Compositions and methods for detecting group B streptococcus (streptococcus agalactiae) and clindamycin resistance gene determinants

Abstract

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

Description

[0001] Cross Reference to Related Applications

[0002] This application is based on and claims priority to U.S. Provisional Patent Application No. 63 / 488,446, filed March 03, 2023, which is incorporated by reference in its entirety.

[0003] Reference to Sequence Listing

[0004] The instant application contains a Sequence Listing which has been submitted as an electronic text file named “P38225-WOPCT Seq_Listing” having a size of 24,942 bytes and created on February 13, 2024. The information contained in the electronic file is incorporated by reference in its entirety pursuant to 37 CFR § 1.52(e)(5). TECHNICAL FIELD

[0005] The present disclosure relates to the field of bacterial diagnostics, and more specifically to the detection of group B streptococci that are resistant to the antibiotic clindamycin. BACKGROUND

[0006] Group B Streptococcus infection has been a major cause of neonatal infection, caused by the bacterium Streptococcus agalactiae (also known as Group B Streptococcus - GBS). GBS infection in the first week of life, defined as early onset disease (EOD), can cause severe illness such as sepsis, meningitis, pneumonia or even death (Schuchat, Anne, "Group B streptococcus", The Lancet 353.9146 (1999): 51-56). GBS consists of a single species, Streptococcus agalactiae, a Gram-positive commensal bacterium found in the intestinal and genital tract. Bacterial transmission can occur when the fetus passes through the birth canal of a colonized mother during delivery. Prevention of GBS EOD through universal screening at 36 and 38 weeks of gestation and antibiotic prophylaxis has greatly reduced the odds of getting sick ("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 management guidelines, but in 2018 responsibility was transferred 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 clinical laboratory practices. (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).

[0007] According to the ASM guidelines, a vaginal-rectal swab is collected from the patient and placed in overnight enrichment. It can then be subjected to either a plate-based agar identification or a nucleic acid amplification test for GBS identification. A positive GBS colonization result leads to the recommendation of intravenous penicillin prophylaxis. If the patient reports a penicillin allergy with a low risk of anaphylaxis, a first generation cephalosporin is recommended. In the case of a penicillin allergy with a high risk of anaphylaxis, clindamycin is recommended.

[0008] Increasingly, GBS has been found to be resistant to clindamycin, up to 40% of isolates in some regions (CDC. “Antibiotic Resistance Threats in the United States”. 2019). Due to the high rate of resistance, antibiotic susceptibility testing is performed, which is often culture-based and can take several days to get results. Constitutive and inducible clindamycin resistance can be attributed to five different resistance genes: 23S rRNA methylase genes, ermB, ermTR, ermT, and 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 US. (Metcalf, B. J., 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 to rapidly determine GBS clindamycin resistance.

[0009] There are multiple commercially available molecular assays for GBS screening, but most require overnight enrichment culture prior to use. Testing directly from the sample can greatly shorten the time to result, but requires better sensitivity and specificity to robustly detect GBS (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 the sample or from enrichment culture. Similar direct from sample assays 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), but it is difficult to distinguish if the antibiotic resistance genes are from GBS when other gram positive bacteria in the sample contain the antibiotic resistance genes. SUMMARY

[0010] A multiplex PCR assay is disclosed that allows for reduced limit of detection (LoD) for a direct from sample workflow that utilizes dual GBS targets: a single copy target and a multiple copy target. The assay also adds oligonucleotide primer / oligonucleotide probe combinations that detect the five genes responsible for clindamycin resistance, which allows for rapid acquisition of clindamycin resistance information. The use of dual GBS targets allows for determination of if the clindamycin resistance genes are from GBS. In one aspect, 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.

[0011] Provided herein are methods for rapid detection of Group B Streptococcus (GBS, Streptococcus agalactiae or S. agalactiae) and the presence or absence of the most common clindamycin resistance genes in GBS in a biological or non-biological sample. This is achieved, for example, by multiplex detection of the multi-copy 23S ribosomal RNA (23S rRNA) gene and single copy cAMP factor (cfb) gene of GBS, and the ermTR, ermB, ermT, lsaC, and lsaE genes that confer resistance to clindamycin by real-time polymerase chain reaction in a single tube. Accordingly, methods of detecting the 23S rRNA and cfb genes and clindamycin resistance genes are provided, the methods comprising performing at least one cycling step, which can include an amplification step and a hybridization step. Further, oligonucleotide primers, oligonucleotide probes, and kits are provided that are designed for detection of the GBS 23S rRNA gene, the GBS cfb gene, and the clindamycin resistance genes ermTR, ermB, ermT, lsaC, and lsaE in a single tube. The detection methods are designed to target these genes, which allows one to detect the presence of GBS and the mechanism of clindamycin resistance in a single test.

[0012] Provided herein is a method for detecting GBS with a mechanism of clindamycin resistance in a sample, the method comprising performing an amplification step 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 produce an amplification product if any of these target genes is present in the sample; performing a hybridization step comprising contacting the amplification product with one or more detectable GBS 23S rRNA oligonucleotide probes, one or more detectable GBS cfb oligonucleotide probes, 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 the amplification product, wherein the presence of the amplification product indicates the presence of GBS and / or a mechanism of clindamycin resistance in the sample, and wherein the absence of the amplification product indicates the absence of GBS and / or a mechanism of clindamycin resistance in the sample.

[0013] In one aspect, a method for detecting Group B Streptococcus (GBS) is provided, the method comprising: contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce an amplification product comprising a representative nucleic acid of each of the targets present in the sample; combining the amplification product with a detectable oligonucleotide probe directed to each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification product, wherein the presence or absence of each of the representative nucleic acids in the amplification product is indicative of the presence or absence of each of the GBS strains and a clindamycin resistance mechanism in the sample. In one embodiment, the method further distinguishes GBS from other species of Streptococcus. In one embodiment, the sample is selected from one of an enriched sample and a sample directly from a specimen. In one embodiment, the defined set of targets comprises: i) a 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: measuring the cycle threshold (Ct) of each of the GBS specific gene (Ct GBS ) and the at least one clindamycin resistance gene (Ct ClinR ); comparing the Ct GBS and the Ct ClinRthe absolute value of ΔCt is less than or equal to 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 certain embodiments, the GBS-specific gene is a 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 a set of 23s rRNA oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 22, 23, or 24; a 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 at least one set of clindamycin resistance gene oligonucleotide primers is selected from the group consisting of 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 Isac oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 42 or 43; and a set of Isae 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 to 23s rRNA comprises the nucleic acid sequence of SEQ ID NO: 27 or its complement; the detectable oligonucleotide probe to a GBS-specific gene comprises the nucleic acid sequence of SEQ ID NO: 30 or 64 or its complement; and the detectable oligonucleotide probe to the at least one clindamycin resistance gene is selected from the group consisting of: a detectable oligonucleotide probe to ermTR comprising the nucleic acid sequence of SEQ ID NO: 40 or 41 or its complement; a detectable oligonucleotide probe to ermB comprising the nucleic acid sequence of SEQ ID NO: 35 or 36 or its complement; a detectable oligonucleotide probe to ermT comprising the nucleic acid sequence of SEQ ID NO: 37 or its complement; a detectable oligonucleotide probe to lsaC comprising the nucleic acid sequence of SEQ ID NO: 44 or its complement; and a detectable oligonucleotide probe to lsaE comprising the nucleic acid sequence of SEQ ID NO: 45 or its complement. In some embodiments, the detectable oligonucleotide probe to each of the targets is labeled with a donor moiety and a corresponding acceptor moiety, and wherein 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 one of the targets 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 having 5' to 3' nuclease activity.

