Compositions and methods for detecting carbapenem resistant acinetobacter calcoaceticus-acinetobacter baumannii (CRAB)

Abstract

The present invention relates to compositions and methods for the detection of Acinetobacter calcoaceticus-Acinetobacter baumannii complex species and the identification of the most prevalent carbapenem enzymes found in carbapenem resistant Acinetobacter calcoaceticus-Acinetobacter baumannii (CRAB), such as a blaOXA-23 sample, a blaOXA-24 sample, a blaOXA-58 sample, and a blaNDM sample, by multiplex real-time PCR assays.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 435,267, filed on December 25, 2022, which is hereby incorporated by reference in its entirety.

[0003] Reference to sequence listing

[0004] This application contains a sequence listing submitted as an electronic text file named "P38059 - WO_Seq_Listing", which is 6,025 bytes in size and was created on December 5, 2023. The information contained in this electronic file is hereby 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 particularly, to the detection of carbapenem - resistant bacteria in the Acinetobacter calcoaceticus - baumannii complex. Background art

[0006] According to the Centers for Disease Control and Prevention (CDC) of the United States, the prevalence of carbapenem - resistant Acinetobacter calcoaceticus - baumannii (CRAB) is increasing, and treatment options are few, making it an urgent threat to public health. According to the World Health Organization (WHO), CRAB is also a global and critical priority. Therefore, novel treatments for Acinetobacter baumannii, as well as frequent monitoring and preventive healthcare activities, are a top priority in healthcare.

[0007] The development of novel antibiotics targeting CRAB is an urgent unmet medical need being addressed by pharmaceutical companies. A major challenge in their planned pathogen-focused randomized controlled trials is the accurate and rapid screening of eligible patients. Although molecular methods are available for viral detection, bacterial culture remains the gold standard for diagnosing bacterial infections from lower respiratory tract infections (LRTIs). The most common types of respiratory samples in traditional microbiological culture methods are bronchoalveolar lavage (BAL) fluid and sputum; however, the Infectious Diseases Society of America (IDSA) and American Thoracic Society (ATS) guidelines also recommend routine blood culture (BC) for healthcare-associated pneumonia because this pneumonia is considered to have a greater risk of bacteremia, especially for multidrug-resistant organisms. Traditional microbiological methods recommended for diagnosing pneumonia typically can take at least 48 to 72 hours to obtain a complete identification and susceptibility result by culture. In addition, because many respiratory pathogens can be components of the normal microbiota, it is necessary to determine the relative abundance of pathogens to commensals in LRTI samples by quantitative or semi-quantitative culture. Improved methods for rapid diagnosis of LRTI may involve molecular methods such as quantitative real-time polymerase chain reaction (qRT-PCR), which targets specific genes against the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, and the most prevalent carbapenem resistance mechanisms found in CRAB. Currently, there are multiple FDA-approved syndrome panels for detecting and identifying the most common pathogens and antimicrobial resistance (AMR) markers from LRTI samples (UNYVERO LRT panel and BIOFIRE FILMARRAY PN) and positive BCs (BIOFIRE FILMARRAY BCID2, UNYVERO BCU, LUMINEX VERIGENE BC-GP / BC-GN, and GENMARK EPLEX BCID-GP / BCID-GN / BCID-FP). Only two syndrome panels (UNYVERO LRT and VERIGENE BC-GN) target the most prevalent carbapenemases (OXA-23-like, OXA-24-like, OXA-58-like) found in CRAB, but neither of them has a specific interpretation for CRAB.

[0008] Rapid molecular screening of CRAB LRTI or positive BC samples can help with earlier patient enrollment, reduce the enrollment numbers, reduce the confounding effects of prior antibiotic treatment, and overall reduce the clinical trial cost / time of pathogen-focused clinical trials. Therefore, there is an urgent need to develop a molecular detection method for rapid detection of CRAB directly from BAL and / or sputum samples and from positive blood cultures obtained from LRTI patients, preferably in an automated and high-throughput platform. Summary of the Invention

[0009] Certain aspects of the present invention relate to methods for rapidly detecting the presence or absence of ACB complex species and the most prevalent carbapenemases found in CRAB in biological or non-biological samples. For example, this is achieved by multiplex detection of the gyrB gene of Acinetobacter baumannii, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like alleles of the OXA carbapenemase gene, and blaNDM-like carbapenemase genes by real-time polymerase chain reaction in a single test tube. Embodiments include methods for detecting the gyrB gene and carbapenemase genes, which include performing at least one cycling step that can include an amplification step and a hybridization step. Additionally, embodiments include primers, probes, and kits designed for detecting the gyrB gene of Acinetobacter baumannii, blaOXA-23-like, blaOXA-24-like, and bla-OXA-58-like alleles of the OXA carbapenemase gene, and blaNDM carbapenemase gene in a single tube. The detection method is designed to target these genes, which allows for the detection of the presence of Acinetobacter baumannii and the mechanism of carbapenem resistance in one test.

[0010] In one aspect, there is provided a method for detecting Acinetobacter baumannii with a carbapenem resistance mechanism in a sample, the method comprising: performing an amplification step that includes contacting the sample with a set of gyrB forward and reverse primers, a set of blaOXA-23-like forward and reverse primers, a set of blaOXA-24-like forward and reverse primers, a set of blaOXA-58-like forward and reverse primers, and a set of blaNDM forward and reverse primers to produce an amplification product if any of these target genes are present in the sample; performing a hybridization step that includes contacting the amplification product with one or more detectable gyrB probes, one or more detectable blaOXA-23-like probes, one or more detectable blaOXA-24-like probes, one or more detectable blaOXA-58-like probes, and one or more detectable blaNDM probes; and detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of Acinetobacter baumannii and / or a carbapenem resistance mechanism in the sample, and wherein the absence of the amplification product indicates the absence of Acinetobacter baumannii and / or a carbapenem resistance mechanism in the sample. In one embodiment, a set of gyrB primers comprises or consists of a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:1, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:2, and the detectable gyrB probe comprising the nucleic acid sequence of SEQ ID NO:3 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-23-like primers comprises or consists of a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:4, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:5, and the detectable blaOXA-23-like probe comprising the nucleic acid sequence of SEQ ID NO:6 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-24-like primers comprises the nucleic acid sequence of SEQ ID NO:7 and a reverse primer, or consists of the same, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:8, and the detectable blaOXA-24-like probe comprising the nucleic acid sequence of SEQ ID NO:9 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-58-like primers comprises or consists of a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:10, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:11, and the detectable blaOXA-58-like probe comprising the nucleic acid sequence of SEQ ID NO:12 or its complementary sequence, or consisting of the same.In one embodiment, a set of blaNDM primers comprises, or consists of, a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:13, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:14, and the detectable blaNDM probe comprising, or consisting of, the nucleic acid sequence of SEQ ID NO:15 or its complementary sequence. In one embodiment, the amplification can be carried out using a polymerase having 5'-to-3' nuclease activity. In some embodiments of the method, the hybridization step comprises contacting the amplification product with a detectable probe, the detectable probe being labeled with a donor fluorophore and a corresponding acceptor fluorophore; and the detection step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorophore and the acceptor fluorophore of the probe, wherein the presence or absence of fluorescent FRET indicates the presence or absence in the sample. In some embodiments, the first and second fluorophores may be no more than 8 nucleotides apart from each other along the length of the probe. According to the method, the second fluorophore on the probe may be a quencher. In some embodiments, the donor fluorophore and the corresponding acceptor fluorophore are no more than 8 nucleotides apart from each other on the probe. Herein, the acceptor fluorophore may be a quencher. In some embodiments, detecting the presence or absence of the amplification product further comprises: detecting the presence or absence of the amplification product of gyrB in a first optical detection channel; detecting the presence or absence of the amplification products of blaOXA-23-like alleles, blaOXA-24-like alleles, and / or blaOXA-58-like alleles in a second optical detection channel; and detecting the presence or absence of the amplification product of blaNDM in a third optical detection channel. In certain embodiments, the detectable gyrB probe comprises a first donor fluorophore and a corresponding first acceptor fluorophore, wherein each of the one or more detectable blaOXA-23-like probes, one or more detectable blaOXA-24-like probes, and one or more detectable blaOXA-58-like probes comprises a second donor fluorophore and a corresponding second acceptor fluorophore, and wherein the one or more detectable blaNDM probes comprises a third donor fluorophore and a corresponding third acceptor fluorophore. In other embodiments, the gyrB, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and / or blaNDM probes comprise nucleic acid sequences that allow the formation of secondary structures. Such secondary structure formation generally results in spatial proximity between the first fluorophore and the second fluorophore. According to the method, the second fluorophore on the probe may be a quencher. In some embodiments, any one or more of the oligonucleotide primers and / or probes used in the method comprise at least one modified nucleotide, for example, to alter nucleic acid hybridization stability relative to unmodified nucleotides.

