22 complete GBS sequences. Thereafter, 559 GBS whole genome sequencing data sets (unassembled, lllumina) were downloaded from the Genbank Short Read Archive. These were mapped to the SG-M1 genome using bwa, then coverage was calculated using bedtools. Regions originally identified from the 22 complete genomes were further filtered based on coverage from these 559 short read data sets, leaving only those sequences that were fully unique to SG-M1 from this data set of 559 lllumina data sets + 22 full genomes.

Primer Design

One of these SG-M1 unique regions was selected (see Figure 2, top row). Primers were designed based on manual examination and general PCR guidelines similar to those found at https://www.sigmaaldrich.com/technical- documents/articles/biology/pcr-qpcr-dpcr-assay-design.html (Figure 1 and 2). The initial assay was tested on a panel of 1 10 strains (55 ST283, 55 non-ST283) from Singapore and Hong Kong that were previously typed as ST283 or not using traditional MLST (Figure 3 shows data for non-ST283 and 1 ST283 with 2 additional internal controls).

Modification of primers

Due to several false positives in a more extensive screening of 100 additional human GBS isolates from Singapore, primers were further manually modified (Figure 1 ). These were retested on a panel of previously tested positive and negative controls as well as on those strains that previously were giving false positive amplification with the initial primers (See Figure 4, 5, 6).

PCR Conditions

Results

Our studies show it is possible to identify a target region from the genome of Streptococcus agalactiae of strain Sequence Type 283 (ST283) that enables this particular strain of bacteria to be reliably and specifically identified within a sample.

Probe Design

Probes were designed to recognise sequences within said target region and whilst this produced a no. of primers pairs that could be used to work the assay our preferred primer pairs were FP404-A or FP404-A-7 (5'- GGTAAGGATTGTCGATAGTTT or GTAAGGATTGTCGAGAGTTT-3') SEQ ID NO:1 or 38 when used with RP39 (5'- TTTGCTTGGTCTTAGGGGC-3') SEQ ID NO:2.

Further, where Taqman detection is undertaken, the primer SGB-P23 (5'-

CTCCATTAGTGTTTAACATCAAA-3') SEQ ID NO:3 was also used.

Our data shows that FP404-A-7 produced the best results as it was the most discriminate.

Summary

We have therefore identified a highly specific target region in Streptococcus agalactiae of strain Sequence Type 283 (ST283) which can be used to provide a highly sensitive and accurate test for the presence of the organism in a sample. Detection of this specific target region is strain specific and most suitably employs the use of a forward and reverse primer pair, one of which is ideally modified to improve its performance.

