Compositions and methods for the detection of group B streptococci (Streptococcus agalactiae) and clindamycin resistance genetic determinants

Abstract

The present invention relates to compositions and methods for detecting Group B Streptococcus (GBS, Streptococcus agalactiae) and identifying the most prevalent genes involved in clindamycin-resistant GBS, such as ermB, ermTR, ermT, lsaC, and lsaE, by multiplex real-time PCR assays.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on and claims priority to U.S. Provisional Patent Application No. 63 / 488,446, filed March 3, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been submitted as an electronic text file entitled "P38225-WO PCT Seq_Listing," which is 24,942 bytes in size and created on February 13, 2024. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5).

[0003] The present disclosure relates to the field of bacterial diagnostics, and more particularly to the detection of group B streptococcus resistant to the antibiotic clindamycin. [Background technology]

[0004] Background of the Invention Group B streptococcal infection, caused by the bacterium Streptococcus agalactiae (also known as group B streptococcus—GBS), is a major cause of neonatal infection. GBS infection during the first week of life, defined as early-onset disease (EOD), can lead to severe illness, including sepsis, meningitis, pneumonia, and even death (Schuchat, Anne, “Group B streptococcus,” The Lancet 353.9146 (1999):51-56). GBS consists of a single species, group B streptococcus, a Gram-positive commensal bacterium found in the intestinal and genital tracts. Transmission of the bacteria during labor can occur when the fetus passes through the maternal birth canal, which is overgrown with bacteria. Prevention of GBS EOD through universal screening at 36 and 38 weeks of gestation and antibiotic prophylaxis significantly reduces the likelihood of disease ("Prevention of Group B Streptococcal Early-Onset Disease in Newborns ACOG COMMITTEE OPINION Number 797," Obstetrics & Gynecology, 135(2)(2020):e51-72). The Centers for Disease Control and Prevention (CDC) previously provided stewardship guidelines, but in 2018, responsibility shifted to three professional organizations. The American College of Obstetricians and Gynecologists and the American Academy of Pediatrics provide guidelines for prevention and treatment, while the American Society of Microbiology (ASM) provides guidelines for standard laboratory testing.(American College of Obstetricians and Gynecologists Committee on Obstetric Practice. “Prevention of early-onset group B streptococcal disease in newborns: ACOG committee opinion no 782”, Obstet Gynecol 134(2019):e19-e40).

[0005] According to ASM guidelines, a vaginal / rectal swab is collected from the patient and allowed to concentrate overnight. It can then be subjected to plate-based agar or nucleic acid amplification testing for GBS identification. A positive GBS colonization result leads to the recommendation of intravenous penicillin prophylaxis. If the patient is reported to have a penicillin allergy with a low risk of anaphylaxis, a first-generation cephalosporin is recommended. For patients with a penicillin allergy who are at high risk of anaphylaxis, clindamycin is recommended.

[0006] GBS has been found to be increasingly resistant to clindamycin, with up to 40% of isolates in certain regions (CDC. "Antibiotic Resistance Threats in the United States" 2019). Due to the high resistance rate, antibiotic susceptibility testing is performed, which is typically culture-based and can result in several days of delay. Constitutive and inducible clindamycin resistance can be attributed to five different resistance genes: the 23S rRNA methylase genes, ermB, ermTR, and ermT, and the ATP-binding cassette (ABC) transporter genes, lsaC and lsaE. ermB and ermTR have been shown to account for nearly 90% of resistant isolates in the United States (Metcalf, BJ, et al. "Short-read whole genome sequencing for determination of antimicrobial resistance mechanisms and capsular serotypes of current invasive Streptococcus agalactiae recovered in the USA," Clinical Microbiology and Infection 23.8(2017):574-e7). There is currently no molecular assay for the rapid determination of GBS clindamycin resistance.

[0007] While several commercially available molecular assays exist for GBS screening, most require overnight enrichment culture before use. While testing directly from a sample can significantly reduce the time to results, robust detection of GBS requires better sensitivity and specificity (Filkins, L., et al., "Guidelines for the detection and identification of group B streptococcus," Am Soc Microbiol). Furthermore, there are currently no known commercially available assays for GBS and clindamycin resistance testing directly from a sample or enrichment culture. While similar assays directly from a sample have been attempted (Gygax et al., "Detection of erythromycin and clindamycin resistance genes in Group B Streptococcal clinical isolates and cervicovaginal-rectal swabs," Microb Drug Resist., 2007 Summer;13(2):119-23), it has been difficult to identify whether antibiotic resistance genes originate from GBS when they are present in other Gram-positive bacteria contained in the sample. Summary of the Invention

[0008] Summary of the Invention The present invention discloses a multiplex PCR assay utilizing dual GBS targets, i.e., single-copy and multi-copy targets, which allows for a lower limit of detection (LoD) directly from the sample workflow. The assay also adds an oligonucleotide primer / oligonucleotide probe combination to detect five genes involved in clindamycin resistance, enabling rapid clindamycin resistance information. The use of dual GBS targets allows for the determination of whether the clindamycin resistance gene is derived from GBS. In one embodiment, the algorithm for this determination is based on the difference in Ct values ​​between the single-copy GBS gene and at least one clindamycin resistance gene.

[0009] Provided herein is a method for rapidly detecting group B streptococcus (GBS, group B hemolytic streptococcus, or S. agalactiae) in biological or non-biological samples and the presence or absence of the most prevalent clindamycin resistance gene in GBS. This is achieved, for example, by multiplex detection of the GBS multicopy 23S ribosomal RNA (23S rRNA) gene and single-copy cAMP factor (cfb) gene, as well as the ermTR, ermB, ermT, lsaC, and lsaE genes that confer resistance to clindamycin, by real-time polymerase chain reaction in a single test tube. Thus, the method for detecting the 23S rRNA and cfb genes and the clindamycin resistance gene involves performing at least one cycling step, which may include an amplification step and a hybridization step. Additionally, provided are oligonucleotide primers, oligonucleotide probes, and kits designed to detect the GBS 23S rRNA gene, the GBS cfb gene, and the clindamycin resistance genes ermTR, ermB, ermT, lsaC, and lsaE in a single tube. Detection methods are designed to target these genes, making it possible to detect the presence of GBS and the clindamycin resistance mechanism in a single test.

[0010] Provided herein is a method for detecting GBS having a clindamycin resistance mechanism in a sample, if these target genes are present in the sample, comprising contacting the sample with a set of GBS 23S rRNA forward and reverse oligonucleotide primers, a set of GBS cfb forward and reverse oligonucleotide primers, a set of ermTR forward and reverse oligonucleotide primers, a set of ermB forward and reverse oligonucleotide primers, a set of ermT forward and reverse oligonucleotide primers, a set of lsaC forward and reverse oligonucleotide primers, and a set of lsaE forward and reverse oligonucleotide primers to generate amplification products; and coupling the amplification products to one or more detectable GBS 23S rRNA oligonucleotide probes, one or more detectable GBS cfb forward and reverse oligonucleotide primers. cfb oligonucleotide probe, one or more detectable ermTR oligonucleotide probes, one or more detectable ermB oligonucleotide probes, one or more detectable ermT oligonucleotide probes, one or more detectable lsaC oligonucleotide probes, and one or more detectable lsaE oligonucleotide probes; and detecting the presence or absence of an amplification product, wherein the presence of an amplification product indicates the presence of GBS and / or clindamycin resistance mechanisms in the sample and the absence of an amplification product indicates the absence of GBS and / or clindamycin resistance mechanisms in the sample.