[0011] In another aspect, an oligonucleotide is provided that comprises or consists of a sequence of nucleotides selected from SEQ ID NOs: 1 to 15 or its complementary sequence, and the oligonucleotide has 100 or fewer nucleotides. Additionally, the present disclosure provides an oligonucleotide that comprises a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%, etc.) with one of SEQ ID NOs: 1 to 15 or its complementary sequence, and the oligonucleotide has 100 or fewer nucleotides. Generally, in these embodiments, the oligonucleotides as disclosed herein can be primer nucleic acids, probe nucleic acids, etc. In certain of these embodiments, the oligonucleotide has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides, 20 or fewer nucleotides, 15 or fewer nucleotides, etc.). In some embodiments, the oligonucleotide comprises at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability relative to an unmodified nucleotide. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide includes at least one conservatively modified variant. A "conservatively modified variant" or simply "conservative variant" of a particular nucleic acid sequence refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, in the case where the nucleic acid does not encode an amino acid sequence, refers to substantially the same sequences. Those skilled in the art will recognize that individual substitutions, deletions, or additions that change, add, or delete a single amino acid or a small portion of amino acids (generally less than 5%, more typically less than 4%, 2%, or 1%) in a coding sequence are "conservatively modified variants", where such changes result in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid.

[0012] In a further aspect, the present invention provides a kit for detecting Acinetobacter baumannii and provides one or more nucleic acids of the carbapenem resistance mechanism, wherein the kit at least comprises: a set of Acinetobacter baumannii gyrB gene primers specific for the amplification of the gyrB gene, and one or more detectable gyrB probes specific for the detection of the amplification product of the gyrB gene; a set of blaOXA-23-like gene primers specific for the amplification of the blaOXA-23-like gene, and one or more detectable blaOXA-23-like probes specific for the detection of the amplification product of the blaOXA-23-like gene; a set of blaOXA-24-like gene primers specific for the amplification of the blaOXA-24-like gene, and one or more detectable blaOXA-24-like probes specific for the detection of the amplification product of the blaOXA-24-like gene; a set of blaOXA-58-like gene primers specific for the amplification of the blaOXA-58-like gene, and one or more detectable blaOXA-58-like probes specific for the detection of the amplification product of the blaOXA-58-like gene; and a set of blaNDM gene primers specific for the amplification of the blaNDM gene, and one or more detectable blaNDM probes specific for the detection of the amplification product of the blaNDM gene. In some embodiments, the kit may comprise: multiple sets of Acinetobacter baumannii gyrB gene primers specific for the amplification of the gyrB gene, and one or more detectable gyrB probes specific for the detection of the amplification product of the gyrB gene; multiple sets of blaOXA-23-like gene primers specific for the amplification of the blaOXA-23-like gene, and one or more detectable blaOXA-23-like probes specific for the detection of the amplification product of the blaOXA-23-like gene; multiple sets of blaOXA-24-like gene primers specific for the amplification of the blaOXA-24-like gene, and one or more detectable blaOXA-24-like probes specific for the detection of the amplification product of the blaOXA-24-like gene; multiple sets of blaOXA-58-like gene primers specific for the amplification of the blaOXA-58-like gene, and one or more detectable blaOXA-58-like probes specific for the detection of the amplification product of the blaOXA-58-like gene; and multiple sets of blaNDM gene primers specific for the amplification of the blaNDM gene, and one or more detectable blaNDM probes specific for the detection of the amplification product of the blaNDM gene.In one embodiment, a set of gyrB primers comprises, or consists of, a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:1, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:2, and the detectable gyrB probe comprising the nucleic acid sequence of SEQ ID NO:3 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-23-like primers comprises, or consists of, a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:4, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:5, and the detectable blaOXA-23-like probe comprising the nucleic acid sequence of SEQ ID NO:6 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-24-like primers comprises the nucleic acid sequence of SEQ ID NO:7 and a reverse primer, or consists of the same, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:8, and the detectable blaOXA-24-like probe comprising the nucleic acid sequence of SEQ ID NO:9 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaOXA-58-like primers comprises, or consists of, a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:10, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:11, and the detectable blaOXA-58-like probe comprising the nucleic acid sequence of SEQ ID NO:12 or its complementary sequence, or consisting of the same. In one embodiment, a set of blaNDM primers comprises, or consists of, a forward primer and a reverse primer, the forward primer comprising the nucleic acid sequence of SEQ ID NO:13, the reverse primer comprising the nucleic acid sequence of SEQ ID NO:14, and the detectable blaNDM probe comprising the nucleic acid sequence of SEQ ID NO:15 or its complementary sequence, or consisting of the same. In some embodiments, the detectable probe comprises a donor fluorophore moiety and a corresponding acceptor fluorophore moiety. In certain embodiments, the acceptor fluorophore moiety is a quencher. In some embodiments, the detectable gyrB probe comprises a first donor fluorophore moiety and a corresponding first acceptor fluorophore moiety, wherein each of the one or more detectable blaOXA-23-like probes, one or more detectable blaOXA-24-like probes, and one or more detectable blaOXA-58-like probes comprises a second donor fluorophore moiety and a corresponding second acceptor fluorophore moiety, and wherein the one or more detectable blaNDM probes comprises a third donor fluorophore moiety and a corresponding third acceptor fluorophore moiety.In certain embodiments, the first, second, and third donor fluorophore moieties are different from one another and are selected from the group consisting of HEX (hexachlorofluorescein), FAM (6-carboxyfluorescein), and JA270 (1-(2-hydroxyethyl-6-(2,3,4,5-tetrachlorophenyl)11-ethyl-2,2,4,8,10,11-hexamethyl-10,11-dihydro-2H-13-oxa-11-aza-1-azaperi-pentacene perchlorate). In certain embodiments, the first donor fluorophore moiety is HEX, the second donor fluorophore moiety is FAM, and the third donor fluorophore moiety is JA270. In one embodiment, the kit may include probes that have been labeled with donor and corresponding acceptor fluorophore moieties, or may include fluorophore moieties for labeling the probes. The kit may also include nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase. The kit may also include package instructions and instructions for using the primers, probes, and fluorophore moieties to detect the presence or absence of the gyrB gene and / or bla gene in a sample.

