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

Abstract

The present invention relates to compositions and methods for detecting Acinetobacter calcoaceticus-Acinetobacter baumannii complex species and identifying the most common carbapenemases found in carbapenem-resistant Acinetobacter calcoaceticus baumannii (CRAB), such as blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM-like, by multiplex real-time PCR assays.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is based on and claims priority to U.S. Provisional Patent Application No. 63 / 435,267, filed December 25, 2022, which is incorporated herein by reference in its entirety.

[0002] Reference sequence listing This application contains a Sequence Listing that has been submitted as an electronic text file entitled "P38059-WO_Seq_Listing," which is 6,025 bytes in size and created on December 5, 2023. The information contained in this electronic file is hereby incorporated by reference in its entirety in accordance with 37 CFR § 1.52(e)(5).

[0003] 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 technology]

[0004] Carbapenem-resistant Acinetobacter calcoaceticus baumannii (CRAB) is increasing in prevalence and has few treatment options, making it an urgent threat to public health according to the U.S. Centers for Disease Control and Prevention (CDC). CRAB is also a global priority according to the World Health Organization (WHO). Therefore, the need for novel treatments for Acinetobacter baumannii (A. baumannii) and frequent surveillance and preventive care activities are top medical priorities.

[0005] The development of new antibiotics targeting CRAB is an urgent unmet medical need that pharmaceutical companies are addressing. A key challenge for the pathogen-focused randomized controlled trials planned by pharmaceutical companies is the accurate and rapid screening of eligible patients. While molecular methods may be used 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 specimens for traditional microbiological culture are bronchoalveolar lavage (BAL) fluid and sputum. However, routine blood cultures (BCs) are also recommended by the Infectious Diseases Society of America (IDSA) and American Thoracic Society (ATS) guidelines for healthcare-associated pneumonia due to the recognized greater risk of bacteremia, especially for multidrug-resistant organisms. Traditional microbiological methods recommended for diagnosing pneumonia often require at least 48–72 hours to obtain complete identification and susceptibility results by culture. Furthermore, because many respiratory pathogens can be found as components of the normal microbiota, the relative abundance of pathogens relative to commensal organisms must be determined for LRTI specimens by quantitative or semiquantitative culture. Improved approaches to rapid diagnosis of LRTIs may include molecular methods such as quantitative real-time polymerase chain reaction (qRT-PCR) that target specific genes for the most common carbapenem resistance mechanisms found in the Acinetobacter calcoaceticus-baumannii (ACB) complex and CRAB. Currently, there are multiple FDA-approved syndromic panels for detecting and identifying the most common pathogens and antimicrobial resistance (AMR) markers from LRTI specimens (UNYVERO LRT Panel and BIOFIRE FILMARRAY PN) and positive BC specimens (BIOFIRE FILMARRAY BCID2, UNYVERO BCU, LUMINEX VERIGENE BC-GP / BC-GN, and GENMARK EPLEX BCID-GP / BCID-GN / BCID-FP).Only two syndromic panels (UNYVERO LRT and VERIGENE BC-GN) target the most common carbapenemases found in CRAB (OXA-23-like, OXA-24-like, OXA-58-like), but neither of them has a specific interpretation for CRAB.

[0006] Rapid molecular screening of CRAB LRTI specimens or positive BC specimens could facilitate earlier patient enrollment, reduce enrollment numbers, reduce the confounding effect of prior antibiotic treatment, and lower overall clinical trial costs / time for pathogen-focused clinical trials. Therefore, there is an urgent need to develop molecular assays for rapid detection of CRAB directly from BAL-like and / or sputum-like specimens and positive blood cultures obtained from patients with LRTI, preferably in an automated, high-throughput platform. Summary of the Invention

[0007] Certain aspects of the present invention relate to methods for rapidly detecting the presence or absence of the most common carbapenemases found in ACB complex species and CRAB in biological or non-biological samples. This is accomplished, for example, by multiplexed detection of the A. baumannii gyrB gene, blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like alleles of the OXA carbapenemase gene, and blaNDM-like carbapenemase gene by real-time polymerase chain reaction in a single test tube. Embodiments include methods for detecting the gyrB gene and carbapenemase genes, which involve performing at least one cycling step, which may include an amplification step and a hybridization step. Furthermore, embodiments include primers, probes, and kits designed for single-tube detection of the A. baumannii gyrB gene, blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like alleles of the OXA carbapenemase gene, and the blaNDM carbapenemase gene. Detection methods have been designed to target these genes, allowing the presence of A. baumannii and mechanisms of carbapenem resistance to be detected in a single test.