[0011] In one aspect, a method for detecting Group B Streptococcus (GBS) is provided, comprising contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; combining the amplification products with a detectable oligonucleotide probe for each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification products, wherein the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of each of the GBS strains and clindamycin resistance mechanisms in the sample. In one embodiment, the method further distinguishes GBS from other streptococcal species. In one embodiment, the sample is selected from one of an enriched sample and a sample obtained directly from a specimen sample. In one embodiment, the defined set of targets includes i) the GBS 23s ribosomal RNA gene (23s rRNA), ii) a GBS-specific gene, and iii) at least one clindamycin resistance gene. In one embodiment, the method further comprises detecting a GBS-specific gene (Ct GBS ) and at least one clindamycin resistance gene (Ct ClinR ) measuring the cycle threshold (Ct) for each of the GBS and Ct ClinRand calculating the difference between the absolute value of ΔCt and the clindamycin resistance gene as ΔCt; and identifying the sample as containing GBS carrying at least one clindamycin resistance gene if the absolute value of ΔCt is equal to or less than a threshold value (x). In one embodiment, x≦2. In one embodiment, the GBS-specific gene is selected from the group consisting of a CAMP factor-encoding gene (cfb), a surface immunogenic protein-encoding gene (sip), a glycosyltransferase protein-encoding gene, and a lysR family protein-encoding gene. In a specific embodiment, the GBS-specific gene is the CAMP factor-encoding gene (cfb). In one embodiment, the at least one clindamycin resistance gene is selected from the group consisting of ermTR, ermB, ermT, IsaC, and IsaE. In one embodiment, the plurality of oligonucleotide primers comprises a set of oligonucleotide primers for amplifying at least a portion of each of the targets, wherein the set of 23s rRNA oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 22, 23, or 24, and the set of GBS-specific gene oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 28, 29, 31, or 32; and the at least one set of clindamycin resistance gene oligonucleotide primers comprises a set of ermTR oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 38 or 39, a set of ermB oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 33 or 34, a set of ermT oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 13 or 14, a set of lsaC oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 42 or 43, and a set of lsaE oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 19 or 20.In one embodiment, the detectable oligonucleotide probe for 23s rRNA comprises the nucleic acid sequence of SEQ ID NO: 27 or its complement, the detectable oligonucleotide probe for the GBS-specific gene comprises the nucleic acid sequence of SEQ ID NO: 30 or 64 or its complement, and the detectable oligonucleotide probes for at least one clindamycin resistance gene include a detectable oligonucleotide probe for ermTR comprising the nucleic acid sequence of SEQ ID NO: 40 or 41 or its complement, a detectable oligonucleotide probe for ermB comprising the nucleic acid sequence of SEQ ID NO: 35 or 36 or its complement, a detectable oligonucleotide probe for ermT comprising the nucleic acid sequence of SEQ ID NO: 37 or its complement, a detectable oligonucleotide probe for lsaC comprising the nucleic acid sequence of SEQ ID NO: 44 or its complement, and a detectable oligonucleotide probe for lsaE comprising the nucleic acid sequence of SEQ ID NO: 45 or its complement. In some embodiments, the detectable oligonucleotide probe for each target is labeled with a donor moiety and a corresponding acceptor moiety, and the detecting step further comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety of the detectable oligonucleotide probe, wherein the presence or absence of a fluorescent signal from the detectable oligonucleotide probe indicates the presence or absence of the corresponding moiety of the target in the sample. In some embodiments, the donor moiety and the corresponding acceptor moiety are separated by at least 7 nucleotides on the detectable oligonucleotide probe. In one embodiment, the acceptor moiety is a quencher. In one embodiment, the contacting step further comprises a polymerase enzyme having 5' to 3' nuclease activity.

[0012] In one embodiment, the set of GBS 23S rRNA oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 22 or 23, and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 24; and / or the detectable GBS 23S rRNA oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 27, or a complement thereof. In one embodiment, the set of GBS cfb oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 28 or 31, and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 29 or 32; and / or the detectable GBS cfb oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 30 or 64, or a complement thereof. In one embodiment, the set of ermTR oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 38 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 39; and the detectable ermTR oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 40 or 41, or a complement thereof. In one embodiment, the set of ermB oligonucleotide primers comprises a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 33 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 34; and / or the detectable ermB oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 35 or 36, or a complement thereof. In one embodiment, the set of ermT oligonucleotide primers comprises a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 13 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 14; and / or the detectable ermT oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 37, or a complement thereof.In one embodiment, the set of lsaC oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 42 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 43; the detectable lsaC oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 44 or its complement. In one embodiment, the set of lsaE oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 19 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 20; and the detectable lsaE oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 45 or its complement.

[0013] In another embodiment, the set of GBS 23S rRNA oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 2; and / or the detectable GBS 23S rRNA oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 3 or its complement. In one embodiment, the set of GBS cfb oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 4 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 5; and / or the detectable GBS cfb oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 6 or its complement. In one embodiment, the set of ermTR oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 7 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 8; and the detectable ermTR oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 9 or its complement. In one embodiment, the set of ermB oligonucleotide primers comprises a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 10, and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 11; and / or the detectable ermB oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 12, or its complement. In one embodiment, the set of ermT oligonucleotide primers comprises a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 13, and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 14; and / or the detectable ermT oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 15, or its complement.In one embodiment, the set of lsaC oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 17; the detectable lsaC oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 18 or its complement. In one embodiment, the set of lsaE oligonucleotide primers comprises or consists of a forward primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 19 and / or a reverse primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 20; and the detectable lsaE oligonucleotide probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 21 or its complement.

[0014] In another aspect, a method for detecting group B streptococcus (GBS) is provided, comprising contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products containing a representative nucleic acid for each of the targets present in the sample; combining the amplification products with a detectable oligonucleotide probe for each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification products, wherein the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample and distinguishes GBS from other streptococcal species. In one embodiment, the sample is selected from one of an enriched sample and a sample obtained directly from a specimen sample. In one embodiment, the defined set of targets includes the GBS 23s ribosomal RNA gene (23s rRNA). In one embodiment, the amplification products are further combined with a blocking oligonucleotide. In one embodiment, the defined set of targets further includes a GBS-specific gene. In one embodiment, the GBS-specific gene is the CAMP factor-encoding gene (cfb). In one embodiment, the method distinguishes GBS from at least one of Streptococcus urinalis, Streptococcus thermophilus, and Streptococcus anginosus. In one embodiment, the defined set of targets further comprises at least one clindamycin resistance gene. In some embodiments, the at least one clindamycin resistance gene is selected from the group consisting of ermTR, ermB, ermT, IsaC, and IsaE. In one embodiment, any of the oligonucleotide primers and / or probes or sets of oligonucleotide primers and / or probes described above may be used.

[0015] In one embodiment, amplification can be performed using a polymerase enzyme with 5' to 3' nuclease activity. Thus, the first and second fluorescent moieties can be within 8 nucleotides of each other along the length of the oligonucleotide probe. In another embodiment, the 23S rRNA, cfb, ermTR, ermB, ermT, lsaC, and lsaE oligonucleotide probes contain nucleic acid sequences that allow for secondary structure formation. The formation of such secondary structures generally results in spatial proximity between the first and second fluorescent moieties. According to this method, the second fluorescent moiety on the oligonucleotide probe can be a quencher.