[0013] Unless otherwise defined, 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 invention 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.

[0014] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and the detailed description and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1A and 1B shows the inclusivity and exclusivity assessment of the CRAB multiplex assay of the invention demonstrating the workflow using a high-throughput PCR system. In the presence of human genomic DNA at 130 ng / reaction, multiple ACB strains (inclusive species) were tested at 10 4 colony forming units per milliliter (CFU / mL), and at 10 7CFU / mL was tested for other relevant carbapenem-resistant species (limited species). Inclusive species (n=18) included Acinetobacter baumannii (n=10), Acinetobacter nosocomialis (n=2), Acinetobacter pittii (n=2), and Acinetobacter calcoaceticus (n=1). The restricted species (n=18) included Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, P. mirabilis, Staphylococcus aureus, Streptococcus pneumoniae, Citrobacter koseri, E. cloacae, K. aerogenes, Pseudomonas aeruginosa, S. maltophilia, S. agalactiae, S. pyogenes, E. faecium, E. faecalis, H. influenzae, and Serratia marcescens. Figure 1A Shown are PCR amplification curves for representative inclusive species compared to each of the defined species, for which no amplification was observed. Figure 1B PCR amplification curves for each of the inclusive species compared to a no-template control are shown.

[0016] Figure 2A and 2B Graphical representations of two interpretations of the CRAB multiplex assay of the present invention are provided for distinguishing pathogenic from commensal ACB in LRTI samples. Figure 2A Shown in the concentration of 1*10 3 CFU / mL and 1*10 4 PCR amplification curves of primers and probes targeting gyrB in samples with titers of CFU / mL and A. baumannii control. The cycle threshold (Ct) cutoff is shown as a vertical dashed line. Figure 2B Shown in the concentration of 1*10 3 CFU / mL, 5*10 3 CFU / mL, 1*10 4 CFU / mL and 5*10 4PCR amplification data of primers and probes targeting gyrB measured in the first detection channel and internal control measured in the second detection channel in samples of Acinetobacter baumannii control with a titer of CFU / mL. For each of the test concentrations, the measured Ct values and the Ct value difference (ΔCt) between the two detection channels are shown.

[0017] Figure 3 Performance of the prototype 3-channel (JA270, HEX, and FAM) CRAB multiplex assay on samples of one of five different strains of Acinetobacter baumannii encoding one or more targets selected from the following: gyrB (detected in the HEX channel), blaOXA-23-like, blaOXA-24-like, and bla-OXA-58-like (detected in the FAM channel), and blaNDM (detected in the JA270 channel) is shown. Relative fluorescence intensity (RFI) and Ct values of reactions carried out in two different PCR media (CPM and MIS) are reported. CPM samples were tested in a sample volume of 850 μL at concentrations of 1*10 2 、1*10 3 and 1*10 4 CFU / mL, corresponding to 2*10 1 、2*10 2 and 2*10 3 CFU / reaction, while MIS samples were tested in a sample volume of 400 μL at concentrations of 1*10 2 、1*10 3 and 1*10 4 CFU / mL, corresponding to 1*10 1 、1*10 2 and 1*10 3 CFU / reaction. The target composition present in each of the test samples is described in Table 7.

[0018] Figure 4 A graphical representation of the CRAB multiplex assay workflow for suspected healthcare-associated bacterial pneumonia (HABP), ventilator-associated bacterial pneumonia (VABP), or bloodstream infection (BSI) caused by CRAB is provided.

[0019] Figures 5A to 5C The complete assay map ( Figure 5A ) and data ( Figure 5B and 5C ) of CRAB spiked into the sample diluent (CPM) cleaning system or combined bronchoalveolar lavage fluid (BAL) and / or sputum (SPU) samples are shown. Figure 5B The assay performance using Acinetobacter baumannii titer controls spiked into a negative BAL matrix at different concentrations is shown.Figure 5C The assay performance is shown for Acinetobacter baumannii titer controls spiked at different concentrations into a negative SPU matrix.

[0020] Figure 6 The complete assay workflow for a preliminary test is shown using negative whole blood (WB) incubated in a commercially available blood culture bottle as the negative matrix and spiked with 3 final CFU / mL concentrations (1*10 4 、1*10 3 、1*10 2 ) of a commercial Acinetobacter baumannii titer control. Data for the CRAB assay performance are additionally shown in Table 8. DETAILED DESCRIPTION

[0021] As used herein, the term "amplifying" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., the gyrB gene of Acinetobacter baumannii). Amplifying a nucleic acid molecule generally involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the primer melting temperature, and enzymatically extending from the primers to produce an amplification product. Amplification generally requires the presence of deoxynucleoside triphosphates, a DNA polymerase (e.g., Taq), and appropriate buffers and / or cofactors (e.g., MgCl2 and / or KCl) for optimizing polymerase activity.

[0022] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound capable of "initiating" DNA synthesis by a template-dependent DNA polymerase, mainly referring to oligonucleotides, but also to modified oligonucleotides, i.e., for example, the 3'-end of the oligonucleotide provides a free 3'-OH group to which additional "nucleotides" can be attached by a template-dependent DNA polymerase, thereby establishing a 3' to 5' phosphodiester bond, where deoxynucleoside triphosphates are used and pyrophosphate is released. Thus, there is no fundamental difference between "primer", "oligonucleotide", or "probe" other than their possible intended functions.

[0023] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions generally include a temperature below the probe melting temperature but avoiding non-specific hybridization of the probe.

[0024] 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.

[0025] The term "thermostable polymerase" refers to a polymerase that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when subjected to elevated temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Typically, synthesis initiates at the 3' end of each primer and proceeds along the template strand in the 5' to 3' direction. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. However, polymerases that are not thermostable can also be used in polymerase chain reaction (PCR) assays provided that the enzyme is supplemented.

[0026] The term "its complementary sequence" refers to a nucleic acid that has the same length as a given nucleic acid and is completely complementary thereto.

[0027] When used in reference to a nucleic acid, the terms "extension" or "elongation" refer 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.

[0028] In the context of two or more nucleic acid sequences, the terms "identical" or "percent identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides when compared and aligned for maximum correspondence (e.g., measured using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection). Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, which are 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.