Table 1

Combination of

primers

Table

2

Combinations of primers that were tested

during assay development

SGBR28 CTCTCGC I I I GCTTGGTC

S3F1 0 TAACAGCAGTATCGTAAT 28

-1 TT

CCGTTCAAGGTTCAGGAA

S3F1 0 TAACAGCAGTATCGTAAT 29 SGBR26

AA

SGBR28 AAG G CT AATG G TTTCTG G

S3F1 0 TAACAGCAGTATCGTAAT 30

-2 GG

GGTAAGGATTGTCGATAG SGBR28 CTCTCGCTTTGCTTGGTC

S3F12 28

CT -1 TT

GGTAAGGATTGTCGATAG CCGTTCAAGGTTCAGGAA

S3F12 29 SGBR26

CT AA

GGTAAGGATTGTCGATAG SGBR28 AAG G CT AATG G TTTCTG G

S3F12 30

CT -2 GG

SGBF2 AAGTTGCCGGTGCAGAAT SGB RP TCGCTTTGCTTGGTCTTA

31

5 AC 1 GG

SGBF2 AAGTTGCCGGTGCAGAAT SGB RP CTTTG CTTG GTCTT AG G G

32

5 AC 3 G

SGBF2 AAGTTGCCGGTGCAGAAT SGB G CTTTG CTTG GTCTT AG G

33

5 AC RP4 G

GGTAAGGATTGTCGATAG TCGCTTTGCTTGGTCTTA

S3F12 31 SGBRP1

CT GG

GGTAAGGATTGTCGATAG SGB RP TTG CTTG GTCTT AG G G G C

S3F12 34

CT 2 A

SGBF2 AAGTTGCCGGTGCAGAAT SGB RP TTG CTTG GTCTT AG G G G C

34

5 AC 2 A

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP40 35 RP38

ATA GCT T GGG CT

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP40 2 RP39

ATA GCT T GGG C

S3F12 GGTAAGGATTGTCGATAA TTT GCT TGG TCT TAG

2 RP39

B CT GGG C

S3F12 GGTAAGGATTGTCGATAG TTT GCT TGG TCT TAG

2 RP39

D GT GGG C

S3F12 GGTAAGGATTGTCGAAAG TTT GCT TGG TCT TAG

2 RP39

E CT GGG C

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP401 2 RP39

ATA GCT GGG C

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP402 2 RP39

ATA GGT T GGG C

GT AAG GAT TGT CGA TTT GCT TGG TCT TAG

FP403 2 RP39

TAG CT GGG C

GG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP404 2 RP39

ATA GGT T GGG C

GG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP405 2 RP39

ATA GGT GGG C

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP406 2 RP39

ATA GGT T GGG C

CTGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP407 2 RP39

ATA GGT T GGG C

CTGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

FP408 2 RP39

ATA GGT GGG C

ACT GCT TTT TCA TCT TTT GCT TGG TCT TAG

FP9 2 RP39

ACA ATG G GGG C

ACT GCT TTT TCA TCT TTT GCT TGG TCT TAG

FP10 2 RP39

ACA ATG G GGG C

CT ACT GCT TTT TCA TCT TTT GCT TGG TCT TAG

FP1 1 2 RP39

ACA ATG GGG C

FP409 GGT AAG GAT TGT CGA TTT GCT TGG TCT TAG

2 RP39

K TAG ATT GGG C TGGT AAG GAT TGT CGA I I I GCT TGG TCT TAG

23 FP41 0 2 RP39

TAA CTT GGG C

GGT AAG GAT TGT CGA TTT GCT TGG TCT TAG

24 FP41 1 2 RP39

TAA CTT GGG C

FP402 TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

25 2 RP39

1 ATA GGT T GGG C

TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

26 FP41 5 2 RP39

ATA GGT GGG C

FP402- TGG TAA GGA TTG TCG TTT GCT TGG TCT TAG

27 2 RP39

A ATA GTT T GGG C

Probe Sequences

Claims

1 . A method for the detection of Group B Streptococcus (GBS) bacteria, strain ST283 in a sample wherein said strain comprises a nucleic acid target region having the following sequence, or a part thereof:

l)TGGTAAGGATTGTCGATAGCTTCTCCATTAGTGTTTAACATCAAAGCATAAGTT GCCGGTGCAGAATACCCCAGTCCGTTAAATGTAGTATAGAAGTCTACACCTACC CCAGCTGAGTTGTAGTTGCCCCTAAGACCAAGCAAA SEQ ID NO:39;

which method comprises exposing said sample to:

b) at least one oligonucleotide that is complementary to at least a part of a degenerate version of said target regions or;

c) an oligonucleotide that has at least 85% sequence identity/homology with the oligonucleotides in a) or b); and

2. The method according to claim 1 wherein said target region comprises bacterial polynucleotide, other than bacterial ribosomal ribonucleic acid (rRNA).

3. The method according to claim 1 wherein said target region comprises no less than about 100 base pairs (bp) and, more ideally no more than about 144 base pairs (bp), including all 1 bp integers there between.

4. The method according to claim 3 wherein said target region comprises said 142 base pairs SEQ ID NO:40.

5. The method according to any one of the preceding claims wherein said oligonucleotide is designed for use in a conventional oligonucleotide binding assay selected from the group comprising or consisting of: Polymerase Chain Reaction (PCR) or Real-Time PCR and Taq-Man assisted PCR.

6. The method according to any one of the preceding claims wherein said oligonucleotide comprises a forward primer and a reverse primer and wherein the forward primer is capable of hybridizing/binding to a first strand of the target region and is capable of priming synthesis of said strand and the reverse primer is capable of hybridizing/binding to a second complementary strand of the target region of and is capable of priming synthesis of said strand.