[0014] In one embodiment, a 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 a detectable GBS 23S rRNA oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 27 or its complement. In one embodiment, a 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 a detectable GBS cfb oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 30 or 64 or its complement. In one embodiment, a 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 / or a detectable ermTR oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 40 or 41 or its complement. In one embodiment, a set of ermB oligonucleotide primers comprises or consists of: 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 a detectable ermB oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 35 or 36 or its complement.In one embodiment, a set of ermT oligonucleotide primers comprises or consists of: 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 a detectable ermT oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 37, or a complement thereof. In one embodiment, a 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; and / or a detectable lsaC oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 44, or a complement thereof. In one embodiment, a 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 / or a detectable lsaE oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 45, or a complement thereof.

[0015] In one embodiment, a 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 a detectable GBS 23S rRNA oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 3, or a complement thereof. In one embodiment, a 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 a detectable GBS cfb oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 6, or a complement thereof. In one embodiment, a 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 / or a detectable ermTR oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 9, or a complement thereof. In one embodiment, a set of ermB oligonucleotide primers comprises or consists of: 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 a detectable ermB oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 12, or a complement thereof. In one embodiment, a set of ermT oligonucleotide primers comprises or consists of: 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 a detectable ermT oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 15, or a complement thereof.In one embodiment, a 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; and / or a detectable lsaC oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 18, or the complement thereof, or the nucleic acid sequence of SEQ ID NO: 44, or the complement thereof. In one embodiment, a 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 / or a detectable lsaE oligonucleotide probe comprising or consisting of the nucleic acid sequence of SEQ ID NO: 21, or the complement thereof, or the nucleic acid sequence of SEQ ID NO: 45, or the complement thereof.

[0016] In another aspect, a method for detecting Group B Streptococcus (GBS) is provided, the method comprising: contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce an amplification product comprising a representative nucleic acid of each of the targets present in the sample; combining the amplification product with a detectable oligonucleotide probe directed to each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification product, wherein the presence or absence of each of the representative nucleic acids in the amplification product is indicative of the presence or absence of a GBS strain in the sample, and wherein the method distinguishes GBS from other species of Streptococcus. In one embodiment, the sample is selected from one of an enriched sample and a sample directly from a specimen. In one embodiment, the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA). In one embodiment, the amplification product is further combined with a blocking oligonucleotide. In one embodiment, the defined set of targets further comprises a GBS-specific gene. In one embodiment, the GBS-specific gene is a 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, a set of the above oligonucleotide primers and / or probes or any one oligonucleotide primer and / or probe can be used.

[0017] In one embodiment, amplification can employ a polymerase with 5' to 3' nuclease activity. Thus, the first fluorescent moiety and the second fluorescent moiety can be no more than 8 nucleotides apart from each other along the length of the oligonucleotide probe. In another aspect, the 23S rRNA, cfb, ermTR, ermB, ermT, lsaC, and lsaE oligonucleotide probes include nucleic acid sequences that allow for secondary structure formation. This secondary structure formation would typically result in a spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the oligonucleotide probe can be a quencher.

[0018] In another aspect, the present disclosure provides an oligonucleotide comprising or consisting of a sequence of nucleotides selected from the group consisting of SEQ ID NOs: 1-64, or the complement thereof, the oligonucleotide having 100 or fewer nucleotides. The present disclosure further provides an oligonucleotide comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%, etc.) to one of SEQ ID NOs: 1-64, or the complement thereof, the oligonucleotide having 100 or fewer nucleotides. Typically, in these embodiments, the oligonucleotides can be primer nucleic acids, probe nucleic acids, etc., and can be used in any of the methods provided herein. In certain of these embodiments, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, etc.). In some embodiments, any of the oligonucleotides can comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability relative 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 variant. A "conservatively modified variation" or "conservative variation" of a particular nucleic acid sequence refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Those of skill will recognize that individual substitutions, deletions or additions to a sequence which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence, but which do not otherwise alter the biological activity of the encoded product are "conservatively modified variations" or simply "conservative variations" where the alterations are ones which result in the deletion of an amino acid, addition of an amino acid or substitution of an amino acid with a chemically similar amino acid.

[0019] 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 to produce amplification products comprising representative nucleic acids of each of the targets present in a sample; wherein the defined set of targets comprises a 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 a GBS 23S rRNA gene, and one or more detectable GBS 23S rRNA oligonucleotide probes specific for detecting GBS 23S rRNA gene amplification products; 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 amplifying an ermTR gene and one or more detectable ermTR oligonucleotide probes specific for detecting ermTR gene amplification products; a set of ermB gene oligonucleotide primers specific for amplifying an ermB gene and one or more detectable ermB oligonucleotide probes specific for detecting ermB gene amplification products; a set of ermT gene oligonucleotide primers specific for amplifying an ermT gene and one or more detectable ermT oligonucleotide probes specific for detecting ermT gene amplification products; a set of lsaC gene oligonucleotide primers specific for amplifying a lsaC gene and one or more detectable lsaC oligonucleotide probes specific for detecting lsaC gene amplification products; and a set of lsaE gene oligonucleotide primers specific for amplifying a lsaE gene and one or more detectable lsaE oligonucleotide probes specific for detecting lsaE gene amplification products. In one embodiment, the defined set of targets further comprises a GBS-specific gene. In one embodiment, the GBS-specific gene is a CAMP factor-encoding gene (cfb).

[0020] In one embodiment, the kit further comprises 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 detection of GBS cfb gene amplification products. In one embodiment, the above-mentioned oligonucleotide primers and / or probes or any set of oligonucleotide primers and / or probes can be included in the kit. In one embodiment, the kit can include oligonucleotide probes that have been labeled with donor and corresponding acceptor fluorescent moieties or can include fluorescent moieties for labeling the oligonucleotide probes. The kit can also include nucleoside triphosphates, nucleic acid polymerase, and buffers necessary for nucleic acid polymerase function. The kit can also include a package insert and instructions for using the oligonucleotide primers, oligonucleotide probes, and fluorescent moieties to detect the presence or absence of GBS and / or clindamycin resistance mechanisms in a sample.

[0021] In another aspect, a method for detecting Group B Streptococcus (GBS) is provided, the method comprising: contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce an amplification product comprising a representative nucleic acid of each of the targets present in the sample; combining the amplification product with a detectable oligonucleotide probe directed to each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification product, wherein the presence or absence of each of the representative nucleic acids in the amplification product is indicative of the presence or absence of a GBS strain in the sample, and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA). In one embodiment, the sample is selected from one of an enriched sample and a sample directly from a specimen. In one embodiment, the defined set of targets further comprises 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 includes a set of oligonucleotide primers for amplifying at least a portion of each target, wherein a 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 directed to 23s rRNA comprises or consists of the nucleic acid sequence of SEQ ID NO: 27, or the complement thereof. In one embodiment, any of the above set of oligonucleotide primers and / or probes or oligonucleotide primers and / or probes can be used in the method.