[0029] "Modified nucleotide" in the context of an oligonucleotide refers to a change in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide, thereby providing the oligonucleotide with desired properties. In the oligonucleotides described herein, exemplary modified nucleotides that can be substituted include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2'-deoxyxanthosine, pyrazolopyrimidine, pseudo-dU, nitropyrrole, nitroindole, 2'-O-methyl ribose-U, 2'-O-methyl ribose-C, N4-ethyl-dC, N6-methyl-dA, and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or are otherwise known in the art. In certain embodiments, the substitution of a modified nucleotide modifies the melting temperature (Tm) of the oligonucleotide relative to the melting temperature of the corresponding unmodified oligonucleotide. For further illustration, 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 the desired target amplicon, and the like. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611 (incorporated herein by reference).

[0030] 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 3'-end of the corresponding sequence of the oligonucleotide. As described above, a primer (and / or probe) can be chemically modified, i.e., the primer and / or probe can contain a modified nucleotide or a non-nucleotide compound. A probe (or primer) is then a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analogue") 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 portion of a "nucleotide", thereby obtaining a "modified nucleotide". The natural base in a "nucleotide" can also be replaced, for example, with 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analogue" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analogue") differs from a natural nucleoside by some modification in the manner outlined above for a "modified nucleotide" (or "nucleotide analogue").

[0031] For example, a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) can be used to design oligonucleotides (including modified oligonucleotides and oligonucleotide analogs) that amplify nucleic acid molecules (e.g., nucleic acid molecules encoding the gyrB gene or the nucleic acid sequences of the blaOXA and blaNDM genes). When designing oligonucleotides to be used as amplification primers, important features include, but are not limited to, an appropriately sized amplification product for easy detection (e.g., by electrophoresis), similar melting temperatures for the members of a pair of primers, and the length of each primer (i.e., the primer needs to be long enough to anneal sequence specifically and initiate synthesis, but not so long that the fidelity is reduced during oligonucleotide synthesis). Generally, the length of an oligonucleotide primer is from 8 to 50 nucleotides (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).

[0032] In addition to a set of primers, these methods can also use one or more probes to detect the presence or absence of the ACB complex and the carbapenem resistance mechanism. The term "probe" refers to a nucleic acid (DNA or RNA) produced synthetically or biologically that, by design or selection, contains a specific nucleotide sequence that allows it to hybridize specifically (i.e., preferentially) to a "target nucleic acid" under defined predetermined stringencies. In this case, it hybridizes to the AbigyrB (target) nucleic acid and / or the blaOXA-23-like, blaOXA-24-like, and bla-OXA-58-like alleles of the OXA carbapenemase and the blaNDM carbapenemase (target) nucleic acid. A "probe" can be referred to as a "detection probe", meaning that it detects the target nucleic acid.

[0033] In some embodiments, the described probes 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).

[0034] Oligonucleotides designed to be used as probes can be designed in a manner similar to primer design. Embodiments can use a single probe or a pair of probes to detect the amplification product. According to an embodiment, the probe used can comprise at least one label and / or at least one quencher moiety. Like primers, probes generally have similar melting temperatures, and the length of each probe must be sufficient to allow sequence-specific hybridization, but not so long that the fidelity is reduced during synthesis. The length of an oligonucleotide probe is generally from 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides.

[0035] The construct can include vectors each containing one of the primer and probe nucleic acid molecules (e.g., SEQ ID NOs: 1 to 15). The construct can be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by methods conventional in the art of recombinant nucleic acid technology. The target nucleic acid molecule can be obtained, for example, by chemical synthesis, direct cloning of a gene, or by PCR amplification.

[0036] In addition to the target nucleic acid molecule (e.g., a nucleic acid molecule containing one or more sequences of SEQ ID NOs: 1 to 15), the constructs suitable for use in the method generally also include sequences encoding a selectable marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system usually depends on several factors, including but not limited to the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0037] The construct containing the target nucleic acid molecule can be propagated in a host cell. As used herein, the term host cell is intended to include prokaryotes and eukaryotes, such as yeast, plant, and animal cells. Prokaryotic hosts can include Escherichia coli, Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeast (such as Saccharomyces cerevisiae, Schizosaccharomyces 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). Any technique known to those of ordinary skill in the art can be used to introduce the construct into the host cell. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be directly delivered to cells (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0038] Polymerase Chain Reaction (PCR)

[0039] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically 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 starting points for nucleic acid synthesis within the described target gene and target allele nucleic acid sequences (e.g., SEQ ID NO: 1, 2, 4, 5, 7, 8, 10, 11, 13, 14). Primers can be purified from restriction digests by conventional methods, or it can be synthesized. For maximum efficiency in amplification, primers are preferably single-stranded, but primers can be double-stranded. First, the double-stranded primer is denatured (i.e., treated) to separate the strands. One method of denaturing double-stranded nucleic acid is by heating.

[0040] If the template nucleic acid is double-stranded, the two strands must be separated before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing 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 required to denature the template nucleic acid will depend on, for example, buffer salt concentration and the length and nucleotide composition of the nucleic acid being denatured, but typically range from about 90°C to about 105°C for a period of time depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0041] If the double-stranded template nucleic acid is denatured by heating, the reaction mixture is cooled to a temperature that promotes annealing of each primer to the target sequence on the described nucleic acid molecule. The temperature for annealing is typically from about 35°C to about 65°C (e.g., about 40°C to about 60°C; about 45°C to about 50°C). The annealing time can be from about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds; about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient to effect extension 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 annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., the temperature for extension typically ranges from about 40°C to about 80°C (e.g., about 50°C to about 70°C; about 60°C). The extension time can be from about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes; about 1 minute to about 3 minutes; about 1 minute 30 seconds to about 2 minutes).

[0042] PCR assays can employ target gene and / or allele nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid need not be purified; it can be a small part of a complex mixture, such as the target nucleic acid contained in a biological sample. The target nucleic acid molecule 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, such as plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms, such as plants or animals.

[0043] Oligonucleotide primers are combined with PCR reagents under reaction conditions that induce primer extension. For example, the strand extension reaction typically includes 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5 to 1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO). The reaction typically contains 150 to 320 μM of each of dATP, dCTP, dTTP, dGTP or one or more analogs thereof.

[0044] The newly synthesized strands form double-stranded molecules that can be used in subsequent reaction steps. The steps of strand separation, annealing, and extension can be repeated as often as needed to produce the desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors in the reaction are the amounts of primers, heat-stable enzyme, and nucleoside triphosphates present in the reaction. Preferably, the cycle step (i.e., denaturation, annealing, and extension) is repeated at least once. For use in detection, the number of cycle steps will depend, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycle steps will be required to amplify the target sequence sufficiently for detection. Typically, the cycle step is repeated at least about 20 times, but can be repeated up to 40, 6Y0, or even 100 times.

[0045] Fluorescence Resonance Energy Transfer (FRET)

[0046] 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 fluorophore and a corresponding acceptor fluorophore are within a certain distance of each other, energy transfer occurs between the two fluorophores, and this energy transfer can be visualized or otherwise detected and / or quantified. When the donor is excited by light radiation of an appropriate wavelength, the donor generally transfers energy to the acceptor. The acceptor generally re-emits the transferred energy in the form of light radiation having a different wavelength. In some systems, non-fluorescent energy can be transferred between the donor and acceptor moieties by a biomolecule comprising a substantially non-fluorescent donor moiety (see, e.g., U.S. Patent No. 7,741,467).