7. The method according to any one of the preceding claims wherein said oligonucleotide comprises a signalling molecule which emits a signal when said oligonucleotide exists in either a bound or an unbound state; or, alternatively, it emits a quantitative signal whose scale is representative of at least one of said states.

8. The method according to any one of the preceding claims wherein said oligonucleotide comprises a part of a signalling system which part interacts with other parts of said system to emit a signal when said oligonucleotide is either in a bound state or an unbound state whereby the binding of said oligonucleotide to said target site can be detected.

9. The method according to claim 7 or claim 8 wherein said signalling molecule or system comprises a fluorescent molecule or a chemiluminescent molecule or a bioluminescent molecule or any molecule that provides a detectable signal when said oligonucleotide exists in either a bound or unbound state.

10. The method according to claim 9 wherein signalling molecule or system comprises a molecule selected from the group comprising or consisting of: SYBR Green, FAM (5-or 6-carboxyfluorescein), VIC, NED, Fluorescein, Digoxigenin, FITC, IRD- 700/800, CY3, CY5, CY3.5, CY5.5, Cy7, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Hydroxycumarin, Aminocoumarin, Lucifer Yellow, TruRed, Rhodamine Red, Texas Red, Yakima Yellow, Alexa Fluor, PET Biosearch Blue(TM), Marina Blue(R), Bothell Blue(R), CAL Fluor(R) Gold, CAL Fluor(R) Red 610, Quasar(TM) 670, LightCycler Red640(R), Quasar(TM) 705, and LightCycler Red705(R).

1 1 . The method according to any one of the preceding claims wherein said oligonucleotides are selected from the group comprising or consisting of: 15 - 30 nucleotides in length; or 15 - 27 nucleotides in length; or 18 - 25 nucleotides in length.

12. The method according to anyone of the preceding claims wherein said oligonucleotide of part c) has at least 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the oligonucleotides in parts a) or b).

13. The method according to any one of the preceding claims wherein said oligonucleotide is selected from the group comprising or consisting of:

SEQ ID NO:1 FP404 -A GG TAA GGA TTG TCG ATA GTT T; SEQ ID NO 2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO 3 SGB-P23 CTC CAT TAG TGT TTA ACA TCA AA;

SEQ ID NO 4 SGBF25 AAG TTG CCG GTG CAG AAT AC;

SEQ ID NO 5 S3F10 TAA CAG CAG TAT CGT AAT;

SEQ ID NO 6 S3F12 GGTAAGGATTGTCGATAGCT;

SEQ ID NO 7 FP40 TGG TAA GGA TTG TCG ATA GCT T;

SEQ ID NO 8 S3F12B GGTAAGGATTGTCGATAACT;

SEQ ID NO 9 S3F12D GGTAAGGATTGTCGATAGGT;

SEQ ID NO 10 S3F12E GGTAAGGATTGTCGAAAGCT;

SEQ ID NO 1 1 FP401 TGG TAA GGA TTG TCG ATA GCT;

SEQ ID NO 12 FP402 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 13 FP403 GT AAG GAT TGT CGA TAG CT;

SEQ ID NO 14 FP404 GG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 15 FP405 GG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 16 FP406 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 17 FP407 CTGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 18 FP408 CTGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 19 FP9 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 20 FP10 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG;

SEQ ID NO 22 FP409K GGT AAG GAT TGT CGA TAG ATT;

SEQ ID NO 23 FP410 TGGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 24 FP41 1 GGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 25 FP4021 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 26 FP415 TGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 27 FP402-A TGG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT;

SEQ ID NO 29 SGBR26 CCGTTCAAGGTTCAGGAAAA;

SEQ ID NO 30 SGBR28-2 AAG GCT AAT GGT TTC TGG GG;

SEQ ID NO 31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID NO 32 SGB_RP3 CTT TGC TTG GTC TTA GGG G;

SEQ ID NO 33 SGB RP4 GCT TTG CTT GGT CTT AGG G;

SEQ ID NO 34 SGB_RP2 TTG CTT GGT CTT AGG GGC A;

SEQ ID NO 35 RP38 TTT GCT TGG TCT TAG GGG CT; SEQ ID NO:36 SGB-P44 AAG CAT AAG TTG CCG GTG CAG AAT A; SEQ ID NO:37 SGB-P56 CCG GTG CAG AAT ACC CCA GTC CGT TAA; and SEQ ID NO:38 FP404-A-7 GTA AGG ATT GTC GAG AGT TT.