[0022] In another aspect, a method for detecting Group B Streptococcus (GBS) is provided, the method comprising: contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce an amplification product comprising a representative nucleic acid of each of the targets present in the sample; combining the amplification product with a detectable probe directed to each of the targets; and detecting the presence or absence of each of the representative nucleic acids in the amplification product, wherein the presence or absence of each of the representative nucleic acids in the amplification product is indicative of the presence or absence of a GBS strain in the sample, and wherein the sample is a sample directly from a specimen, wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA), and wherein the method distinguishes GBS from other species of Streptococcus. In one embodiment, any of the above set of oligonucleotide primers and / or probes or oligonucleotide primers and / or probes can be used in the method.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and 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 control.

[0024] The details of one or more embodiments of the application are set forth in the accompanying drawings and the detailed description below. Other features, objects, and advantages of the application will be apparent from the drawings and detailed description, and from the claims. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Two sets of qPCR amplification curves for detecting GBS 23S rRNA or Ureaplasma urealyticum 23S rRNA according to the present disclosure are shown. The set of oligonucleotide primers and oligonucleotide probe used in the reaction amplify only the GBS 23S rRNA target (left), no amplification for U. urealyticum 23S rRNA is observed (right). The fluorescence signal is plotted as a function of the number of cycles for three template concentrations, including 1*10 2 , 1*10 3 , and 1*10 4 copies for each reaction for either GBS 23S rRNA template or U. urealyticum 23S rRNA template. DETAILED DESCRIPTION

[0026] As used herein, the term "amplification" refers to the process of synthesizing nucleic acid molecules 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 produce amplification products. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase, e.g., Taq, and appropriate buffers and / or cofactors for optimizing polymerase activity, e.g., MgCl2and / or KC1.

[0027] As used herein, the term "primer" is known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, but also a modified oligonucleotide, i.e., an oligonucleotide whose 3'-end provides a free 3'-OH group, which enables initiation of DNA synthesis by a template-dependent DNA polymerase, wherein in addition "nucleotides" can be attached to this group by the template-dependent DNA polymerase, thereby establishing 3' to 5' phosphodiester linkages, wherein deoxyribonucleoside triphosphates are used and pyrophosphate is thereby released. Thus, there is no fundamental difference between "primer", "oligonucleotide", "oligonucleotide primer", "probe" or "oligonucleotide probe" other than the possible intended function.

[0028] The term "hybridization" 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 probes but avoiding non-specific hybridization of the probes.

[0029] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase, typically associated with nucleic acid strand synthesis, whereby nucleotides are removed from the 5' end of a nucleic acid strand.

[0030] The term "thermostable polymerase" refers to a polymerase that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to elevated temperatures for the time required to achieve denaturation of double-stranded template nucleic acids. Typically, synthesis is initiated at the 3' end of each primer and proceeds in the 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. However, not thermostable polymerases can also be used in a PCR assay, provided that the enzyme is supplemented.

[0031] ​The term "complement" refers to a nucleic acid having the same length and being completely complementary to a given nucleic acid.

[0032] The term "extend" or "elongate" when used in reference to a nucleic acid refers to the incorporation of additional nucleotides (or other similar molecules) into the nucleic acid. For example, a nucleic acid is optionally extended by a biocatalyst that incorporates nucleotides, such as a polymerase that typically adds nucleotides to the 3' end of a nucleic acid.

[0033] As used herein, the term "identical" or percent "identity," in the context of two or more nucleic acids sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms available to persons of skill or by visual inspection. Exemplary algorithms are the BLAST program described in, 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.

[0034] A "modified nucleotide" in the context of an oligonucleotide refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide, thereby providing the oligonucleotide with a desired property. Exemplary modified nucleotides that can be substituted into 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-deoxyxanthine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, and the like. Numerous other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or otherwise known in the art. In certain embodiments, a modified nucleotide substitution modifies the melting temperature (Tm) of an oligonucleotide relative to the melting temperature of a corresponding unmodified oligonucleotide. To further illustrate, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of a desired target amplicon, and the like. Examples of these types of nucleic acid modifications are described in, for example, U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0035] A "variant" of a given oligonucleotide can contain one or more nucleotide additions, deletions, or substitutions, such as one or more nucleotide additions, deletions, or substitutions at the 5' end and / or the 3' end of the corresponding sequence of the oligonucleotide. As noted above, a primer (and / or a probe) can be chemically modified, i.e., the primer and / or probe can comprise modified nucleotides or non-nucleotide compounds. A probe (or primer) is then a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, 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 moiety of a "nucleotide", thereby obtaining a "modified nucleotide". A natural base in a "nucleotide" can also be replaced by, for example, a 7-deaza purine, wherein also a "modified nucleotide" is obtained. The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in the present application. A "modified nucleoside" (or "nucleoside analog") differs from a natural nucleoside by some modification in the manner outlined above for "modified nucleotides" (or "nucleotide analogs").

[0036] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, used to amplify nucleic acid molecules (e.g., nucleic acid molecules encoding GBS 23S rRNA gene, GBS cfb gene, or clindamycin resistance gene (e.g., ermTR or lsaC) nucleic acid sequences) can be designed using, for example, computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.). Important features when designing oligonucleotides for use as amplification primers include, but are not limited to, an appropriate size of amplification product for ease of detection (e.g., by electrophoresis), similar melting temperatures for members of a pair of primers, and length of each primer (i.e., primers need to be long enough to anneal to the sequence specifically and initiate synthesis, 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).

[0037] In addition to a set of primers, these methods can use one or more probes in order to detect the presence or absence of GBS and clindamycin resistance mechanisms. The term "probe" refers to synthetic or biologically produced nucleic acids (DNA or RNA) that are designed or selected to contain specific nucleotide sequences that allow them to hybridize specifically (i.e., preferentially) to "target nucleic acids" (in the case of the present application, to GBS 23S rRNA or GBS cfb (GBS target) nucleic acids and / or to ermTR, ermB, ermT, lsaC, lsaE (clindamycin resistance target) nucleic acids) under defined, predetermined stringencies. A "probe" can be referred to as a "detection probe," meaning that it detects a target nucleic acid.

[0038] In some embodiments, the probe can be labeled with at least one fluorescent label. In one embodiment, the probe can be labeled with a donor fluorescent moiety (e.g., a fluorescent dye) and a corresponding acceptor fluorescent moiety (e.g., a quencher).

[0039] Oligonucleotides designed for use as probes can be designed in a similar manner to primer design. Embodiments can use a single probe or a pair of probes to detect an amplification product. Depending on the embodiment, the probe used can comprise at least one label and / or at least one quencher moiety. Like primers, probes typically 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 typically 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0040] The constructs can include vectors, each vector containing one of the primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1-21). The constructs can be used, for example, as control template nucleic acid molecules. Suitable vectors are commercially available and / or produced by methods of recombinant nucleic acid technology that are routine in the art. The target nucleic acid molecules can be obtained by, for example, chemical synthesis, direct cloning of a gene, or by PCR amplification.