[0047] In one example, an oligonucleotide probe can contain a donor fluorophore and a corresponding quencher, which may or may not be fluorescent and dissipates the transferred energy in a form different from light. When the probe is intact, energy transfer generally occurs between the two fluorophores such that the fluorescence emission from the donor fluorophore is quenched. During the extension step of a polymerase chain reaction, the probe bound to the amplification product is cleaved by the 5' to 3' nuclease activity of, e.g., Taq polymerase, such that the fluorescence emission of the donor fluorophore is no longer quenched. Exemplary probes for this purpose are described in, e.g., 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 BlackHole 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.).

[0048] In another example, two oligonucleotide probes (each containing a fluorophore) can hybridize to the amplification product at specific positions 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 positions, 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.

[0049] Fluorescence analysis can be carried out using, for example, a photon counting epifluorescence microscope system (containing an appropriate dichroic mirror and filters for monitoring fluorescence emission in a specific range), a photon counting photomultiplier tube system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of fluorophores can be carried out using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a fiber optic light source, or other high-intensity light sources that are appropriately filtered to excite in the desired range.

[0050] As used herein with respect to the donor and corresponding acceptor fluorescence moieties, "corresponding" means an acceptor fluorescence moiety or a dark quencher having an absorption spectrum that overlaps the emission spectrum of the donor fluorescence moiety. The maximum wavelength of the emission spectrum of the acceptor fluorescence moiety should be at least 100 nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescence moiety. Thus, efficient non-radiative energy transfer can occur between them.

[0051] Fluorescent donor and corresponding acceptor moieties are typically selected for: (a) high efficiency Forster energy transfer; (b) a large final Stokes shift (>100 nm); (c) emission shifted as far as possible into the red portion of the visible spectrum (>600 nm); and (d) emission shifted to a wavelength higher than the Raman water fluorescence emission generated by excitation at the donor excitation wavelength. For example, the following donor fluorescence moieties can be selected: those having their excitation maximum near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), having a high extinction coefficient, a high quantum yield, and whose fluorescence emission overlaps well with the excitation spectrum of the corresponding acceptor fluorescence moiety. Corresponding acceptor fluorescence moieties can be selected having a high extinction coefficient, a high quantum yield, good overlap of their excitation with the emission of the donor fluorescence moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0052] Representative donor fluorophores that can be used with various acceptor fluorophores in FRET techniques include fluorescein, eosin, B-phycoerythrin, 9-acridine isothiocyanate, eosin VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorophores, depending on the donor fluorophore used, include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosin isothiocyanate, fluorescein, chelates of diethylenetriaminepentaacetic acid or other lanthanide ions (e.g., europium or terbium). Donor and acceptor fluorophores can be obtained from, for example, Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0053] Donor and acceptor fluorophores can be attached to a suitable probe oligonucleotide via a linker arm. The length of each linker arm is important because the linker arm affects the distance between the donor and acceptor fluorophores. The length of the linker arm is the distance from the nucleobase to the fluorophore in angstroms units. Typically, the linker arm is about to about Linker arms can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching linker arms to specific nucleobases and for attaching fluorophores to linker arms.

[0054] An acceptor fluorophore such as LC Red 640 can be combined with an oligonucleotide containing an amino linker (e.g., C6-amino phosphoramidite available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA)) to produce, for example, an LC Red 640-labeled oligonucleotide. Linkers commonly used to couple a donor fluorophore such as fluorescein to an oligonucleotide include thiourea linkers (FITC-derived, e.g., fluorescein-CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide linkers (fluorescein-NHS-ester-derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPGs that require coupling of fluorescein-NHS-ester after oligonucleotide synthesis.

[0055] Detection of Target Genes and Alleles in CRAB

[0056] The present disclosure provides methods for detecting the presence or absence of the gyrB gene of Acinetobacter baumannii, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like alleles of the OXA carbapenemase gene, and blaNDM-like NDM carbapenemase gene in biological or non-biological samples. The provided methods avoid problems of sample contamination, false negatives, and false positives. The method includes 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 multiple pairs of target primers. Multiple cycling steps are performed, preferably in a thermal cycler. Target primers and probes can be used to perform the method to detect the presence of a target gene, and detection of the amplification product in the assay indicates the presence of the target gene and / or target allele in the sample.

[0057] As described herein, labeled hybridization probes utilizing FRET technology can be used to detect the amplification product. One form of FRET utilizes technology to detect the presence or absence of the amplification product and thus the presence or absence of CRAB. The technology utilizes a single-stranded hybridization probe that is labeled with, for example, a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the FRET principle. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension phase by, for example, the 5' to 3' nuclease activity of Taq polymerase. Thus, the fluorescent moiety and the quencher moiety become spatially separated from each other. Therefore, fluorescence emission from the first fluorescent moiety can be detected after excitation of the first fluorescent moiety in the absence of the quencher. By way of example, ABI 7700 Sequence Detection System (Applied Biosystems) uses technology and is suitable for performing the methods described herein for detecting the presence or absence of CRAB in a sample.

[0058] It is also possible to use molecular beacons conjugated with FRET to detect the presence of amplification products using the real-time PCR method. Molecular beacon technology uses hybridization probes labeled with a first fluorophore and a second fluorophore. The second fluorophore is typically a quencher, and the fluorescent labels are generally located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow the formation of a secondary structure (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, the two fluorophores are spatially close when the probe is in solution. After hybridization with the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted, and the fluorophores become separated from each other, such that upon excitation with light of an appropriate wavelength, the emission of the first fluorophore can be detected.

[0059] Another common form of FRET technology is to use two hybridization probes. Each probe can be labeled with a different fluorophore and is typically designed to hybridize very close to each other in the target DNA molecule (e.g., the amplification product). The donor fluorophore, such as fluorescein, is excited by the light source of the instrument at 470 nm. During FRET, fluorescein transfers its energy to the acceptor fluorophore, such as -Red 640 (LC Red 640) or -Red705 (LC Red 705). The acceptor fluorophore then emits light at a longer wavelength, which is detected by the optical detection system of the instrument. Effective FRET occurs only when the fluorophores are directly locally close and when the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore. The intensity of the emission signal can be correlated with the number of original target DNA molecules. If amplification of the target nucleic acid occurs and amplification products are generated, the hybridization step produces a detectable signal based on FRET between the probe pair members.

[0060] 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, insufficient sample collection, transport delays, improper transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results. Using the methods disclosed herein, the detection of FRET within, for example, 45 cycle steps indicates CRAB infection.

[0061] Representative biological samples that can be used to practice the method include, but are not limited to, dermal swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. The biological sample can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release the target gene nucleic acid, or in some cases, the biological sample can be directly contacted with the PCR reaction components and appropriate oligonucleotides.

[0062] Melting curve analysis is an additional step that can be included in the cycling curve. 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 duplex separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature of the probe from the amplification product can confirm the presence or absence of the target sequence in the sample.