[0016] In another aspect, the present disclosure provides oligonucleotides comprising or consisting of a nucleotide sequence selected from SEQ ID NOs: 1-64 or its complement, the oligonucleotide comprising 100 or fewer nucleotides. The present disclosure also provides oligonucleotides comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%) to one of SEQ ID NOs: 1-64 or its complement, the oligonucleotide comprising 100 or fewer nucleotides. Generally, these oligonucleotides, in these embodiments, can be primer nucleic acids, probe nucleic acids, etc., and can be used in any of the methods provided herein. In some of these embodiments, the oligonucleotides have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, etc.). In some embodiments, any one of the oligonucleotides can comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. The oligonucleotide can comprise at least one conservatively modified mutation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Those skilled in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in an encoded sequence are "conservatively modified variations," which result in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid.

[0017] In another aspect, a kit for detecting Group B Streptococcus (GBS) and at least one clindamycin resistance mechanism is provided, the kit comprising a plurality of oligonucleotide primers for a defined set of targets for generating amplification products comprising a representative nucleic acid for each of the targets present in a sample, the defined set of targets comprising the GBS 23s ribosomal RNA gene (23s rRNA). In one embodiment, the defined set of targets further comprises at least one of a GBS-specific gene and / or a clindamycin resistance gene. In one embodiment, the kit comprises a plurality of oligonucleotide primers and oligonucleotide probes, including a set of GBS 23S rRNA gene oligonucleotide primers specific for amplifying the GBS 23S rRNA gene, and one or more detectable GBS 23S rRNA gene oligonucleotide primers and oligonucleotide probes specific for detecting the GBS 23S rRNA gene amplification product. rRNA oligonucleotide probes; and at least one set of clindamycin resistance gene oligonucleotide primers selected from the group consisting of a set of ermTR gene oligonucleotide primers specific for amplification of the ermTR gene and one or more detectable ermTR oligonucleotide probes specific for detection of ermTR gene amplification products; a set of ermB gene oligonucleotide primers specific for amplification of the ermB gene and one or more detectable ermB oligonucleotide probes specific for detection of ermB gene amplification products; a set of ermT gene oligonucleotide primers specific for amplification of the ermT gene and one or more detectable ermT oligonucleotide probes specific for detection of ermT gene amplification products; a set of lsaC gene oligonucleotide primers specific for amplification of the lsaC gene and one or more detectable lsaC oligonucleotide probes specific for detection of lsaC gene amplification products; and a set of lsaE gene oligonucleotide primers specific for amplification of the lsaE gene and one or more detectable lsaE oligonucleotide probes specific for detection of lsaE gene amplification products. In one embodiment, the set of defined targets further comprises a GBS-specific gene.In one embodiment, the GBS-specific gene is the CAMP factor-encoding gene (cfb).

[0018] In one embodiment, the kit further includes a set of GBS cfb gene oligonucleotide primers specific for amplification of the GBS cfb gene and one or more detectable GBS cfb oligonucleotide probes specific for detecting GBS cfb gene amplification products. In one embodiment, any of the oligonucleotide primers and / or probes or sets of oligonucleotide primers and / or probes described above may be included in the kit. In one embodiment, the kit may include oligonucleotide probes already labeled with donor and corresponding acceptor fluorescent moieties, or may include fluorescent dye moieties for labeling the oligonucleotide probes. The kit may also include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for nucleic acid polymerase function. The kit may also include a package insert and instructions for using the oligonucleotide primers, oligonucleotide probes, and fluorescent dye moieties to detect the presence or absence of GBS and / or clindamycin resistance mechanisms in a sample.

[0019] In another aspect, a method for detecting Group B Streptococcus (GBS) is provided, comprising contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products containing a representative nucleic acid for each of the targets present in the sample; combining the amplification products with a detectable oligonucleotide probe for each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification products, wherein the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample, and the defined set of targets includes the GBS 23s ribosomal RNA gene (23s rRNA). In one embodiment, the sample is selected from one of an enriched sample and a sample obtained directly from a specimen sample. In one embodiment, the defined set of targets further includes at least one of a GBS-specific gene and a clindamycin resistance gene. In one embodiment, the method distinguishes GBS from at least one of Streptococcus urinalis, Streptococcus thermophilus, and Streptococcus anginosus. In one embodiment, the plurality of oligonucleotide primers comprises a set of oligonucleotide primers for amplifying at least a portion of each of the targets, and the set of 23s rRNA oligonucleotide primers comprises at least one primer comprising or consisting of the nucleic acid sequence of SEQ ID NO: 22, 23, or 24. In one embodiment, the detectable oligonucleotide probe for 23s rRNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 27, or its complement. In one embodiment, any of the above-described oligonucleotide primers and / or probes, or sets of oligonucleotide primers and / or probes, may be used in the method.

[0020] In another aspect, a method for detecting Group B Streptococcus (GBS) is provided, comprising contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; combining the amplification products with a detectable probe for each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification products, wherein the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample, wherein the sample is obtained directly from a specimen sample, and the defined set of targets comprises the GBS 23s ribosomal RNA gene (23s rRNA), thereby distinguishing GBS from other streptococcal species. In one embodiment, any of the oligonucleotide primers and / or probes or sets of oligonucleotide primers and / or probes described above can be used in this method.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this subject, and suitable methods and materials are described below.In addition, materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0022] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0023] [Figure 1]Two sets of qPCR amplification curves are shown for the detection of GBS 23S rRNA or Streptococcus urinalis 23S rRNA according to the present disclosure. The set of oligonucleotide primers and probes used in the reactions exclusively amplified the GBS 23S rRNA target (left), and no amplification was observed for Streptococcus urinalis 23S rRNA (right). Fluorescence signal is plotted as a function of cycle number for three template concentrations containing 1*102, 1*103, and 1*104 copies per reaction of either the GBS 23S rRNA template or the S. urinalis 23S rRNA template. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description of the Invention As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., a GBS 23S rRNA gene). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors (e.g., MgCl and / or KCl) for optimal activity of the polymerase enzyme.

[0025] The term "primer" is used herein as known to those skilled in the art to refer to oligomeric compounds, primarily oligonucleotides, but also modified oligonucleotides that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by the template-dependent DNA polymerase, establishing a 3' to 5' phosphodiester linkage, thereby using a deoxynucleoside triphosphate and releasing pyrophosphate. Thus, there is no fundamental difference between a "primer," an "oligonucleotide," an "oligonucleotide primer," a "probe," or an "oligonucleotide probe," except perhaps in terms of intended function.

[0026] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0027] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0028] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when subjected to high temperatures for the time required to cause denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in a 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, non-thermostable polymerases can be used in PCR assays if the enzyme is supplemented.

[0029] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0030] The term "extension" or "lengthening" when used in reference to nucleic acids refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds a nucleotide to the 3' end of the nucleic acid.

[0031] The terms "identical" or percent "identity" in the context of two or more nucleic acid sequences refer to two or more sequences or subsequences that have the same or a specified percentage of the same nucleotides when compared and aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. An exemplary algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST program, see, e.g., Altschul et al. (1990) "Basic local alignment search tool" J. Mol. Biol. 215:403-410, Gish et al. (1993) "Identification of protein coding regions by database similarity search" Nature Genet. 3:266-272, Madden et al. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131-141, Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs" Nucleic Acids Res. 25:3389-3402, and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation" Genome Res. 7:649-656, each of which is incorporated herein by reference.