[0008] In one aspect, a method for detecting A. baumannii having a carbapenem resistance mechanism in a sample is provided, comprising: performing an 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 generate amplification product(s) if any of these target genes are present in the sample; and identifying the amplification product(s) as one or more detectable gyrB promoters. and detecting the presence or absence of amplification product(s), wherein the presence of the amplification product(s) indicates the presence of A. baumannii and / or carbapenem resistance mechanisms in the sample, and the absence of the amplification product(s) indicates the absence of A. baumannii and / or carbapenem resistance mechanisms in the sample. In one embodiment, the gyrB primer set comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 1 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 2, and the detectable gyrB probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 3, or a complement thereof. In one embodiment, the set of blaOXA-23-like primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 5, and the detectable blaOXA-23-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 6, or its complement. In one embodiment, the set of blaOXA-24-like primers comprises or consists of the nucleic acid sequence of SEQ ID NO: 7 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 8, and the detectable blaOXA-24-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, or its complement.In one embodiment, the set of blaOXA-58-like primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 10 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 11, and the detectable blaOXA-58-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 12, or its complement. In one embodiment, the set of blaNDM primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 13 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 14, and the detectable blaNDM probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 15, or its complement. In one embodiment, amplification can use a polymerase enzyme with 5' to 3' nuclease activity. In some embodiments of this method, the hybridizing step involves contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, and the detecting step involves detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor fluorescent moiety of the probe, where the presence or absence of fluorescent FRET indicates the presence or absence of the target molecule in the sample. In some embodiments, the first and second fluorescent moieties can be within 8 nucleotides of each other along the length of the probe. According to this method, the second fluorescent moiety on the probe can be a quencher. In some embodiments, the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within 8 nucleotides of each other on the probe. As used herein, the acceptor fluorescent moiety can be a quencher. In some embodiments, detecting the presence or absence of the amplification product(s) further comprises detecting the presence or absence of an amplification product of gyrB in a first optical detection channel, detecting the presence or absence of an amplification product(s) of a blaOXA-23-like allele, a blaOXA-24-like allele, and / or a blaOXA-58-like allele in a second optical detection channel, and detecting the presence or absence of an amplification product of blaNDM in a third optical detection channel.In certain embodiments, the detectable gyrB probe comprises a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety; 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 each comprise a second donor fluorescent moiety and a corresponding second acceptor fluorescent moiety; and one or more detectable blaNDM probes comprise a third donor fluorescent moiety and a corresponding third acceptor fluorescent moiety. In other embodiments, the gyrB, blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and / or blaNDM probes comprise nucleic acid sequences that enable secondary structure formation. Formation of such secondary structures generally results in spatial proximity between the first and second fluorescent moieties. According to this method, the second fluorescent moiety on the probe can be a quencher. In some embodiments, any one or more of the oligonucleotide primers and / or probes used in the methods comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides.

[0009] In another aspect, an oligonucleotide is provided that comprises or consists of a nucleotide sequence selected from SEQ ID NOs: 1-15, or its complement, and has 100 or fewer nucleotides. Additionally, the present disclosure provides an oligonucleotide comprising a nucleic acid having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90%, or 95%) to one of SEQ ID NOs: 1-15, or its complement, and has 100 or fewer nucleotides. Generally, the oligonucleotides disclosed herein can be primer nucleic acids, probe nucleic acids, etc. in these embodiments. 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 compared to unmodified nucleotides. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation. A "conservatively modified variation" or simply "conservative variation" of a particular nucleic acid sequence refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to an essentially identical sequence. 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 percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence are "conservatively modified variations," where the change results 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.