[0063] In each thermal cycler run, control samples can also be cycled. Positive control samples 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. Positive control samples can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such plasmid controls can be amplified internally (e.g., within the sample) or in a separate sample run in parallel with the patient sample using the same primers and probes as those used to detect the expected target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reactions. Each thermal cycler run can also include a negative control, e.g., lacking the target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, control reactions can easily determine, for example, the ability of the primers to anneal and initiate extension sequence specifically, and the ability of the probes to hybridize and undergo FRET sequence specifically.

[0064] In one embodiment, the method includes steps to avoid contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermal cycler run and the next.

[0065] These methods can be practiced using conventional PCR methods that incorporate FRET technology. In one embodiment, use instrument. The following patent applications describe as Real-time PCR used in the technology: WO97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0066] It can be operated using a PC workstation and the Windows NT operating system can be used. When the machine places the capillary tubes on the optical unit in sequence, signals from the samples can be obtained. The software can display the fluorescence signal in real time immediately after each measurement. The fluorescence acquisition time is 10 - 100 milliseconds (msec). After each cycle step, the quantitative display of fluorescence versus the number of cycles can be continuously updated for all samples. The data generated can be stored for further analysis.

[0067] As an alternative to FRET, double-stranded DNA-binding dyes such as fluorescent DNA-binding dyes (e.g., Green or Gold (Molecular Probes)) can be used to detect the amplification products. When interacting with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescence signal when excited with light of a suitable wavelength. Double-stranded DNA-binding dyes such as nucleic acid intercalating dyes can also be used. When using double-stranded DNA-binding dyes, melting curve analysis is usually performed to confirm the presence of the amplification products.

[0068] It should be understood that the embodiments of the present disclosure are not limited by the configuration of one or more commercially available instruments.

[0069] Article / kits

[0070] The embodiments of the present disclosure further provide an article or kit for detecting the gyrB gene of Acinetobacter baumannii, the blaOXA-23-like, blaOXA-24-like, blaOXA-58-like alleles of the OXA carbapenemase gene, and the blaNDM-like NDM carbapenemase gene (i.e., the genes and alleles responsible for CRAB). The article can include primers and probes for detecting CRAB, as well as suitable packaging materials. Representative primers and probes for detecting CRAB are capable of hybridizing with the target nucleic acid molecule. In addition, the kit can also include reagents and materials required for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes for amplifying and hybridizing with the target nucleic acid molecule are provided.

[0071] The article can also include one or more fluorescent moieties for labeling the probe, or alternatively, can label the probe provided with the kit. For example, the article can include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0072] The article of manufacture may also include a package insert or package label having instructions for using the target primers and probes to detect CRAB in a sample. The article of manufacture may additionally include reagents for practicing the methods disclosed herein (e.g., buffers, polymerases, cofactors, or agents to prevent contamination). Such reagents may be specifically designed for use with one of the commercially available instruments described herein.

[0073] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0074] Examples

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

[0076] Example 1

[0077] Example 1 partially illustrates primer and probe sequences used in accordance with the present disclosure. Table 1 shows primers and probes used in a multiplex PCR assay for detecting ACB complex and carbapenem resistance mechanisms.

[0078] Table 1:

[0079]

[0080]

[0081] Example 2

[0082] Example 2 partially describes the PCR experimental conditions according to the present disclosure. 4800 system or Real-time PCR detection of target genes was performed using a 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents are shown in Table 2.

[0083] Table 2:

[0084]

[0085]

[0086] Table 3 shows an example of a thermal curve for a PCR amplification reaction according to the present disclosure.

[0087] Table 3:

[0088]

[0089] The pre-PCR program includes an initial denaturation and incubations at 55 °C, 60 °C, and 65 °C for reverse transcription of the RNA template. Incubating at the three temperatures has the following beneficial 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, making transcription more efficient. The PCR cycles are divided into two measurements, where the two measurements apply a one-step setting (combining annealing and extension). The first 5 cycles at 55 °C allow for increased inclusivity by pre-amplifying slightly mismatched target sequences, while the 45 cycles of the second measurement provide increased specificity by using an annealing / extension temperature of 58 °C.

[0090] Example 3

[0091] Example 3 partially illustrates the performance of a multiplex PCR assay for detecting CRAB according to the present disclosure. A prototype multiplex PCR assay was performed using the primer and probe sequences described in Table 1 to target the ACB complex species as well as the most prevalent carbapenemases found in CRAB (blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM-like). The most prevalent carbapenemases found in CRAB include the previously described OXA enzyme group as well as the New Delhi metallo-β-lactamase (blaNDM) enzyme group (see, e.g., Ramirez MS et al., “Carbapenemases: Transforming Acinetobacter baumannii into a Yet More Dangerous Menace”. Biomolecules 10, 720 (2020) and Hujer, AM. et al., “A Comprehensive and Contemporary ‘Snapshot’ of β-Lactamases in Carbapenem Resistant Acinetobacter Baumannii.” Diagnostic Microbiology and Infectious Disease 99, no. 2 (2021): 115242). Using the experimental conditions described in Example 2, the performance of the assay was evaluated using CDC strains AR-0036 (gyrB and blaOXA-24 targets), AR-0052 (gyrB and blaOXA-58 targets), and AR-0083 (gyrB, blaOXA-23, and blaNDM targets), and the resulting linear and dynamic ranges of the assay are shown in Table 4. The multiplex assay was effective and linear from 10 2 to 10 6 copies / reaction, and no significant interference from human genomic DNA was observed.

[0092] Table 4:

[0093]

[0094] 6OM: 10 6 to 10 1 Cp / Rxn, 5OM: 10 6 to 10 2 Cp / Rxn

[0095] Example 4

[0096] Example 4 partially illustrates the CRAB assay analysis and interpretation according to the present disclosure. The CRAB assay interpretation scheme is shown in Table 5 and includes the detection of ACB and CRAB. The assay includes performing real-time PCR detection of target genes using a 6800 / 8800 system with 5 independent detection channels. Channel 1, which is not listed in Table 5, is left blank but can be used to detect additional targets (e.g., novel resistance mechanisms). Channel 2 is used to detect the main CRAB-specific resistance mechanism (blaOXA-23 / 24 / 58-like). Channel 3 is used to detect the conserved region of the ACB gyrB gene to identify the presence of Acinetobacter (also used to distinguish pathogens from symbionts in LRTI samples). Channel 4 is used to detect emerging CRAB resistance mechanisms (blaNDM-like) and needs to be associated with Channel 3 to indicate CRAB versus other carbapenem-resistant organisms (CROs). Channel 5 is used to detect a universal internal control (GIC), which is a control for sample preparation and PCR amplification used to distinguish valid from invalid samples.

[0097] Table 5:

[0098]

[0099] * Detection of the ACB gene on Channel 3 indicates the presence of ACB or a CRAB result if the CRAB resistance mechanism is also detected on Channel 2 and / or 4.

[0100] ** Detection of CRAB with NDM requires an association between Channel 3 and Channel 4.