[0041] In addition to the target nucleic acid molecules (e.g., nucleic acid molecules comprising one or more of the sequences of SEQ ID NOs: 1-21), the constructs suitable for use in the methods generally include sequences encoding a selection marker (e.g., an antibiotic resistance gene) for selection of the desired constructs and / or transformants and an origin of replication. The choice of vector system is generally dependent on several factors, including but not limited to, the choice of host cell, the efficiency of replication, selectivity, inducibility, and ease of recovery.

[0042] The constructs containing the target nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is intended to include prokaryotic and eukaryotic organisms, such as yeast, plant, and animal cells. Prokaryotic hosts can include E. coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeast such as S. cerevisiae, S. pombe, 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. The constructs can be introduced into the host cells using any technique known to one of ordinary skill 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. Additionally, naked DNA can be delivered directly to cells (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).

[0043] The term "enriched sample" or "enriched sample" refers to a sample or specimen that has been treated to increase the amount or concentration of a target of interest suspected to be present in the sample. Specimens obtained from patients can be enriched for one or more microorganisms present in the sample, such as GBS and other strains of Streptococcus. There are a number of culture- and molecule-based methods for enriching for Streptococcus species, including GBS and other microorganisms, in a specimen. In one example, a swab used to collect a specimen from a patient is placed into an elution medium (e.g., Liquid Amies). Then, a LIM enrichment broth (see, e.g., Lim, D.V., et al. 1982. Current Microbiol.; 7:99-101) is inoculated with the elution medium. The enrichment broth is then incubated for a period of time sufficient to selectively enrich for Streptococcus species, including GBS (e.g., 18-24 hours). Subsequently, the resulting enriched specimen can be used to detect GBS, one or more clindamycin resistance markers, and / or other targets of interest that can be present in the enriched sample. Commercially available swabs, such as ESWAB (COPAN), can be used to collect patient specimens, including vaginal and rectal specimens.

[0044] The term "directly from a specimen" refers to a sample that is directly treated upon collection without further enrichment. Samples directly from a specimen include vaginal and rectal samples collected with a swab and optionally placed in an elution medium. The swab and / or elution medium can be tested directly without enriching for microorganisms that can be present in the elution medium or on the swab.

[0045] Polymerase chain reaction (PCR)

[0046] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR generally employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides capable of serving as a point of initiation of nucleic acid synthesis within the nucleic acid sequence of the target gene 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 it can be produced synthetically. Primers are preferably single-stranded for maximum efficiency in the amplification, but primers can be double-stranded. Double-stranded primers are first denatured (i.e., treated) to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0047] If the template nucleic acid is double-stranded, the two strands must be separated before it can be used as a template in a PCR. Strand separation can be accomplished by any suitable denaturation method, including physical, chemical, or enzymatic methods. One method of separating nucleic acid strands involves heating the nucleic acid until it is mostly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions necessary to denature the template nucleic acid will depend on, for example, the buffer salt concentration as well as the length and nucleotide composition of the denatured nucleic acid, but generally range from about 90 °C to about 105 °C for a period of time that depends on the characteristics of the reaction, such as the temperature and the length of the nucleic acid. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0048] If the double-stranded template nucleic acid is denatured by heating, the reaction mixture is then cooled to a temperature that promotes annealing of each primer to a target sequence on the described nucleic acid molecule. The temperature for annealing is typically from about 35 °C to about 65 °C (e.g., from about 40 °C to about 60 °C; from about 45 °C to about 50 °C). The annealing time can be from about 10 seconds to about 1 minute (e.g., from about 20 seconds to about 50 seconds; from 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 to occur from the annealed primers to generate a product complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer that is annealed to a nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., the temperature for extension is typically in the range of about 40 °C to about 80 °C (e.g., from about 50 °C to about 70 °C; about 60 °C). The extension time can be from about 10 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes; from about 1 minute to about 3 minutes; from about 1 minute and 30 seconds to about 2 minutes).

[0049] PCR assays can employ target gene and / or allelic nucleic acids such as RNA or DNA (cDNA). The template nucleic acid need not be purified; it can be a small fraction of a complex mixture, such as the target nucleic acids contained in a biological sample. The target nucleic acid molecules can be extracted from a biological sample by conventional techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds.), 1993, American Society for Microbiology, Washington D.C.). Nucleic acids can be obtained from many sources, for example, plasmids, or naturally occurring sources, including bacteria, yeast, viruses, organelles, or higher organisms, for example, plants or animals.

[0050] The oligonucleotide primers are combined with PCR reagents under reaction conditions that induce primer extension. For example, a strand extension reaction typically includes 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 15 mM MgC12, 0.001% (w / v) gelatin, 0.5-1.0 μg denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U Taq polymerase, and 10% DMSO). The reaction typically contains 150 to 320 μΜ of each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0051] The newly synthesized strands form double-stranded molecules that are available for subsequent reaction steps. The steps of strand separation, annealing, and extension can be repeated as many times as desired to produce the desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factor in the reaction is the amount of primers, heat-stable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For use in detection, the number of cycling steps will depend on, for example, the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be needed to amplify the target sequences sufficient for detection. Typically, the cycling steps are repeated at least about 20 times, but can be repeated as many as 40, 60, or even 100 times. Fluorescence resonance energy transfer (FRET)

[0052] FRET technology (see, e.g., U.S. Patent 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 located within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. When the donor is excited by light radiation having a suitable wavelength, the donor will typically transfer energy to the acceptor. The acceptor will typically re-emit the transferred energy in the form of light radiation having a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties by a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Patent No. 7,741,467).

[0053] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher, which can or can not be fluorescent, and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, such that the fluorescent emission from the donor fluorescent moiety is quenched. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved by the 5' to 3' nuclease activity of, for example, Taq polymerase, such that the fluorescent emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described in, for example, U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include Black Hole Quenchers TM (BHQ) (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black TM (Integrated DNA Tech., Inc., Coralville, Iowa), BlackBERRY TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0054] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at specific locations determined by the complementarity of the oligonucleotide probes to the target nucleic acid sequence. After the oligonucleotide probes hybridize to the amplification product nucleic acid at the appropriate locations, 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.

[0055] Fluorescent analysis can be performed using, for example, a photon counting epi-fluorescence microscope system containing appropriate dichroic mirrors and filters for monitoring a specific range of fluorescent emissions, a photon counting photomultiplier tube system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed with an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered to excite in the desired range.

[0056] As used herein with respect to a donor and a corresponding acceptor fluorescent moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher having an absorption spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The maximum wavelength of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-radiative energy transfer can occur between them.

[0057] Fluorescent donors and corresponding acceptor fractions are typically selected for the following reasons: (a) high Forster energy transfer efficiency; (b) a large final Stokes shift (>100 nm); (c) emission shifting as far as possible into the red portion of the visible spectrum (>600 nm); and (d) emission shifting to a wavelength higher than that produced by excitation at the donor excitation wavelength. For example, a donor fluorescent fraction may be selected that has its excitation maxima near the laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and whose fluorescence emission good overlaps with the excitation spectrum of the corresponding acceptor fluorescent fraction. A corresponding acceptor fluorescent fraction with a high extinction coefficient, high quantum yield, good overlap of its excitation with the donor fluorescent fraction's emission, and emission in the red portion of the visible spectrum (>600 nm) may also be selected.