14. The method according to any one of the preceding claims wherein said oligonucleotide comprises at least one pair of primers selected from the group comprising or consisting of:

SEQ ID NO:1 FP404 -A GG TAA GGA TTG TCG ATA GTT T and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:2 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:30 SGBR28-2 AAG G CT AATG GTTTCTG G GG ;

SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:28 SGBR28-

1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:30 SGBR28-

2 AAGGCTAATGGTTTCTGGGG ;

SEQ ID NO:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID NO:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ;

SEQ ID NO:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID NO:30 SGBR28-2 AAGGCTAATGGTTTCTGGGG ;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:32 SGB_RP3 CTTTGCTTGGTCTTAGGGG ;

SEQ ID NO:4SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:33 SGB RP4 GCTTTGCTTGGTCTTAGGG SEQ ID NO:6 S3F12 GGTAAGGATTGTCGATAGCT and SEQ ID N0:31 SGBRP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID N0:7 FP40 TGG TAA GGA TTG TCG ATA GCT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:8 S3F12B GGTAAGGATTGTCGATAACT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID N0:19 FP9 ACT GCT TTT TCA TCT ACA ATG G and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ; SEQ ID NO:20 FP10 ACT GCT TTT TCA TCT ACA ATG G and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:24 FP41 1 GGT AAG GAT TGT CGA TAA CTT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

SEQ ID NO:25 FP4021 TGG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;

15. The method according to claim 14 wherein said primer pair comprises:

a) 5'- GGTAAGGATTGTCGATAGTTT-3' SEQ ID NO:1 or

b) 5'-GTAAGGATTGTCGAGAGTTT-3'; SEQ ID No: 38; and

c) RP39 (5'-TTTGCTTGGTCTTAGGGGC-3'). SEQ ID NO:2.

16. The method according to any one of the preceding claims wherein Taqman detection is undertaken and the primer is selected from the group comprising or consisting of:

SEQ IDNO:3 SGB-P23 CTCCATTAGTGTTTAACATCAAA ;

SEQ ID NO:36 SGB-P44 AAGCATAAGTTGCCGGTGCAGAATA ; and

SEQ ID NO:37 SGB-P56 CCGGTGCAGAATACCCCAGTCCGTTAA.

17. The method according to claim 16 wherein the primer SEQ ID NO:3 SGB-P23 (5'-CTCCATTAGTGTTTAACATCAAA-3') is used.

18. The method according to any one of the preceding claims wherein the concentration of said oligonucleotide is typically and without limitation 0.2uM to

0.00002uM.

19. The method according to any one of the preceding claims wherein said sample is selected from the group comprising or consisting of: an inanimate object, a food sample, earth, soil, a gut sample, body fluid, food, laboratory cultures, hospital equipment, or wound dressings, blood, serum, plasma, enriched peripheral blood mononuclear cells, fecal material, urine, neoplastic or other tissue obtained from biopsies, cerebrospinal fluid, saliva, fluids collected from the ear, eye, mouth, and respiratory airways, sputum, stool, skin, gastric secretions, oropharyngeal swabs, nasopharyngeal swabs, throat swabs, peri-rectal swabs, rectal swabs, vaginal swabs, nasal aspirates, nasal wash, renal tissue, and fluid therefrom including perfusion media, pure cultures of bacterial fungal isolates, fluids and cells obtained by the perfusion of tissues of both human and non-human origin, and fluids and cells derived from culturing of human cells, including human stem cells and human cartilage or fibroblasts, pure cultures of bacterial fungal isolates, and swabs or washes of environmental surfaces, other samples derived from environmental surfaces, samples derived from fish, samples derived from raw fish and samples from food.