[0101] In silico analysis was performed and the reactivity of clinically relevant ACB species and known variants of blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM-like available in the National Center for Biotechnology Information (NCBI) database was predicted to be 100%. The predicted (in silico) reactivity is shown in Table 6. Based on wet laboratory tests performed at 10 4 and 10 7 CFU / mL, respectively, in a clean system, the assay inclusivity for common ACB species (n = 15) in respiratory samples and exclusivity for other bacterial species (n = 18) were also confirmed ( Figure 1A and 1B ).

[0102] Table 6:

[0103]

[0104] Reference Figure 2A and 2B, additional assay interpretation is required to distinguish pathogens from symbionts of LRTI sample types with Ct thresholds of channel 3 (gyrB) or ΔCt thresholds calculated between Ct of channel 3 (gyrB) and channel 5 (internal control). Due to the characteristics of LRTI samples such as heterogeneity and viscosity, LRTI samples are also a challenging sample type for bacterial DNA recovery. With this in mind, various sample diluents such as microbial inactivation solution (MIS) and PCR culture medium (CPM) were tested for the CRAB assay, but no significant differences were observed, as Figure 3 shown in and Table 7. Therefore, the best sample transfer method for the LRTI sample workflow was determined using uniswab (with CPM dilution tubes for direct loading into the workflow).

[0105] Table 7

[0106]

[0107] Reference Figure 4 , method 100 for detecting CRAB in a sample includes a first step 101 of identifying a subject suspected of having hospital-acquired bacterial pneumonia (HABP) or ventilator-associated bacterial pneumonia (VABP). In the next step 102, a primary sample is obtained from the subject identified in step 101. The sample can be a residual LRTI sample such as a sputum sample, an endotracheal aspirate (ETA) sample, or a BAL sample. Step 102 further includes processing the residual LRTI sample to prepare the sample for downstream processing. In the next step 103, a portion of the primary sample from step 102 is transferred to a secondary tube. In this example, a swab or other similar tool is used to transfer a portion of the primary sample. Alternatively or additionally, method 100 can include a step 104 of identifying a subject suspected of having bloodstream infection (BSI). After step 104, the method can include a step 105 of obtaining a primary sample from the subject of step 104 in a blood culture bottle. Step 105 can further include incubating or otherwise processing the blood culture bottle under suitable conditions to provide an initial indication as to whether the primary sample is positive or negative for BSI. In the case where the blood culture bottle result is positive, the next step 106 of method 100 includes transferring a portion of the primary sample from step 102 to a secondary tube. In the next step 107 of method 100, the secondary tube obtained from one or both of step 103 and step 106 is loaded onto a high-throughput PCR system and processed using the system. An example of a suitable high-throughput PCR system includes one from ROCHE Series of X800 (such as 4800, 5800, 6800, and 8800) instruments. In the next step 108, the result is determined based on the information derived from step 107. In one aspect, the result is a qualitative determination result. Possible results include the sample being positive for CRAB, negative for CRAB, and the result being indeterminate.

[0108] Go to Figures 5A to 5C , and use Figure 5A shown in the uniswab transfer method to screen negative and spiked BAL and sputum samples with a titer Acinetobacter baumannii control or a CRAB strain to verify the performance of the CRAB assay. Continuing to refer to Figure 5A , method 200 for detecting CRAB according to this example includes a first step 201 of preparing a titer control containing Acinetobacter baumannii at a known concentration (e.g., CFU / mL). In this example, a commercially available titer control of Acinetobacter baumannii was used. In the next step 203, a portion of the spiked sample from step 201 is transferred to a secondary tube. In one aspect, a swab (as in this example) or other similar tool can be used to transfer a portion of the spiked sample. Alternatively or additionally, method 200 includes a step 204 of preparing a pooled matrix sample. The pooled matrix sample can be prepared by combining one or more BAL samples from different subjects, one or more sputum samples from different subjects, or a combination thereof. Each of the BAL and sputum samples should be free (i.e., negative) of Acinetobacter species to provide a clean background for testing. After step 204, a spiked pooled sample is prepared by diluting the titer control from step 201 in the product of step 204. In this example, the dilution is prepared at a ratio of 1 part titer control to 20 parts of the pooled BAL sample or the pooled sputum sample. Method 200 can further include a step 206 of transferring a portion of the primary sample from step 205 to a secondary tube. In the next step 207 of method 200, the secondary tubes obtained from one or both of step 203 and step 206 are loaded onto a high-throughput PCR system and processed using the system. The high-throughput PCR system used in this example is the 6800 instrument. In the next step 208, the result is determined based on the information derived from step 207. In this example, the result is a qualitative determination result. Possible results include the sample being positive for CRAB, negative for CRAB, and the result being indeterminate.

[0109] Go to Figure 5B and Figure 5C , which shows the spiked pooled sample for BAL ( Figure 5B ) or the spiked pooled sample for sputum ( Figure 5C) Execution result of method 200 according to this example. It is worth noting that at all tested Acinetobacter baumannii concentrations, for each of the combined BAL and sputum samples, a difference in Ct was observed between the results measured in the first channel (gyrB) and the results measured in the second channel (internal control).

[0110] Reference Figure 6 , for the BC sample type, the complete CRAB assay workflow (method 300) was tested using a negative VERSATREK TM blood culture bottle medium and potassium dihydrogen ethylenediaminetetraacetate (K2-EDTA) whole blood (WB), and a control containing a pure titer of Acinetobacter baumannii culture was spiked. This automated prototype assay can be used to screen for ACB / CRAB from LRTI and BSI samples.

[0111] Specifically, method 300 includes a first step 301 of collecting a whole blood sample from a subject in a collection tube containing EDTA. In the next step 302, a portion of the EDTA whole blood sample is transferred or otherwise inoculated into a blood culture bottle. In this example, 5 mL of whole blood was transferred to a 40 mL VERSATREK TM blood culture bottle. The next step 303 includes incubating or otherwise treating the blood culture bottle under suitable conditions to provide an initial indication in step 304 as to whether the primary sample is positive or negative for BSI. In the case where the blood culture bottle result is negative, the next step 305 of method 300 includes transferring a portion of the sample from the treated blood culture bottle to a first tube containing a dilution medium. In this example, 0.1 mL of negative or spiked blood culture was transferred to a first tube containing 1 mL of CPM. In the next step 306 of method 300, a portion of the material from the first tube is transferred to a secondary tube. In this example, at least 0.6 mL of the material from the first tube was transferred to the secondary tube.

[0112] Continue to refer to Figure 6, the method 300 for detecting CRAB according to this example includes a step 308 of preparing a titer control containing Acinetobacter baumannii at a known concentration (e.g., CFU / mL). In this example, a commercially available titer control of Acinetobacter baumannii was used. In the next step 309, a spiked matrix sample is prepared by diluting the titer control from step 308 in a portion of the negative whole blood sample obtained from step 304. In this example, the mixture is prepared by transferring 0.1 mL of the negative or spiked blood culture to 1 mL of CPM. In the next step 310, a portion of the spiked sample from step 308 is transferred to a secondary tube. In one aspect, a swab or other similar tool can be used to transfer a portion of the spiked sample. In this example, at least 0.6 mL of the material from the first tube in step 309 is transferred to the second tube in step 310.