[0058] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, fluorescein yellow, β-phycoerythrin, 9-acridine isothiocyanate, fluorescein VS, 4-acetamido-4'-isothiocyanate violet-2,2'-disulfonic acid, 7-diethylamino-3-(4'-phenyl isothiocyanate)-4-methylcoumarin, succinimide 1-pyrene butyrate, and 4-acetamido-4'-isothiocyanate violet-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties, depending on the donor fluorescent moieties used, include LC Red 640, LC Red 705, Cy5, Cy5.5, rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or chelates of other lanthanides (e.g., europium or terbium). The fluorescent components of the donor and acceptor can be obtained from, for example, MolecularProbes (Junction City, OR) or Sigma Chemical Co. (St. Louis, MO).

[0059] The fluorescent portions of the donor and acceptor can be ligated to a suitable probe oligonucleotide via adapter arms. The length of each adapter arm is important because it affects the distance between the fluorescent portions of the donor and acceptor. The distance from the nucleotide base to the fluorescent portion is measured in units of [unit]. Typically, the linker arm is approximately [length]. to approximately The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching linker arms to specific nucleotide bases and methods for attaching fluorescent portions to linker arms.

[0060] Acceptor fluorescent moieties (such as LC Red 640) can be combined with oligonucleotides containing an amino linker (e.g., C6-amino phosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA) to produce, for example, LC Red 640 labeled oligonucleotides. Frequently used linkers for coupling donor fluorescent moieties (such as fluorescein) to oligonucleotides include thiourea linkers (FITC-derivatized, such as fluorescein-CPG's from Glen Research or ChemGene (Ashland, MA)), amide linkers (fluorescein-NHS-ester derivatized, such as CX-fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG which requires coupling of fluorescein-NHS-ester after oligonucleotide synthesis.

[0061] Detection of target GBS genes and clindamycin resistance genes

[0062] The present disclosure provides methods for detecting the presence or absence of GBS 23S rRNA genes, GBS cfb genes, and ermTR, ermB, ermT, lsaC, and lsaE genes in a biological or non-biological sample. The methods provided avoid problems of sample contamination, false negatives, and false positives. The methods include performing at least one cycling step and a FRET detection step, the cycling step including amplifying a portion of a target nucleic acid molecule from a sample using a plurality of target primers. Multiple cycling steps are performed, preferably in a thermal cycler. The methods can be performed using target primers and probes that detect the presence of the target genes, and detection of the amplified product in the assay indicates the presence of the target genes and / or target alleles in the sample.

[0063] As described herein, labeled hybridization probes utilizing FRET technology can be used to detect the amplified product. One FRET format utilizes technology to detect the presence or absence of the amplified product, and thus the presence or absence of GBS and / or clindamycin resistance genes. The technique utilizes a single-stranded hybridization probe labeled with, for example, a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent portion is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent portion according to the FRET principle. The second fluorescent portion is typically the 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 degraded by the 5' to 3' nuclease activity of, for example, Taq polymerase during the subsequent extension phase. Therefore, the fluorescent portion and the quencher portion become spatially separated from each other. Thus, fluorescence emission from the first fluorescent portion can be detected after excitation of the first fluorescent portion in the absence of the quencher. For example, ABI... 7700Sequence DetectionSystem (Applied Biosystems) use The technology is applicable to performing the methods described herein for detecting the presence or absence of GBS and / or clindamycin resistance genes in a sample.

[0064] Molecular beacons bound to FRET can also be used to detect the presence of amplification products using real-time PCR methods. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is typically a quencher, and the fluorescent label is generally located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow for secondary structure formation (e.g., hairpins). As a result of secondary structure formation within the probe, the two fluorescent moieties are spatially close when the probe is in solution. After hybridization with the target nucleic acid (i.e., the amplification product), the probe's secondary structure is disrupted, and the fluorescent moieties become separated from each other, allowing detection of the emission of the first fluorescent moiety upon excitation with light of an appropriate wavelength.

[0065] Another common form of FRET technology uses two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is typically designed to hybridize very close to each other in the target DNA molecule (e.g., the amplification product). The donor fluorescent moiety, such as fluorescein, is detected at 470 nm. The instrument is excited by a light source. During FRET, the fluorescein transfers its energy to the fluorescent portion of the acceptor, for example... -Red 640 (LC Red 640) or -Red705 (LC Red 705). Then the fluorescent portion of the acceptor emits longer wavelength light, which is then... The optical detection system of the instrument detects. Effective FRET occurs only 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 to the amount of original target DNA molecule. If amplification of the target nucleic acid occurs and produces an amplification product, then the hybridization step produces a detectable signal based on FRET between the members of the probe pair.

[0066] Generally, the presence of FRET indicates the presence of the target sequence in the sample, and the absence of FRET indicates the absence of the target sequence in the sample. However, inadequate sample collection, transport delays, improper transport conditions, or use of certain collection swabs (calcium alginate or aluminum shafts) are conditions that can affect the success and / or accuracy of the test results. Using the methods disclosed herein, detection of FRET within, for example, 45 cycle steps indicates GBS infection.

[0067] Representative biological samples that can be used to practice these methods include, but are not limited to, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods of collection and storage of biological samples are known to those of skill in the art. The biological sample can be treated (e.g., by nucleic acid extraction methods and / or kits known in the art) to release the target genetic nucleic acid, or in some cases, the biological sample can be directly contacted with the PCR reaction components and appropriate oligonucleotides.

[0068] 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 half of the DNA duplexes separate into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Thus, DNA molecules that are rich in G and C nucleotides have a higher Tm than DNA molecules that have a rich A and T nucleotide. 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 produced, 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.

[0069] A control sample can also be cycled in each thermocycler run. A positive control sample can amplify a target nucleic acid control template (different from the amplification product of the target gene) using, for example, control primers and control probes. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within a sample) or in a separate sample run alongside the patient samples using the same primers and probes used to detect the intended target. Such a control is an indicator 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 the target template DNA. The negative control can measure for contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reactions can readily determine, for example, the ability of primers to anneal and initiate extension in a sequence-specific manner, and the ability of probes to hybridize in a sequence-specific manner and undergo FRET.

[0070] In one embodiment, the method includes a step to avoid contamination. For example, enzymatic methods using uracil-DNA glycosylase to reduce or eliminate contamination between one thermocycler run and the next are described in U.S. Patent Nos. 5,035,996; 5,683,896; and 5,945,313.

[0071] These methods can be practiced using conventional PCR methods in conjunction with FRET technology. In one embodiment, the TaqMan® instrument. The following patent applications describe the use of real-time PCR in the TaqMan® technology: WO 97 / 46707; WO 97 / 46714; and WO 97 / 46712.

[0072] A PC workstation can be used for operation, and the Windows NT operating system can be used. The signal from the sample can be obtained as the machine places the capillary sequentially on the optical unit. The software can display the fluorescent signal in real time immediately after each measurement. The fluorescence acquisition time is 10-100 milliseconds (msec). After each cycle step, a quantitative display of fluorescence versus cycle number can be constantly updated for all samples. The data generated can be stored for further analysis.