[0032] "Modified nucleotide" in the context of oligonucleotides refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide that provides desired properties to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, the modified nucleotide substitutions modify the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further explain, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of the intended target amplicon, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0033] A "variant" of a given oligonucleotide may contain one or more nucleotide additions, deletions, or substitutions, for example, one or more nucleotide additions, deletions, or substitutions at the 5' and / or 3' ends of each sequence of the oligonucleotide. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modifications but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" can be attached to the base portion of a "nucleotide" to thereby obtain a "modified nucleotide." A natural base in a "nucleotide" can also be replaced, for example, by 7-desazapurine, thereby similarly obtaining a "modified nucleotide." The terms "modified nucleotide" and "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analogue") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogues").

[0034] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules, such as nucleic acid molecules encoding the nucleic acid sequence of the GBS 23S rRNA gene, the GBS cfb gene, or a clindamycin resistance gene (e.g., ermTR or lsaC), can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures of the members of the pair of primers, and the length of each primer (i.e., the primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is reduced during oligonucleotide synthesis). Typically, oligonucleotide primers are 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0035] In addition to the set of primers, the present method may use one or more probes to detect the presence or absence of GBS and the clindamycin resistance mechanism. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to, in the present case, GBS 23S rRNA or GBS cfb (GBS target) nucleic acid and / or ermTR, ermB, ermT, lsaC, lsaE (clindamycin resistance mechanism) nucleic acid under a defined, predetermined stringency. A "probe" may also be referred to as a "detection probe," which means that it detects a target nucleic acid.

[0036] In some embodiments, the described probes can be labeled with at least one fluorescent label. In one embodiment, the probes can be labeled with a donor fluorescent moiety, e.g., a fluorescent dye, and a corresponding acceptor fluorescent moiety, e.g., a quencher.

[0037] The design of oligonucleotides used as probes can be carried out in the same manner as the design of primers. In embodiments, a single probe or a pair of probes may be used to detect amplification products. Depending on the embodiment, the probe(s) used may contain at least one label and / or at least one quencher moiety. Like primers, probes usually have similar melting temperatures, and the length of each probe must be sufficient to allow sequence-specific hybridization to occur, but not so long that fidelity is reduced during synthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0038] The constructs may include vectors each containing one of the primer and probe nucleic acid molecules (e.g., SEQ ID NOS: 1-21). The constructs may be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. Target nucleic acid molecules may be obtained, for example, by chemical synthesis, direct gene cloning, or PCR amplification.

[0039] Constructs suitable for use in the present methods typically include, in addition to the target nucleic acid molecule (e.g., a nucleic acid molecule containing one or more of the sequences of SEQ ID NOs: 1-21), a sequence encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, as well as an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0040] Constructs containing target nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotes and eukaryotes, such as yeast, plant, and animal cells. Prokaryotic hosts may include E. coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris; mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells; insect cells; and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Constructs can be introduced into host cells using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and viral-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be delivered directly to cells (see, eg, US Pat. Nos. 5,580,859 and 5,589,466).

[0041] The term "enriched sample" or "enriched specimen" refers to a sample or specimen that has been processed to increase the amount or concentration of a target of interest suspected to be present in the sample. A specimen obtained from a patient may be enriched for one or more microorganisms present in the sample, such as GBS and other streptococcal strains. Several culture-based molecular methods exist for enriching streptococcal species, including GBS and other microorganisms, in a specimen. In one example, a swab used to collect a specimen from a patient is placed in elution medium (e.g., Liquid Amies). The elution medium is then inoculated into LIM enrichment broth (see, e.g., Lim, DV, et al. 1982. Current Microbiol.; 7:99-101). The enrichment broth is then incubated for a period of time (e.g., 18-24 hours) sufficient to selectively enrich streptococcal species, including GBS. The resulting enriched specimen may then be used to detect GBS, one or more clindamycin resistance markers, and / or other targets of interest that may be present in the enriched sample. Patient specimens, including vaginal and rectal specimens, can be collected using commercially available swabs such as ESWAB (COPAN).

[0042] The term "obtained directly from a specimen" refers to a sample that is processed immediately after collection without further concentration, as opposed to a concentrated sample. Samples obtained directly from a specimen include vaginal and rectal samples obtained with a swab and optionally placed in an elution medium. The swab and / or elution medium can be tested directly without concentrating microorganisms that may be present on the elution medium or swab.

[0043] polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the nucleic acid sequences of the described target genes and target alleles (e.g., SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10, 11, and 13, 14). Primers can be purified from restriction digests by conventional methods or produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, but primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method for denaturing double-stranded nucleic acids is by heating.

[0044] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method for separating nucleic acid strands involves heating the nucleic acid until it is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0045] If the double-stranded template nucleic acid is denatured by heat, the reaction mixture can be cooled to a temperature that promotes annealing of each primer to its target sequence on the described nucleic acid molecule. The annealing temperature is typically about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient for extension of the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but not so high as to denature the extension products from their complementary templates (e.g., the temperature for extension is generally in the range of about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (eg, from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0046] PCR assays can use nucleic acids of target genes and / or alleles, such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified; it can be a small portion of a complex mixture, such as the target nucleic acid contained in a biological sample. Target nucleic acid molecules can be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0047] Oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, and dGTP, or one or more of their analogs.

[0048] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The strand separation, annealing, and extension steps can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or even 100 times.

[0049] Fluorescence Resonance Energy Transfer (FRET) FRET technology (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, energy transfer occurs between two fluorescent moieties that can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule containing a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0050] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, that dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, resulting in quenching of the fluorescence emission from the donor fluorescent moiety.During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched.Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785.A commonly used donor-acceptor pair includes the FAM-TAMRA pair.Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™, (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Mich.).

[0051] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0052] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a specific range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0053] As used herein, "corresponding" to a donor fluorescent moiety and a corresponding acceptor fluorescent moiety refers to an acceptor fluorescent moiety having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety, thereby allowing efficient non-radiative energy transfer between them.

[0054] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) emission shift as far as possible into the red portion of the visible spectrum (>600 nm), and (d) emission shift to wavelengths higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good excitation overlap with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0055] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, OR) or Sigma Chemical Co. (St. Louis, MO).

[0056] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the angstrom (Å) distance from the nucleotide base to the fluorescent moiety. Typically, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0057] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to produce, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, MA)), amide linkers (derived from fluorescein-NHS-ester, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0058] Detection of target GBS genes and clindamycin resistance genes The present disclosure provides a method for detecting the presence or absence of the GBS 23S rRNA gene, GBS cfb gene, and ermTR, ermB, ermT, lsaC, and lsaE genes in a biological or non-biological sample. The provided method avoids problems of sample contamination, false negatives, and false positives. The method includes at least one cycling step involving amplifying a portion of a target nucleic acid molecule from the sample using multiple target primer pairs, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed to detect the presence of a target gene using the target primers and probes, and detection of the amplification product in the assay indicates the presence of the target gene and / or target allele in the sample.

[0059] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and thus the presence or absence of GBS and / or clindamycin resistance genes. TaqMan® technology utilizes a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of the appropriate wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is subsequently degraded during the extension phase by, for example, the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for performing the methods described herein for detecting the presence or absence of GBS and / or clindamycin resistance genes in a sample.

[0060] Molecular beacons combined with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and fluorescent labels are typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows secondary structure formation (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, both fluorescent moieties are spatially close together when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted, separating the fluorescent moieties from each other, allowing the emission of the first fluorescent moiety to be detected after excitation with light of an appropriate wavelength.