[0113] In the next step 307, the secondary tube obtained from either or both of step 306 and step 310 is loaded onto a high-throughput PCR system and processed using the system. The high-throughput PCR system used in this example is the 6800 instrument from ROCHE. In the next step 311, the result is determined based on the information derived from step 307. In one aspect, the result is a qualitative determination result. Possible results include the sample being positive for CRAB, negative for CRAB, and the result being inconclusive. The performance of the CRAB assay for the negative and spiked blood culture samples in CPM (BC-WB:CPM) is shown in Table 8.

[0114] Table 8

[0115]

Claims

1. A method for detecting Acinetobacter baumannii with a carbapenem resistance mechanism in a sample, the method comprising: - performing an amplification step, the amplification step comprising contacting the sample with a set of gyrB forward and reverse primers, a set of blaOXA-23-like forward and reverse primers, a set of blaOXA-24-like forward and reverse primers, a set of blaOXA-58-like forward and reverse primers, and a set of blaNDM forward and reverse primers to produce amplification products if any of these target genes are present in the sample; - performing a hybridization step, the hybridization step comprising contacting the amplification products with one or more detectable gyrB probes, one or more detectable blaOXA-23-like probes, one or more detectable blaOXA-24-like probes, one or more detectable blaOXA-58-like probes, and one or more detectable blaNDM probes; and - detecting the presence or absence of the amplification products, wherein the presence of the amplification products indicates the presence of Acinetobacter baumannii and / or a carbapenem resistance mechanism in the sample, and wherein the absence of the amplification products indicates the absence of Acinetobacter baumannii and / or a carbapenem resistance mechanism in the sample.

2. The method according to claim 1, wherein a set of gyrB primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:1, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:2, and the detectable gyrB probe comprises the nucleic acid sequence of SEQ ID NO:3 or its complementary sequence; and / or wherein a set of blaOXA-23-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:4, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:5, and the detectable blaOXA-23-like probe comprises the nucleic acid sequence of SEQ ID NO:6 or its complementary sequence; and / or wherein a set of blaOXA-24-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:7, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:8, and the detectable blaOXA-24-like probe comprises the nucleic acid sequence of SEQ ID NO:9 or its complementary sequence; and / or wherein a set of blaOXA-58-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:10, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:11, and the detectable blaOXA-58-like probe comprises the nucleic acid sequence of SEQ ID NO:12 or its complementary sequence; and / or wherein a set of blaNDM primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:13, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:14, and the detectable blaNDM probe comprises the nucleic acid sequence of SEQ ID NO:15 or its complementary sequence.

3. The method according to any one of claims 1 to 2, wherein the amplification step employs a polymerase having 5' to 3' nuclease activity.

4. The method according to any one of claims 1 to 3, wherein: - The hybridization step comprises contacting the amplification product with a detectable probe, the detectable probe being labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; And - The detection step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the probe, wherein the presence or absence of fluorescent FRET indicates the presence or absence in the sample.

5. The method according to claim 4, wherein the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are no more than 8 nucleotides apart from each other on the probe.

6. The method according to any one of claims 4 to 5, wherein the acceptor fluorescent moiety is a quencher.

7. The method according to any one of claims 1 to 6, wherein detecting the presence or absence of the amplification product further comprises: - Detect the presence or absence of the amplification product of gyrB in the first detection channel; - Detect the presence or absence of the amplification product of blaOXA-23-like allele, blaOXA-24-like allele, and / or blaOXA-58-like allele in the second detection channel; and - Detect the presence or absence of the amplification product of blaNDM in the third detection channel.

8. The method according to claim 7, wherein the detectable gyrB probe comprises a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety, wherein each of the one or more detectable blaOXA-23-like probes, the one or more detectable blaOXA-24-like probes, and the one or more detectable blaOXA-58-like probes comprises a second donor fluorescent moiety and a corresponding second acceptor fluorescent moiety, and wherein the one or more detectable blaNDM probes comprise a third donor fluorescent moiety and a corresponding third acceptor fluorescent moiety.

9. The method according to claim 8, wherein the first donor fluorescent moiety is HEX, the second donor fluorescent moiety is FAM, and the third donor fluorescent moiety is JA270.

10. An oligonucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 to 15.

11. A kit for detecting one or more nucleic acids of Acinetobacter baumannii and carbapenem resistance mechanisms, the kit comprising: - A set of Acinetobacter baumannii gyrB gene primers specific for the amplification of the gyrB gene, and one or more detectable gyrB probes specific for the detection of the gyrB gene amplification product; - A set of blaOXA-23-like gene primers specific for the amplification of the blaOXA-23-like gene, and one or more detectable blaOXA-23-like probes specific for the detection of the blaOXA-23-like gene amplification product; - A set of blaOXA-24-like gene primers specific for the amplification of the blaOXA-24-like gene, and one or more detectable blaOXA-24-like probes specific for the detection of the blaOXA-24-like gene amplification product; - A set of blaOXA-58-like gene primers specific for the amplification of the blaOXA-58-like gene, and one or more detectable blaOXA-58-like probes specific for the detection of the blaOXA-58-like gene amplification product; and - A set of blaNDM gene primers specific for the amplification of the blaNDM gene, and one or more detectable blaNDM probes specific for the detection of the blaNDM gene amplification product.

12. The kit according to claim 11, wherein the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety.

13. The kit according to claim 12, wherein the acceptor fluorescent moiety is a quencher.

14. The kit according to any one of claims 11 to 13, wherein a set of gyrB primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:1, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:2, and the detectable gyrB probe comprises the nucleic acid sequence of SEQ ID NO:3 or its complementary sequence; and / or wherein a set of blaOXA-23-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:4, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:5, and the detectable blaOXA-23-like probe comprises the nucleic acid sequence of SEQ ID NO:6 or its complementary sequence; and / or wherein a set of blaOXA-24-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:7, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:8, and the detectable blaOXA-24-like probe comprises the nucleic acid sequence of SEQ ID NO:9 or its complementary sequence; and / or wherein a set of blaOXA-58-like primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:10, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:11, and the detectable blaOXA-58-like probe comprises the nucleic acid sequence of SEQ ID NO:12 or its complementary sequence; and / or wherein a set of blaNDM primers comprises a forward primer and a reverse primer, the forward primer comprises the nucleic acid sequence of SEQ ID NO:13, the reverse primer comprises the nucleic acid sequence of SEQ ID NO:14, and the detectable blaNDM probe comprises the nucleic acid sequence of SEQ ID NO:15 or its complementary sequence.

15. The kit according to any one of claims 11 to 14, wherein the detectable gyrB probe comprises a first donor fluorophore and a corresponding first acceptor fluorophore, each of the one or more detectable blaOXA-23-like probes, the one or more detectable blaOXA-24-like probes, and the one or more detectable blaOXA-58-like probes comprises a second donor fluorophore and a corresponding second acceptor fluorophore, and wherein the one or more detectable blaNDM probes comprises a third donor fluorophore and a corresponding third acceptor fluorophore.

16. The kit according to claim 15, wherein the first donor fluorophore is HEX, the second donor fluorophore is FAM, and the third donor fluorophore is JA270.

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