20. The method according to any one of the preceding claims wherein said sample is treated to extract DNA therefrom.

21 . The method according to any one of the preceding claims wherein said sample is from a human, mammal, reptile, avian, fish, or amphibian.

22. A kit for the detection of Group B Streptococcus (GBS) bacteria, strain ST283 in a sample wherein said strain comprises a nucleic acid target region having the following sequence:

I)

TGGTAAGGATTGTCGATAGCTTCTCCATTAGTGTTTAACATCAAAGCATA AGTTGCCGGTGCAGAATACCCCAGTCCGTTAAATGTAGTATAGAAGTCTACACC TACCCCAGCTGAGTTGTAGTTGCCCCTAAGACCAAGCAAA (SEQ IDNO:39) which kit comprises:

optionally, reagents and/or instructions for practicing said detection.

23. A kit according to claim 22 wherein said oligonucleotide is selected from the group comprising or consisting of, including any and all combination(s) thereof:

SEQ ID NO 3 SGB-P23 CTC CAT TAG TGT TTA ACA TCA AA;

SEQ ID NO 4 SGBF25 AAG TTG CCG GTG CAG AAT AC;

SEQ ID NO 5 S3F10 TAA CAG CAG TAT CGT AAT;

SEQ ID NO 6 S3F12 GGTAAGGATTGTCGATAGCT;

SEQ ID NO 7 FP40 TGG TAA GGA TTG TCG ATA GCT T;

SEQ ID NO 8 S3F12B GGTAAGGATTGTCGATAACT;

SEQ ID NO 9 S3F12D GGTAAGGATTGTCGATAGGT;

SEQ ID NO 10 S3F12E GGTAAGGATTGTCGAAAGCT;

SEQ ID NO 1 1 FP401 TGG TAA GGA TTG TCG ATA GCT;

SEQ ID NO 12 FP402 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 13 FP403 GT AAG GAT TGT CGA TAG CT;

SEQ ID NO 14 FP404 GG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 15 FP405 GG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 16 FP406 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 17 FP407 CTGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 18 FP408 CTGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 19 FP9 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 20 FP10 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO 21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG;

SEQ ID NO 22 FP409K GGT AAG GAT TGT CGA TAG ATT;

SEQ ID NO 23 FP410 TGGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 24 FP41 1 GGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 25 FP4021 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 26 FP415 TGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 27 FP402-A TGG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT;

SEQ ID NO 29 SGBR26 CCGTTCAAGGTTCAGGAAAA;

SEQ ID NO 30 SGBR28-2 AAG GCT AAT GGT TTC TGG GG;

SEQ ID NO 31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID NO 32 SGB_RP3 CTT TGC TTG GTC TTA GGG G;

SEQ ID NO 33 SGB RP4 GCT TTG CTT GGT CTT AGG G;

SEQ ID NO 34 SGB_RP2 TTG CTT GGT CTT AGG GGC A;

SEQ ID NO 35 RP38 TTT GCT TGG TCT TAG GGG CT; SEQ ID NO:36 SGB-P44 AAG CAT AAG TTG CCG GTG CAG AAT A;

SEQ ID NO:37 SGB-P56 CCG GTG CAG AAT ACC CCA GTC CGT TAA; and

SEQ ID NO:38 FP404-A-7 GTA AGG ATT GTC GAG AGT TT.

24. The kit according to claim 22 or claim 23 wherein said oligonucleotide comprises a signalling molecule which emits a signal when said oligonucleotide exists in either a bound or an unbound state; or, alternatively, it emits a quantitative signal whose scale is representative of at least one of said states.

25. The kit according to claim 22 or claim 23 wherein said oligonucleotide comprises a part of a signalling system which part interacts with other parts of said system to emit a signal when said oligonucleotide is either in a bound state or an unbound state whereby the binding of said oligonucleotide to said target site can be detected.