[0061] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize closely to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, such as fluorescein, is excited at 470 nm by the LightCycler® instrument's light source. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, such as LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the LightCycler® instrument's optical detection system. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules. When amplification of the target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable FRET-based signal between the members of the probe pair.

[0062] Generally, the presence of FRET indicates the presence of target sequence in the sample, and the absence of FRET indicates the absence of target sequence in the sample.However, insufficient specimen collection, delayed transport, improper transport conditions, or the use of some collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and / or accuracy of test results.Using the method disclosed herein, for example, detecting FRET within 45 cycle steps indicates GBS infection.

[0063] Representative biological samples that can be used to carry out this method include, but are not limited to, skin swabs, nasal swabs, wound swabs, blood cultures, and skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. Biological samples can be processed (for example, by nucleic acid extraction methods and / or kits known in the art) to release target gene nucleic acid, or in some cases, biological samples can be directly contacted with PCR reaction components and suitable oligonucleotides.

[0064] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA duplex separates into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature of the probe from the amplification product can confirm the presence or absence of the target sequence in the sample.

[0065] Control samples can be cycled similarly during each thermocycler run. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the listed target gene) using, for example, control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermocycler run can also include a negative control, for example, lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false-positive signals. Thus, control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate elongation, and the ability of probes to hybridize with sequence specificity and allow FRET to occur.

[0066] In one embodiment, the method includes a step of avoiding contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.

[0067] The method can be implemented using conventional PCR methods combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0068] The LightCycler® can be operated using a PC workstation and utilizes the Windows® NT operating system. Signals from samples are acquired as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The generated data can be saved for further analysis.

[0069] As an alternative to FRET, amplification products can be detected using double-stranded DNA-binding dyes, such as fluorescent DNA-binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Upon interaction with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescent signal after excitation with light of a suitable wavelength. Double-stranded DNA-binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA-binding dyes, a melting curve analysis is usually performed to confirm the presence of amplification products.

[0070] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0071] Manufactured Products / Kits Embodiments of the present disclosure further provide articles of manufacture or kits for detecting GBS 23S rRNA and cfb genes, as well as ermTR, ermB, ermT, lsaC, and lsaE genes (i.e., genes involved in clindamycin-resistant GBS). The articles of manufacture may include primers and probes used to detect clindamycin-resistant GBS, along with suitable packaging. Representative primers and probes for detecting clindamycin-resistant GBS are hybridizable to target nucleic acid molecules. Furthermore, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to target nucleic acid molecules are provided.

[0072] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or the probes provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0073] The article of manufacture may include a package insert or packaging label with instructions for using the primers and probes to detect clindamycin-resistant GBS in a sample.The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents for preventing contamination) for carrying out the methods disclosed herein.Such reagents may be specific to one of the commercially available instruments described herein.

[0074] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. [Example]

[0075] The following examples and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0076] Example 1: Primer and probe sequences Table 1 shows the primers and probes used in the multiplex PCR assay for the detection of GBS and clindamycin resistance mechanisms according to the present disclosure. [Table 1]

[0077] Where indicated, the primer and probe sequences in Table 1 are exemplified to include the indicated modifications according to the following scheme: J = t-butylbenzyl-dA;<HEX_Thr> =HEX dye, Q=BHQ2,<Spc_C3> = 3' spacer, K = t-butylbenzyl-dC,<FAM_Thr> = FAM dye, and<JA270_Thr> =JA270 dye.

[0078] Example 2: PCR experimental conditions Real-time PCR detection of gene targets was performed using a LightCycler® 480 system (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents and the thermal profile used for the PCR amplification reactions are shown in Table 2. [Table 2]

[0079] The pre-PCR program included incubations at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. The three-temperature incubation combined the beneficial effects of lower temperatures, which allow transcription of even slightly mismatched target sequences (e.g., genetic variants of an organism), and higher temperatures, which suppress the formation of RNA secondary structures and thus result in more efficient transcription. PCR cycling was divided into two runs, each using a single-step setup (combined annealing and extension). The first five cycles at 55°C allowed comprehensive amplification by preamplifying the slightly mismatched target sequences, while the second run (45 cycles) increased specificity by using an annealing / extension temperature of 58°C.

[0080] Example 3: Performance of PCR assays for detection of GBS and clindamycin resistance genes The results of PCR assays using primers and probes for detecting GBS and ermTR in a multiplex are shown in Table 3. The master mix used forward primers SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, reverse primers SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, and probes SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9. The template for GBS detection was genomic DNA extracted from a clinical isolate of Group B Streptococcus pyogenes at concentrations ranging from 1.00E+06 copies per reaction to 1.00E+00 copies per reaction. Template plasmids carrying the ermTR gene were tested at concentrations ranging from 1.52E+05 copies per reaction to 1.53E+00 copies per reaction. Initial testing demonstrated that these primer / probe pairings produced detection in the multiplex assay.

[0081] The primer / probe pairings for ermB in singleplex and ermT in singleplex are shown in Table 4. The master mix used forward primer SEQ ID NO: 10, reverse primer SEQ ID NO: 11, and probe SEQ ID NO: 12 for ermB. The master mix used forward primer SEQ ID NO: 13, reverse primer SEQ ID NO: 14, and probe SEQ ID NO: 15 for ermT. The primer / probe pairings for lsaC in singleplex and lsaE in singleplex are shown in Table 5. The master mix used forward primer SEQ ID NO: 16, reverse primer SEQ ID NO: 17, and probe SEQ ID NO: 18 for lsaC. The master mix used forward primer SEQ ID NO: 19, reverse primer SEQ ID NO: 20, and probe SEQ ID NO: 21 for lsaE. Template plasmids carrying ermB, lsaC, or lsaE were tested at concentrations ranging from 1.00E+06 copies per reaction to 1.00E+00 copies per reaction. The template plasmid carrying the ermT gene was tested at concentrations ranging from 7.95E+03 copies per reaction to 7.95E+00 copies per reaction. [Table 3] [Table 4] [Table 5]

[0082] In Tables 3-5, Test Concentration (cp / rxn) is the test concentration in units of copy number per reaction, Mean Ct is the mean cycle threshold (i.e., the mean number of cycles required for the fluorescent signal to exceed the background fluorescent signal threshold), and Mean RFI is the mean relative fluorescent intensity.

[0083] Example 4: Additional primer and probe sequences Table 6 shows additional primers and probes used in multiplex PCR assays for the detection of GBS and clindamycin resistance mechanisms according to the present disclosure. [Table 6-1] [Table 6-2]

[0084] Where indicated, primer and probe sequences in Table 6 are exemplified to include the indicated modifications according to the following scheme: J = t-butylbenzyl-dA, K = t-butylbenzyl-dC, L = 7-deaza-dG, M = 2'-O-methyl-rU, N = D_LNA_A, O = N4-ethyl-dC, P = D_LNA_G, R = D_LNA_T, S = 2'-O-methyl-riboU, V = D_LNA_5-methyl-C,<Spc_C3> =3'spacer,<Coum_Thr> = coumarin dyes,<FAM_Thr> =FAM dye,<HEX_Thr> =HEX dye,<JA270_Thr> = JA270 dye, and Q = BHQ2.

[0085] Example 5: Performance of the assay for the detection of GBS The current standard of care for GBS detection and identification is an enrichment-based approach characterized by a long turnaround time of at least a full day. This approach generally involves specimen collection using a swab, release of the specimen from the swab in liquid medium, enrichment by culturing the specimen in liquid medium for 18–24 hours, followed by either further culturing of the enriched specimen on agar plates or the use of a nucleic acid amplification test (NAAT).