[0073] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g. SYBR® Green or Gold (Molecular Probes)), which can detect amplification products. Such fluorescent DNA binding dyes emit a fluorescent signal upon excitation with light of the appropriate wavelength when interacting with double stranded nucleic acid. Double stranded DNA binding dyes such as intercalating dyes can also be used. When a double stranded DNA binding dye is used, a melting curve analysis is typically performed to confirm the presence of amplification products.

[0074] It should be understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available instruments.

[0075] Articles / Kit

[0076] Embodiments of the present disclosure also provide an article of manufacture or a kit for detecting the 23S rRNA and cfb genes of GBS and the ermTR, ermB, ermT, lsaC, and lsaE genes (i.e., the genes responsible for clindamycin resistant GBS). The article of manufacture can include primers and probes for detecting clindamycin resistant GBS, as well as suitable packaging materials. The representative primers and probes for detecting clindamycin resistant GBS are capable of hybridizing to the target nucleic acid molecules. In addition, the kit can also include, in suitable packaging, reagents and materials needed for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods of designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to the target nucleic acid molecules are provided.

[0077] The article of manufacture can also include one or more fluorescent moieties for labeling the probes, or alternatively, the probes provided with the kit can be labeled. For example, the article of manufacture can include donor and / or acceptor fluorescent moieties for labeling the probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0078] The article of manufacture can also contain a package insert or package label having instructions for using the target primers and probes to detect clindamycin resistant GBS in a sample. The article of manufacture can additionally include reagents for carrying out the methods disclosed herein (e.g., buffers, polymerases, co-factors, or agents to prevent contamination). Such reagents can be specific to one of the commercially available instruments described herein.

[0079] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the application described in the claims.

[0080] Example

[0081] The following examples and drawings are provided to aid the understanding of the present application, 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 application.

[0082] Example 1: Primer and probe sequences

[0083] Table 1 shows the primers and probes used in the multiplex PCR assay for detecting GBS and clindamycin resistance mechanisms according to the present disclosure.

[0084] Table 1:

[0085]

[0086]

[0087] The primer and probe sequences in Table 1 are shown, if indicated, to include modifications represented 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.

[0088] Example 2: PCR experimental conditions

[0089] Using Real-time PCR detection of the genetic targets was performed using a LightCycler® 480 System (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents and thermal profiles for the PCR amplification reactions are shown in Table 2:

[0090] Table 2: Mg reagent (R1)

[0091]

[0092] Master Mix (R2) - Universal

[0093]

[0094] PCR thermal cycling parameters

[0095]

[0096] The pre-PCR procedure includes initial denaturation and incubation at 55°C, 60°C and 65°C to perform reverse transcription of the RNA template. Incubation at three temperatures has the following advantageous effects: at lower temperatures, slightly mismatched target sequences, such as genetic variants of an organism, are also transcribed, while at higher temperatures, the formation of RNA secondary structures is inhibited, thus making transcription more efficient. The PCR cycles are divided into two measurements, where both measurements apply a one-step setup (annealing and extension combined). The first 5 cycles at 55°C allow for increased inclusivity by pre-amplification of slightly mismatched target sequences, while the 45 cycles of the second measurement provide increased specificity by using an annealing / extension temperature of 58°C.

[0097] Example 3: Performance of PCR assays for detection of GBS and clindamycin resistance genes

[0098] Results of PCR assays using primers and probes for detection of GBS and ermTR in 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 S. agalactiae clinical isolates at a concentration of 1.00E+06 copies per reaction to 1.00E+00 copies per reaction. Template plasmids carrying the ermTR gene were tested at a concentration of 1.52E+05 copies per reaction to 1.53E+00 copies per reaction. Preliminary testing indicated that these primer / probe pairs produced detection results in a multiplex assay.

[0099] Primer / probe pairings for the singleplex for ermB and the singleplex for ermT are shown in Table 4. For ermB, the master mix uses forward primer SEQ ID NO: 10, reverse primer SEQ ID NO: 11, and probe SEQ ID NO: 12. For ermT, the master mix uses forward primer SEQ ID NO: 13, reverse primer SEQ ID NO: 14, and probe SEQ ID NO: 15. Primer / probe pairings for the singleplex for lsaC and the singleplex for lsaE are shown in Table 5. For lsaC, the master mix uses forward primer SEQ ID NO: 16, reverse primer SEQ ID NO: 17, and probe SEQ ID NO: 18. For lsaE, the master mix uses forward primer SEQ ID NO: 19, reverse primer SEQ ID NO: 20, and probe SEQ ID NO: 21. Template plasmids carrying the ermB, lsaC, or lsaE genes were tested at concentrations ranging from 1.00E+06 copies per reaction to 1.00E+00 copies per reaction. Template plasmids carrying the ermT gene were tested at concentrations ranging from 7.95E+03 copies per reaction to 7.95E+00 copies per reaction.

[0100] Table 3:

[0101]

[0102] Table 4:

[0103]

[0104] Table 5:

[0105]

[0106] In Tables 3-5, test concentration (cp / rxn) is the test concentration in units of copies per reaction, average Ct. is the average cycle threshold (i.e., the average number of cycles required for the fluorescent signal to cross the threshold of background fluorescent signal), and average RFI is the average relative fluorescence intensity.

[0107] Example 4: Additional primer and probe sequences

[0108] Table 6 shows additional primers and probes used in the multiplex PCR assay for detecting GBS and clindamycin resistance mechanisms according to the present disclosure.

[0109] Table 6:

[0110]

[0111]

[0112]

[0113]

[0114] The primer and probe sequences in Table 6 are shown including modifications represented according to the following scheme: J = t-butylbenzyl-dA, K = t-butylbenzyl-dC, L = 7-deaza-dG, M = 2'-0-methyl-rU, N = D_LNA_A, O = N4-ethyl-dC, P = D_LNA_G, R = D_LNA_T, S = 2'-0 methyl-riboU, V = D_LNA_5-methyl-C, <Spc_C3> = 3' spacer, <Coum_Thr> = Coumarin dye, <FAM_Thr> = FAM dye, <HEX_Thr> = HEX dye, <JA270_Thr> = JA270 dye, and Q = BHQ2, if indicated.

[0115] Example 5: Performance of assays for detection of GBS

[0116] The current standard of care for detection and identification of GBS is an enrichment-based method characterized by a long turnaround time of at least one full day. This method typically involves collection of the sample with a swab, release of the sample from the swab in a liquid broth, enrichment by culturing the sample in the liquid broth for 18 to 24 hours, and then further culturing the enriched sample on agar plates or using a nucleic acid amplification test (NAAT).

[0117] The current understanding in the field is that the sensitivity of GBS detection is strongly influenced by culture enrichment. Therefore, it is strongly recommended to incubate GBS screening samples in an enrichment broth prior to selection of the final detection method, whether agar medium plating or NAAT is intended. This recommendation is based at least in part on a report that incubation in broth media prior to plating increases the sensitivity of a screening method by about two-fold compared to plating directly from the sample. Likewise, after culture enrichment, the sensitivity of NAAT is increased. For example, the American Society for Microbiology Committee on Clinical and Public Health Microbiology, Subcommittee on Laboratory Practices (ASM) recommends that "enrichment broth culture must be performed first, regardless of whether culture or NAAT is chosen as the primary method for GBS detection". The ASM guideline continues by stating that "it is important to note that commercial NAATs are available that are performed directly (without enrichment) from the sample, but they are not currently recommended due to high false negative rates of 6.3% to 22%".