26. An oligonucleotide selected from the group comprising or consisting of, including any and all combination(s) thereof:

SEQ ID NO: :1 FP404 -A GG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO: :2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO: :3 SGB-P23 CTC CAT TAG TGT TTA ACA TCA AA;

SEQ ID NO: :4 SGBF25 AAG TTG CCG GTG CAG AAT AC;

SEQ ID NO: :5 S3F10 TAA CAG CAG TAT CGT AAT;

SEQ ID NO: :6 S3F12 GGTAAGGATTGTCGATAGCT;

SEQ ID NO: :7 FP40 TGG TAA GGA TTG TCG ATA GCT T;

SEQ ID NO: :8 S3F12B GGTAAGGATTGTCGATAACT;

SEQ ID NO: :9 S3F12D GGTAAGGATTGTCGATAGGT;

SEQ ID NO: :10 S3F12E GGTAAGGATTGTCGAAAGCT;

SEQ ID NO: :1 1 FP401 TGG TAA GGA TTG TCG ATA GCT;

SEQ ID NO: :12 FP402 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO: :13 FP403 GT AAG GAT TGT CGA TAG CT;

SEQ ID NO: :14 FP404 GG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO: :15 FP405 GG TAA GGA TTG TCG ATA GGT;

SEQ ID NO: :16 FP406 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO: :17 FP407 CTGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO: :18 FP408 CTGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO: :19 FP9 ACT GCT TTT TCA TCT ACA ATG G;

SEQ ID NO: ^■20 FP10 ACT GCT TTT TCA TCT ACA ATG G; SEQ ID NO 21 FP1 1 CT ACT GCT TTT TCA TCT ACA ATG;

SEQ ID NO 22 FP409K GGT AAG GAT TGT CGA TAG ATT;

SEQ ID NO 23 FP410 TGGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 24 FP41 1 GGT AAG GAT TGT CGA TAA CTT;

SEQ ID NO 25 FP4021 TGG TAA GGA TTG TCG ATA GGT T;

SEQ ID NO 26 FP415 TGG TAA GGA TTG TCG ATA GGT;

SEQ ID NO 27 FP402-A TGG TAA GGA TTG TCG ATA GTT T;

SEQ ID NO 28 SGBR28-1 CTCTCGCTTTGCTTGGTCTT;

SEQ ID NO 29 SGBR26 CCGTTCAAGGTTCAGGAAAA;

SEQ ID NO 30 SGBR28-2 AAG GCT AAT GGT TTC TGG GG;

SEQ ID NO 31 SGB_RP1 TCGCTTTGCTTGGTCTTAGG ;

SEQ ID NO 32 SGB_RP3 CTT TGC TTG GTC TTA GGG G;

SEQ ID NO 33 SGB RP4 GCT TTG CTT GGT CTT AGG G;

SEQ ID NO 34 SGB_RP2 TTG CTT GGT CTT AGG GGC A;

SEQ ID NO 35 RP38 TTT GCT TGG TCT TAG GGG CT;

SEQ ID NO 36 SGB-P44 AAG CAT AAG TTG CCG GTG CAG AAT A;

SEQ ID NO 37 SGB-P56 CCG GTG CAG AAT ACC CCA GTC CGT TAA; and

SEQ ID NO 38 FP404-A-7 GTA AGG ATT GTC GAG AGT TT.

27. A pair of oligonucleotides selected from the group comprising or consisting of: SEQ ID NO:38 FP404-A-7 GTAAGGATTGTCGAGAGTTT and SEQ ID NO:2 RP39 TTT GCT TGG TCT TAG GGG C;

SEQ ID NO:4 SGBF25 AAGTTGCCGGTGCAGAATAC and SEQ ID NO:30 SGBR28-2 AAG G CT A ATG GTTTCTG G G G ;

SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:28 SGBR28- 1 CTCTCGCTTTGCTTGGTCTT ;

SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:29 SGBR26 CCGTTCAAGGTTCAGGAAAA ; SEQ ID NO:5 S3F10 TAACAGCAGTATCGTAAT and SEQ ID NO:30 SGBR28- 2 AAGGCTAATGGTTTCTGGGG ;

SEQ ID N0:12 FP402 TGG TAA GGA TTG TCG ATA GGT T and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ; SEQ ID NO:13 FP403 GT AAG GAT TGT CGA TAG CT and SEQ ID N0:2 RP39 TTT GCT TGG TCT TAG GGG C ;