[0086] Current understanding in the field is that the sensitivity of GBS detection is strongly influenced by culture enrichment. Therefore, regardless of whether the ultimate detection method is agar plating or NAAT, it is strongly recommended to incubate GBS screening specimens in selective enrichment broth. This recommendation is based, at least in part, on reports that incubation in broth medium before plating increases the sensitivity of screening methods by approximately two-fold compared to direct plating of specimens. Similarly, the sensitivity of NAAT increases after culture enrichment. For example, the American Society for Microbiology Clinical and Public Health Microbiology Committee, Subcommittee on Laboratory Practices (ASM), recommends that "enrichment broth cultures should be performed first, regardless of whether culture or NAAT is selected as the primary method of GBS detection." The ASM guidelines state, "Importantly, although commercially available NAATs (without enrichment) performed directly from specimens are available, their use is not currently recommended due to high false-negative rates of 6.3%–22%."

[0087] Although ASM does not recommend the use of existing commercially available NAATs performed directly on specimens (without enrichment), it may be useful to provide an approach that reduces or eliminates the need for an enrichment step to shorten the overall turnaround time for GBS detection and identification. For example, direct-from-specimen testing is useful for intrapartum testing, where faster results are desirable. Furthermore, current NAATs for GBS do not characterize antibiotic susceptibility, which can inform further treatment if the GBS result is positive. Therefore, improved testing methods are needed that feature reduced turnaround time compared to enrichment-based approaches. Furthermore, there is a need for NAATs for GBS detection that facilitate antibiotic susceptibility characterization.

[0088] To overcome the aforementioned drawbacks of enrichment-based tests and existing NAATs, a novel approach for identifying GBS, as disclosed herein, was devised and developed. In one aspect, the NAAT disclosed herein provides a direct detection from specimen testing without the need for enrichment. To achieve this, several potential markers were evaluated. It was anticipated that relatively abundant nucleic acids could serve as suitable targets for markers useful for direct detection from unenriched specimen samples. Given that the total RNA pool of a typical microbial cell is primarily composed of ribosomal RNA (rRNA), several rRNA components, including GBS 23S rRNA, were evaluated. To further improve the reliability of this NAAT, the cfb gene, which encodes the Christie, Atkins, and Munch-Peterson (CAMP) factors and is recommended for use in identifying GBS by ASM, was included as an additional target.

[0089] To develop a dual-target NAAT directly from specimen testing, primers and probes were designed and tested for the detection of both the cfb gene and a novel GBS 23S rRNA target. The results of a PCR assay for detecting GBS 23S rRNA and cfb in a multiplex setup are shown in Table 7. The assay contains three sets of oligonucleotides, each containing a forward primer, a reverse primer, and a detectable probe: one set for detecting GBS 23S rRNA and two sets for detecting cfb. In one embodiment, accurate detection of cfb in this example is achieved by using two sets of oligonucleotides containing different GBS serotypes of interest, including GBS serotypes IV and V. Specifically, the oligonucleotides tested were forward primers GBS021 (SEQ ID NO:22), GBS031 (SEQ ID NO:28), and GBS039 (SEQ ID NO:31), reverse primers GBS025 (SEQ ID NO:24), GBS033 (SEQ ID NO:29), and GBS041 (SEQ ID NO:32), and probes GBS027 (SEQ ID NO:26), GBS004 (SEQ ID NO:04), and GBS059 (SEQ ID NO:64). The template for GBS or GBS 23S rRNA detection was a purified synthetic target gene amplicon using digital droplet PCR (ddPCR) corrected copy number. [Table 7]

[0090] As can be seen in Table 7, the primers and probes of this example successfully detected both GBS 23S rRNA and cfb at all concentrations tested on the synthetic template. However, an important consideration in GBS 23S rRNA design is the mitigation of background and / or cross-reactive signals. It should be noted that while testing different primers and probes for the detection of GBS 23S rRNA, initial designs resulted in cross-reactivity (i.e., amplification and detection) of other closely related non-GBS streptococcal species, including S. urinalis and S. thermophilus, in Lim broth-enriched clinical samples. Because non-GBS streptococcal species are known to be commensal with humans, the possibility of their presence in the samples was anticipated. However, given that these oligonucleotides were designed to be highly specific for GBS, observing cross-reactivity of the GBS 23S rRNA primers and probes to non-GBS streptococcal species was surprisingly unexpected.

[0091] To overcome the unexpected cross-reactivity observed in the original NAAT design, we explored various modifications to improve the specificity of the GBS 23S rRNA primers and probes. Referring to Figure 1 and Table 8, we developed an improved assay that significantly reduced or eliminated cross-reactivity between selected 23S rRNA GBS targets and the 23S rRNA of closely related non-GBS streptococcal species, including S. urinalis and S. thermophilus. This result was achieved, at least in part, by relocating the annealing site of the GBS 23S rRNA primer to reduce nonspecific amplification due to homology to non-GBS streptococcal species at the 3' end of the primer and by designing a unique 23S rRNA probe containing an N4-ethyl-dC modification. This probe modification was placed at the mismatch position between S. urinalis 23S rRNA and GBS 23S rRNA in the quencher region of the probe. The result was a severely reduced RFI for S. urinalis and S. thermophilus, with loss of so-called cycle threshold (Ct) values, while maintaining Ct calls and RFI for GBS 23S rRNA. Assay results using GBS 23S rRNA forward and reverse primers GBS021 (SEQ ID NO: 22) and GBS025 (SEQ ID NO: 24), and S. urinalis exclusivity probe GBS069B (SEQ ID NO: 27), using GBS 23S and S. urinalis ddPCR quantification templates, are shown in Table 8. [Table 8]

[0092] In Tables 7 and 8, Test Concentration (cp / rxn) is the test concentration in units of copy number per reaction, Mean Ct is the mean cycle threshold (i.e., the mean number of cycles required for the fluorescent signal to exceed the background fluorescent signal threshold), Mean RFI is the mean relative fluorescent intensity, and NTC is no template control.

[0093] An alternative method for distinguishing GBS 23S rRNA from other non-GBS streptococcal signals involves using two probes, each containing one or more locked nucleic acid (LNA) modified bases. In one embodiment, a first probe labeled with a dye is designed to specifically bind to the GBS 23S rRNA target, while a second probe, lacking a dye, is designed to selectively bind to non-GBS targets. In this case, the second probe acts as a blocking probe to minimize non-specific signals due to the presence of closely related non-GBS streptococcal species. Exemplary oligonucleotides containing the LNA modifications listed in Table 6 include GBS084-GBS097 (SEQ ID NOs: 46-59), which include blocking probes GBS085 (SEQ ID NO: 47), GBS0087 (SEQ ID NO: 49), GBS0089 (SEQ ID NO: 51), GBS0091 (SEQ ID NO: 53), GBS0093 (SEQ ID NO: 55), GBS0095 (SEQ ID NO: 57), and GBS0097 (SEQ ID NO: 59). Other approaches involving blocking probes that hybridize preferentially to non-target nucleic acids where cross-reactivity is observed may be applied as well.