[0118] While the ASM recommends against the use of existing commercial NAATs that are performed directly (without enrichment) from the sample, it is useful to provide a method that can reduce or eliminate the need for an enrichment step to reduce the overall turnaround time for detecting and identifying GBS. For example, testing directly from the sample is useful for intrapartum testing where faster results are desired. In addition, current NAATs for GBS do not characterize antibiotic susceptibility, which can provide information for further treatment in the case of a positive GBS result. Thus, there is a need for an improved testing method that is characterized by a shortened turnaround time compared to enrichment-based methods. In addition, there is also a need for a NAAT for detecting GBS that facilitates characterization of antibiotic susceptibility.

[0119] To overcome the shortcomings of the above-described enrichment-based tests and existing NAATs, a new method for identifying GBS disclosed herein was conceived and developed. In one aspect, the NAAT according to the present disclosure provides for testing directly from the sample without the need for enrichment. To achieve this goal, a number of potential markers were evaluated. To make a marker useful for detection in a sample directly from the sample without enrichment, a relatively abundant nucleic acid was expected to serve as a suitable target. Given that the total RNA pool of a typical microbial cell is dominated by ribosomal RNA (rRNA), several rRNA components were evaluated, including GBS 23S rRNA. To further improve the confidence of the NAAT of the present example, the cfb gene was included as an additional target, which encodes the Christie, Atkins, and Munch-Peterson (CAMP) factor and is recommended for use in identifying GBS by the ASM.

[0120] To develop a target for a dual-target NAAT for testing directly from a sample, primers and probes for detecting the cfb gene and the new GBS 23S rRNA target were designed and tested. The results of the multiplexed PCR assay detecting GBS 23S rRNA and cfb are shown in Table 7. The assay includes three sets of oligonucleotides - each set comprising 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 aspect, the accurate detection of cfb according to the present example is achieved by using two sets of oligonucleotides that are inclusive of 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 purified synthetic target gene amplicons, with copy number corrected using digital droplet PCR (ddPCR).

[0121] Table 7:

[0122]

[0123] As shown in Table 7, the primers and probes of the present example successfully detected GBS 23S rRNA and cfb at all concentrations of synthetic template tested. However, one important consideration with respect to the design of the GBS 23S rRNA was the mitigation of background and / or cross-reactive signal. During testing of different primers and probes for detecting GBS 23S rRNA, it was noted that the initial design resulted in cross-reactivity (i.e., amplification and detection) of other closely related non-GBS Streptococcus species, including Ureaplasma urealyticum and Streptococcus thermophilus, of Lim broth-enriched clinical samples. As non-GBS Streptococcus species are known human commensals, it is expected that such non-GBS Streptococcus species can be present in samples; however, given that these oligonucleotides were designed to be highly specific for GBS, the observation of cross-reactivity of the GBS 23S rRNA primers and probes with non-GBS Streptococcus species was unexpected.

[0124] To overcome the unexpected cross-reactivity observed in the initial NAAT design, various modifications were considered to improve the specificity of the GBS 23S rRNA primers and probes. With reference to the initial design of the GBS 23S rRNA primers and probes, the following modifications were considered: Figure 1And Table 8, an improved assay was developed that significantly reduced or eliminated the cross-reactivity of the selected 23S rRNA GBS target with the 23S rRNA of closely related non-GBS Streptococcus species, including Ureaplasma and Streptococcus thermophilus. This result was achieved at least in part by shifting the annealing site of the GBS 23S rRNA primers to reduce non-specific amplification arising from homology at the 3' end of the primers with non-GBS Streptococcus species, and designing a unique 23S rRNA probe that included N4-ethyl-dC modifications. This probe modification was placed at a mismatched position between the Ureaplasma 23S rRNA and the GBS 23S rRNA in the quencher region of the probe. The result was a highly reduced RFI, loss of call cycle threshold (Ct) values for Ureaplasma and Streptococcus thermophilus, while maintaining the Ct call and RFI for GBS 23S rRNA. The results of the assay using GBS 23S rRNA forward and reverse primers GBS021 (SEQ ID NO: 22) and GBS025 (SEQ ID NO: 24) for GBS 23S, and Ureaplasma exclusive probe GBS069B (SEQ ID NO: 27) along with Ureaplasma ddPCR quantification template are shown in Table 8.

[0125] Table 8:

[0126]

[0127] In Tables 7 and 8, test concentration (cp / rxn) is the test concentration in units of copies per reaction, average Ct. is the average cycle threshold (i.e., the average number of cycles required for the fluorescent signal to cross the threshold of background fluorescent signal), average RFI is the average relative fluorescence intensity, and NTC is no template control.

[0128] Alternative methods of differentiating GBS 23S rRNA from other non-GBS streptococcal signals include the use of two probes, each comprising one or more locked nucleic acid (LNA)-modified bases. In one aspect, a first probe labeled with a dye is designed to specifically bind to the GBS 23S rRNA target, while a second probe without 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 caused by the presence of closely related non-GBS streptococcal species. Example oligonucleotides comprising LNA modifications listed in Table 6 include GBS084-GBS097 (SEQ ID NOs:46-59), including 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 methods involving blocking probes that preferentially hybridize to non-target nucleic acids for which cross-reactivity is observed can be similarly applied.

[0129] The assays of the present example can be implemented on a real-time PCR device that supports two or more fluorescence detection channels. In one example, GBS 23S rRNA and cfb can be detected in a first channel and a universal internal control (GIC) can be detected in a second channel, as shown in Table 9, which further indicates how the resulting data can be interpreted based on the presence or absence of each of GBS 23S rRNA and cfb (23S / cfb) and the GIC. 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. Thus, for such assays, GBS 23S rRNA and cfb can be detected in the same channel. However, in certain instances, it can be useful to detect GBS 23S rRNA and cfb in separate channels, as discussed, for example, in Example 6 below.

[0130] Table 9:

[0131] 23S / cfb GIC Explanation + + GBS + - GBS - - Failure - + No GBS

[0132] Example 6: Performance of assays for detecting GBS and clindamycin resistance genes

[0133] Current NAATs for detection and identification of GBS do not provide information on antibiotic resistance. Rather, culture-based methods are required to obtain isolates for susceptibility testing. In one aspect, designing a multiplex NAAT with the necessary sensitivity and specificity to confidently detect and characterize GBS in a sample, and particularly a sample directly from a specimen, is challenging. The present example overcomes these and other challenges by providing primers and probes for detecting and identifying GBS in conjunction with detecting one or more clindamycin resistance genes for use in a NAAT. The present NAAT can be used for samples directly from a specimen as well as enriched samples.

[0134] Templates for clindamycin resistance targets ermB, ermTR, ermT, IsaC, and IsaE were prepared from purified synthetic target gene amplicons, with ddPCR to correct copy number. Nucleic acids extracted from cultured GBS strain cells, quantified by plate count, served as templates for GBS 23S rRNA and cfb genes.