[0094] The assay of this example can be performed on a real-time PCR device that supports two or more fluorescent detection channels. In one example, GBS 23S rRNA and cfb can be detected in one channel, and a general internal control (GIC) can be detected in a second channel, as shown in Table 9, which further illustrates how the resulting data are interpreted based on the presence or absence of GBS 23S rRNA and cfb (23S / cfb) and the GIC, respectively. It will be appreciated that for assays limited to detecting GBS in a sample, identification of either 23S rRNA or cfb is sufficient to characterize the sample as GBS positive. Therefore, in such assays, GBS 23S rRNA and cfb can be detected in the same channel. However, in certain situations, it may be useful to detect GBS 23S rRNA and cfb in separate channels, as discussed in Example 6 below. [Table 9]

[0095] Example 6: Performance of the assay for detection of GBS and clindamycin resistance genes Current NAATs for detecting and identifying GBS do not provide antimicrobial resistance information. Instead, culture-based approaches require obtaining isolates and performing susceptibility testing. In one aspect, it is difficult to design a multiplex NAAT with the sensitivity and specificity required to confidently detect and characterize GBS in samples, especially those obtained directly from specimen samples. This example overcomes these and other challenges by providing primers and probes for use in NAATs for detecting and identifying GBS in conjunction with the detection of one or more clindamycin resistance genes. This NAAT can be used with both specimen samples obtained directly and enriched samples.

[0096] Templates for the clindamycin resistance targets ermB, ermTR, ermT, IsaC, and IsaE were prepared from purified synthetic target gene amplicons with ddPCR-corrected copy numbers. Nucleic acids extracted from cultured GBS strain cells were quantified by plate counting and served as templates for the GBS 23S rRNA and cfb genes.

[0097] The highly multiplexed results for primer and probe sets for each of i) GBS 23s rRNA, ii) the GBS-specific cfb gene, and iii) the clindamycin resistance genes ermB, ermTR, ermT, IsaC, and IsaE are shown in Tables 10 and 11. The master mix contained eight sets of oligonucleotides, each containing a forward primer, a reverse primer, and a detectable probe—one set to detect GBS 23S rRNA, two sets to detect cfb, and one set for each of five different resistance targets (i.e., ermB, ermTR, ermT, IsaC, and IsaE). Specifically, the oligonucleotides tested were forward primers GBS021 (SEQ ID NO: 22), GBS031 (SEQ ID NO: 28), GBS039 (SEQ ID NO: 31), ermB4.F (SEQ ID NO: 33), ermT6.F (SEQ ID NO: 13), ermTR_F_35_69TBB (SEQ ID NO: 38), SEG4091 (SEQ ID NO: 42), and SEGP3910 (SEQ ID NO: 19); reverse primers GBS025 (SEQ ID NO: 24), GBS033 (SEQ ID NO: 29), GBS041 (SEQ ID NO: 32), ermB_R_462_440 (SEQ ID NO: 34); row number 34), ermT6.R (sequence number 14), ermTR_R_203_171TBB (sequence number 39), SEG4092 (sequence number 43), and SEGP3911 (sequence number 20), and probes GBS027 (sequence number 26), GBS004 (sequence number 04), GBS059 (sequence number 64), GBS006 (sequence numbers 35 and 36), GBS010 (sequence number 37), ermTR8.JA270_10 (sequence numbers 40 and 41), GBS012 (sequence number 44), and GBS015 (sequence number 45). [Table 10] [Table 11]

[0098] In Tables 10 and 1, Test Concentration (CFU / Rxn) is the test concentration in units of cell formation per reaction, Mean Ct is the mean cycle threshold (i.e., the mean number of cycles required for the fluorescent signal to exceed the background fluorescent signal threshold), Mean RFI is the mean relative fluorescent intensity, and NTC is no template control.

[0099] The assay of this example can be performed on a real-time PCR device that supports two or more fluorescence detection channels. Preferably, the real-time PCR device supports at least three fluorescence detection channels. More preferably, the real-time PCR device supports at least four fluorescence detection channels. In this example, detection and differentiation of GBS bacteria carrying one or more clindamycin resistance genes from other bacteria carrying clindamycin resistance genes can be achieved by an assay that separates GBS 23S rRNA and cfb targets in different channels, as shown in Table 12, and a rule-based interpretation call, as shown in Table 13. In the example shown in Table 12, GBS 23S rRNA may be detected in the first channel (e.g., channel 3), cfb may be detected in the second channel (e.g., channel 1), one or more clindamycin resistance genes may be detected in the third channel (e.g., channel 4), and a general internal control (GIC) may be detected in the fourth channel (e.g., channel 5).

[0100] Probes GBS80 to GBS83 (SEQ ID NOs: 60 to 63) contain a coumarin dye as the donor moiety and are designed to hybridize to and detect cfb. cfb is present as a single DNA copy in the GBS genome and therefore represents a low-copy target. Here, low copy means five or fewer copies per genome. Similarly, clindamycin resistance genes are also found as either single or low-copy chromosomal gene targets or on low-copy plasmids. By determining the single-target Ct for each of cfb and any resistance genes present in a sample, the ΔCt correlation between each of the resistance genes and cfb can be calculated to better inform whether the level of the resistance gene detected in the sample is similar to the level of the GBS-specific cfb gene. This information can then be utilized to provide more accurate information about whether a given resistance signal is more likely to be from GBS compared to another Gram-positive organism. [Table 12] [Table 13]

[0101] Referring to Table 13, an asterisk (*) indicates that the delta Ct between the Ct of cfb and the Ct of a given resistance gene can be used to further correlate GBS-derived resistance.

[0102] In one example, ΔCt is the time course of a sample (Ct GBS ) for cfb (or another low-copy GBS-specific gene such as surface immunogenic protein-encoding gene, sip, glycosyltransferase protein-encoding gene, and lysR family protein-encoding gene) and single-target clindamycin resistance genes such as ermB, ermTR, ermT, IsaC, and IsaE (Ct ClinR ) is calculated as the difference between the so-called Ct and the GBS -Ct ClinRThe ΔCt can be described as: If the absolute value of ΔCt is equal to or less than a defined threshold (x), the sample can be determined to contain a GBS carrying a clindamycin resistance gene. In one embodiment, when x is small (e.g., 2 or less), the relative amounts of cfb (or another low-copy GBS-specific gene) and the target clindamycin resistance gene can be considered similar or identical, and therefore likely to be derived from the same organism. More specifically, a GBS strain identified by cfb can be inferred to carry a clindamycin resistance gene. In contrast, when x is large, the identification of the clindamycin resistance gene is likely due to the presence of a second non-GBS organism carrying the clindamycin resistance gene. The appropriate value of x can be determined empirically and may depend on the properties of the NAAT, including the choice of instrument, buffer, consumables, etc. In one example, x is 2 or less. In another example, x is 2.5 or less. In another example, x is 3 or less.

[0103] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art from a reading of this disclosure that various changes in form and detail may be made therein without departing from the true scope of the invention. For example, all of the techniques and devices described above may be used in various combinations. All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document was individually indicated to be incorporated by reference for all purposes.

Claims

1. 1. A method for detecting Group B Streptococcus (GBS), comprising: - contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; - combining said amplification products with detectable oligonucleotide probes for each of said targets; - detecting the presence or absence of each of said representative nucleic acids in said amplification products; Including, wherein said presence or absence of each of said representative nucleic acids in said amplification products indicates the presence or absence, respectively, of a GBS strain and clindamycin resistance mechanism in said sample.

2. 10. The method of claim 1, wherein the sample is selected from one of an enriched sample and a sample obtained directly from a specimen sample.

3. 3. The method of claim 1, wherein the set of defined targets comprises i) the GBS 23s ribosomal RNA gene (23s rRNA), ii) a GBS-specific gene, and iii) at least one clindamycin resistance gene.

4. 4. The method of claim 3, wherein the GBS-specific gene is selected from the group consisting of a CAMP factor-encoding gene (cfb), a surface immunogenic protein-encoding gene (sip), a glycosyltransferase protein-encoding gene, and a lysR family protein-encoding gene.

5. The method according to any one of claims 3 to 4, wherein the GBS-specific gene is the CAMP factor-encoding gene (cfb).

6. The method of any one of claims 3 to 5, wherein the at least one clindamycin resistance gene is selected from the group consisting of ermTR, ermB, ermT, IsaC and IsaE.

7. the plurality of oligonucleotide primers comprises a set of oligonucleotide primers for amplifying at least a portion of each of the targets; - the set of 23s rRNA oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 22, 23 or 24; - the set of GBS-specific gene oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 28, 29, 31 or 32; at least one set of clindamycin resistance gene oligonucleotide primers - a set of ermTR oligonucleotide primers, comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 38 or 39; - a set of ermB oligonucleotide primers, including at least one primer comprising the nucleic acid sequence of SEQ ID NO: 33 or 34; a set of ermT oligonucleotide primers, including at least one primer comprising the nucleic acid sequence of SEQ ID NO: 13 or 14; - a set of lsaC oligonucleotide primers, including at least one primer comprising the nucleic acid sequence of SEQ ID NO: 42 or 43, and - a set of lsaE oligonucleotide primers, including at least one primer comprising the nucleic acid sequence of SEQ ID NO: 19 or 20 The method according to any one of claims 3 to 6, wherein the compound is selected from the group consisting of:

8. the detectable oligonucleotide probe for -23s rRNA comprises the nucleic acid sequence of SEQ ID NO: 27 or its complement; - the detectable oligonucleotide probe for the GBS-specific gene comprises the nucleic acid sequence of SEQ ID NO: 30 or 64 or its complement; - said detectable oligonucleotide probe for said at least one clindamycin resistance gene a detectable oligonucleotide probe for ermTR comprising the nucleic acid sequence of SEQ ID NO: 40 or 41 or its complement, a detectable oligonucleotide probe for ermB comprising the nucleic acid sequence of SEQ ID NO: 35 or 36 or its complement, a detectable oligonucleotide probe for ermT comprising the nucleic acid sequence of SEQ ID NO: 37 or its complement, a detectable oligonucleotide probe for lsaC comprising the nucleic acid sequence of SEQ ID NO: 44 or its complement, and - a detectable oligonucleotide probe for lsaE comprising the nucleic acid sequence of SEQ ID NO: 45 or its complement The method of any one of claims 3 to 7, wherein the compound is selected from the group consisting of:

9. 9. The method of claim 1, wherein the detectable oligonucleotide probe for each of the targets is labeled with a donor moiety and a corresponding acceptor moiety, and the detecting step further comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor moiety and the acceptor moiety of the detectable oligonucleotide probe, wherein the presence or absence of a fluorescent signal from the detectable oligonucleotide probe indicates the presence or absence of the corresponding moiety of the target in the sample.

10. 10. The method of claim 9, wherein the donor moiety and the corresponding acceptor moiety are separated by at least 7 nucleotides on the detectable oligonucleotide probe.

11. The method of any one of claims 9 to 10, wherein the acceptor moiety is a quencher.

12. 12. The method of any one of claims 1 to 11, wherein the contacting step further comprises a polymerase enzyme having 5' to 3' nuclease activity.

13. - the GBS-specific gene (Ct ＧＢＳ ) and said at least one clindamycin resistance gene (Ct ＣｌｉｎＲ ) measuring the cycle threshold (Ct) for each of - calculating the difference between CtGBS and CtClinR as ΔCt; - identifying said sample as containing GBS carrying said at least one clindamycin resistance gene if the absolute value of ΔCt is less than or equal to a threshold value (x); The method of any one of claims 3 to 12, further comprising:

14. 14. The method of claim 13, wherein x≦2.

15. An oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-64.

16. 1. A method for detecting Group B Streptococcus (GBS), comprising: - contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; - combining said amplification products with detectable oligonucleotide probes for each of said targets; - detecting the presence or absence of each of said representative nucleic acids in said amplification products; Including, the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample; The method distinguishes GBS from other Streptococcus species.

17. 17. The method of claim 16, wherein the sample is selected from one of an enriched sample and a sample obtained directly from an analyte sample.

18. 18. The method of any one of claims 16 to 17, wherein the set of defined targets comprises the GBS 23s ribosomal RNA gene (23s rRNA).

19. 19. The method of claim 18, wherein the defined set of targets further comprises GBS-specific genes.

20. 20. The method of claim 19, wherein the GBS-specific gene is the CAMP factor-encoding gene (cfb).

21. 21. The method of any one of claims 16 to 20, wherein GBS is distinguished from at least one of Streptococcus urinalis, Streptococcus thermophilus, and Streptococcus anginosus.

22. 22. The method of any one of claims 16 to 21, wherein the set of defined targets further comprises at least one clindamycin resistance gene.

23. 23. The method of claim 22, wherein the at least one clindamycin resistance gene is selected from the group consisting of ermTR, ermB, ermT, IsaC, and IsaE.

24. The method of any one of claims 16 to 23, wherein the amplification product is further combined with a blocking oligonucleotide.

25. 1. A kit for detecting Group B Streptococcus (GBS) and at least one clindamycin resistance mechanism, comprising a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in a sample; A kit wherein the set of defined targets includes the GBS 23s ribosomal RNA gene (23s rRNA).

26. 26. The kit of claim 25, wherein the set of defined targets further comprises at least one of a GBS-specific gene and a clindamycin resistance gene.

27. 1. A method for detecting Group B Streptococcus (GBS), comprising: - contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; - combining said amplification products with detectable oligonucleotide probes for each of said targets; - detecting the presence or absence of each of said representative nucleic acids in said amplification products; Including, the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample; The method, wherein the set of defined targets comprises the GBS 23s ribosomal RNA gene (23s rRNA).

28. 28. The method of claim 27, wherein the sample is selected from one of an enriched sample and a sample obtained directly from an analyte sample.

29. The method of any one of claims 27 to 28, wherein the set of defined targets further comprises at least one of a GBS-specific gene and a clindamycin resistance gene.

30. 30. The method of any one of claims 27 to 29, wherein GBS is distinguished from at least one of Streptococcus urinalis, Streptococcus thermophilus, and Streptococcus anginosus.

31. 31. The method of any one of claims 27-30, wherein the plurality of oligonucleotide primers comprises a set of oligonucleotide primers for amplifying at least a portion of each of the targets, and wherein the set of 23s rRNA oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 22, 23, or 24.

32. 32. The method of any one of claims 27 to 31, wherein the detectable oligonucleotide probe for 23s rRNA comprises the nucleic acid sequence of SEQ ID NO: 27 or its complement.

33. 1. A method for detecting Group B Streptococcus (GBS), comprising: - contacting a sample with a plurality of oligonucleotide primers for a defined set of targets to generate amplification products comprising a representative nucleic acid for each of the targets present in the sample; - combining said amplification products with detectable oligonucleotide probes for each of said targets; - detecting the presence or absence of each of said representative nucleic acids in said amplification products; Including, the presence or absence of each of the representative nucleic acids in the amplification products indicates the presence or absence of a GBS strain in the sample; The sample is a sample obtained directly from a specimen sample, the set of defined targets includes the GBS 23s ribosomal RNA gene (23s rRNA); The method distinguishes GBS from other Streptococcus species.

Images