[0135] The highly multiplexed results for each set of primers and probes for i) GBS 23s rRNA, ii) GBS specific cfb gene, and iii) clindamycin resistance genes ermB, ermTR, ermT, IsaC, and IsaE are shown in Tables 10 and 11. The master mix included eight sets of oligonucleotides - each set containing a forward primer, a reverse primer, and a detectable probe - one set for detecting GBS 23S rRNA, two sets for detecting cfb, and one set for detecting one 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), ermT6.R (SEQ ID NO: 14), ermTR_R_203_171TBB (SEQ ID NO: 39), SEG4092 (SEQ ID NO: 43), and SEGP3911 (SEQ ID NO: 20), and probes GBS027 (SEQ ID NO: 26), GBS004 (SEQ ID NO: 04), GBS059 (SEQ ID NO: 64), GBS006 (SEQ ID NO: 35 and 36), GBS010 (SEQ ID NO: 37), ermTR8.JA270_10 (SEQ ID NO: 40 and 41), GBS012 (SEQ ID NO: 44), and GBS015 (SEQ ID NO: 45).

[0136] Table 10

[0137]

[0138] Table 11

[0139]

[0140] In Tables 10 and 1, test concentration (CFU / Rxn) is the test concentration in units of colony forming units per reaction, average Ct. is the average cycle threshold (i.e., the average number of cycles required for the fluorescent signal to cross the threshold of background fluorescent signal), average RFI is the average relative fluorescence intensity, and NTC is no template control.

[0141] The assays of the present example can be implemented 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 the present example, detecting and differentiating GBS having one or more clindamycin resistance genes from other bacteria carrying clindamycin resistance genes can be achieved by assays that isolate GBS 23S rRNA and cfb targets in different channels as shown in Table 12 and rule-based interpretation calls as shown in Table 13. In the example shown in Table 12, GBS 23S rRNA can be detected in a first channel (e.g., channel 3), cfb can be detected in a second channel (e.g., channel 1), one or more clindamycin resistance genes can be detected in a third channel (e.g., channel 4), and a universal internal control (GIC) can be detected in a fourth channel (e.g., channel 5).

[0142] Probes GBS80-GBS83 (SEQ ID NOs: 60-63) include a coumarin dye as a donor moiety and are designed to hybridize to and detect cfb. Since cfb exists as a single DNA copy in the GBS genome, it represents a low copy target. Here, low copy means less than or equal to 5 copies per genome. Similarly, clindamycin resistance genes were found to also be single copy or low copy chromosomal gene targets, or on low copy plasmids. By determining the single target Ct for cfb and one of any resistance genes present in a sample, the correlation of the ACt between one of the resistance genes and cfb can be calculated in order to better inform whether the level of resistance genes detected in a sample is similar to the level of GBS specific cfb genes. This information can then be used to provide more accurate information as to whether a given resistance signal is likely to have been obtained from GBS versus another gram-positive organism.

[0143] Table 12

[0144]

[0145] Table 13

[0146]

[0147] Referring to Table 13, the asterisk (*) indicates that the ACt between the Ct of cfb and the Ct of a given resistance gene can be used to further correlate with resistance originating from GBS.

[0148] In one example, ΔCt is calculated as the difference between the Ct of the call for cfb (or another low copy GBS-specific gene, such as the surface immunogenic protein encoding gene sip, the glycosyltransferase protein encoding gene, and the lysR family protein encoding gene) in the sample (Ct GBS ) and the Ct of the call for a single target clindamycin resistance gene (such as ermB, ermTR, ermT, IsaC, and IsaE) (Ct ClinR ). This can be written as ΔCt = Ct GBS - Ct ClinR If the absolute value of ΔCt is less than or equal to a defined threshold (x), then the sample can be determined to contain a GBS carrying a clindamycin resistance gene. In one aspect, when x is small (e.g., 2 or less), then 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 thus likely to originate from the same organism. More specifically, it can be inferred that the GBS strain identified by cfb carries a clindamycin resistance gene. In contrast, if x is large, then the identification of a clindamycin resistance gene can be due to the presence of a second, non-GBS organism that carries a clindamycin resistance gene. Suitable values of x can be determined empirically and can depend on the nature of the NAAT, including the choice of instrument, buffers, consumables, etc. In one example, x is less than or equal to 2. In another example, x is less than or equal to 2.5. In another example, x is less than or equal to 3.

[0149] While the foregoing application has been described in some detail for purposes of clarity and understanding, it will be appreciated that certain changes and modifications can be practiced within the scope of the appended claims. For instance, all techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications and / or other documents cited in this application are hereby 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 were individually indicated to be incorporated by reference for all purposes.

Claims

1. A method for detecting Group B Streptococcus (GBS), the method comprising: - contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 is indicative of the presence or absence of each of a GBS strain and a clindamycin resistance mechanism in the sample.

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

3. The method of any one of claims 1-2, wherein the defined set of targets comprises: i) a GBS 23s ribosomal RNA gene (23s rRNA), ii) a GBS specific gene, and iii) at least one clindamycin resistance gene.

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 of any one of claims 3-4, wherein the GBS specific gene is a CAMP factor-encoding gene (cfb).

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

7. The method of any one of claims 3-6, wherein the plurality of oligonucleotide primers comprises a set of oligonucleotide primers for amplifying at least a portion of each of the targets, wherein: - a set of 23s rRNA oligonucleotide primers comprises at least one primer comprising the nucleic acid sequence of SEQ ID NO: 22, 23, or 24; - a 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 - at least one set of clindamycin resistance gene oligonucleotide primers is selected from the group consisting of: - 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 IsaC oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 15 or 16, and - a set of IsaE oligonucleotide primers comprising at least one primer comprising the nucleic acid sequence of SEQ ID NO: 17 or 18. - 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.

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

9. The method according to any one of claims 1 to 8, wherein the detectable oligonucleotide probe for each of the targets is labeled with a donor moiety and a corresponding acceptor moiety, and wherein 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 is indicative of the presence or absence of the corresponding target of the targets in the sample.

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

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

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

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

14. The method according to claim 13, wherein x < 2. ​ 15. An oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 64.

16. A method for detecting Group B Streptococcus (GBS), the method comprising: - contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the method distinguishes GBS from other species of Streptococcus.

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

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

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

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

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

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

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 products are further combined with a blocking oligonucleotide.

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

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

27. A method for detecting Group B Streptococcus (GBS), the method comprising: - contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

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

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

30. The method of any one of claims 27-29, wherein the method distinguishes GBS from at least one of Streptococcus urinalis, Streptococcus thermophilus, and Streptococcus anginosus.

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

33. A method for detecting Group B Streptococcus (GBS), the method comprising: - contacting a sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; wherein the sample is a sample directly from a specimen; wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA); and wherein the method distinguishes GBS from other species of Streptococcus.

34. The method of any one of claims 27-33, wherein the method further comprises: - contacting the sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

35. The method of any one of claims 27-34, wherein the method further comprises: - contacting the sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

36. The method of any one of claims 27-35, wherein the method further comprises: - contacting the sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

37. The method of any one of claims 27-36, wherein the method further comprises: - contacting the sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

38. The method of any one of claims 27-37, wherein the method further comprises: - contacting the sample with a plurality of oligonucleotide primers directed to a defined set of targets to produce 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 directed to 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 is indicative of the presence or absence of a GBS strain in the sample; and wherein the defined set of targets comprises a GBS 23s ribosomal RNA gene (23s rRNA).

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