[0010] In a further aspect, the present invention provides a kit for detecting one or more nucleic acids of A. baumannii and carbapenem resistance mechanisms, the kit comprising a set of Acinetobacter baumannii gyrB gene primers specific for amplification of the gyrB gene and one or more detectable gyrB probes specific for detection of the gyrB gene amplification products, a set of blaOXA-23-like gene primers specific for amplification of a blaOXA-23-like gene and one or more detectable blaOXA-23-like probes specific for detection of the blaOXA-23-like gene amplification products, and a set of blaOXA-24-like gene primers specific for amplification of a blaOXA-24-like gene and one or more detectable blaOXA-24-like probes specific for detection of the blaOXA-24-like gene amplification products. The detection method includes at least a set of specific blaOXA-24-like gene primers and one or more detectable blaOXA-24-like probes specific for detecting blaOXA-24-like gene amplification products, a set of blaOXA-58-like gene primers specific for amplification of blaOXA-58-like genes and one or more detectable blaOXA-58-like probes specific for detecting blaOXA-58-like gene amplification products, and a set of blaNDM gene primers specific for amplification of blaNDM genes and one or more detectable blaNDM probes specific for detecting blaNDM gene amplification products.In some embodiments, the kit includes a plurality of sets of A. baumannii gyrB gene primers specific for amplification of the gyrB gene and one or more detectable gyrB probes specific for detection of gyrB gene amplification products, a plurality of sets of blaOXA-23-like gene primers specific for amplification of a blaOXA-23-like gene, and one or more detectable blaOXA-23-like probes specific for detection of a blaOXA-23-like gene amplification product and a plurality of sets of blaOXA-24-like gene primers specific for amplification of a blaOXA-24-like gene. and one or more detectable blaOXA-24-like probes specific for detecting blaOXA-24-like gene amplification products, a plurality of sets of blaOXA-58-like gene primers specific for amplifying blaOXA-58-like genes and one or more detectable blaOXA-58-like probes specific for detecting blaOXA-58-like gene amplification products, a plurality of sets of blaNDM gene primers specific for amplifying blaNDM genes and one or more detectable blaNDM probes specific for detecting blaNDM gene amplification products. In one embodiment, the set of gyrB primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 1 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 2, and the detectable gyrB probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 3, or a complement thereof. In one embodiment, the set of blaOXA-23-like primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 5, and the detectable blaOXA-23-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 6, or its complement. In one embodiment, the set of blaOXA-24-like primers comprises or consists of the nucleic acid sequence of SEQ ID NO: 7 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 8, and the detectable blaOXA-24-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, or its complement.In one embodiment, the set of blaOXA-58-like primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 10 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 11, and the detectable blaOXA-58-like probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 12, or its complement. In one embodiment, the set of blaNDM primers comprises or consists of a forward primer comprising the nucleic acid sequence of SEQ ID NO: 13 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 14, and the detectable blaNDM probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 15, or its complement. In some embodiments, the detectable probe comprises a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. In certain embodiments, the acceptor fluorescent moiety is a quencher. In some embodiments, the detectable gyrB probe comprises a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety, 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 the one or more detectable blaNDM probes comprises a third donor fluorescent moiety and a corresponding third acceptor fluorescent moiety. In certain embodiments, the first, second, and third donor fluorescent moieties are different from one another and selected from the group consisting of HEX (hexachloro-fluorescein), FAM (6-carboxy-fluorescein), 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-azonia-pentacene perchlorate). In certain embodiments, the first donor fluorescent moiety is HEX, the second donor fluorescent moiety is FAM, and the third donor fluorescent moiety is JA270. In one embodiment, the kit may include probes already labeled with donor and corresponding acceptor fluorescent moieties, or may include fluorogenic moieties for labeling the probes.The kit may also include nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase. The kit may also include a package insert and instructions for using the primers, probes, and fluorescent moieties to detect the presence or absence of the gyrB gene and / or the bla gene in a sample.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this subject, suitable methods and materials are described below.In addition, materials, methods, and examples are only illustrative and are not intended to be limiting.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0012] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0013] [Figure 1A]This study demonstrates the inclusiveness and exclusiveness of the CRAB multiplex assay using a high-throughput PCR system workflow. Multiple ACB strains (inclusive species) were tested at 10 colony-forming units per milliliter (CFU / mL), and other related carbapenem-resistant species (exclusive species) were tested at 10 CFU / mL in the presence of 130 ng / reaction of human genomic DNA. The inclusive species (n=18) included A. baumannii (n=10), A. nosocomialis (n=2), A. pittii (n=2), and A. calcoaceticus (n=1). The exclusive species (n = 18) were C. freundii, Escherichia coli, K. pneumoniae, K. oxytoca, P. mirabilis, S. aureus, S. pneumoniae, C. koseri, E. cloacae, and K. aerogenes. The inclusive species included K. aerogenes, P. aeruginosa, S. maltophilia, S. agalactiae, S. pyogenes, E. faecium, E. faecalis, H. influenzae, and S. marcescens. Figure 1A shows PCR amplification curves for representative inclusive species compared to each of the exclusive species, with no amplification observed for the exclusive species. [Figure 1B] Figure 1B shows the PCR amplification curves for each of the global species compared to the no-template control. [Figure 2A]Figure 2A provides a graphical representation of two interpretations of the CRAB multiplex assay of the present invention for distinguishing pathogenic versus commensal ACB in LRTI samples. Figure 2A shows PCR amplification curves for primers and probes targeting gyrB in samples containing titered A. baumannii controls at concentrations of 1 x 10 CFU / mL and 1 x 10 CFU / mL. The cycle threshold (Ct) cutoff is shown as a vertical dashed line. [Figure 2B] Figure 2B shows PCR amplification data for primers and probes targeting gyrB measured in the first detection channel and an internal control measured in the second detection channel in samples containing titered A. baumannii controls at concentrations of 1 x 10 CFU / mL, 5 x 10 CFU / mL, 1 x 10 CFU / mL, and 5 x 10 CFU / mL. The measured Ct values ​​and the difference in Ct values ​​between the two detection channels (ΔCt) are shown for each of the concentrations tested. [Figure 3] We demonstrate the performance of a prototype three-channel (JA270, HEX, and FAM) CRAB multiplex assay on samples containing one of five different strains of A. baumannii encoding one or more targets selected from gyrB (detected in the HEX channel), blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like (detected in the FAM channel), and blaNDM (detected in the JA270 channel). Relative fluorescence intensity (RFI) and Ct values ​​are reported for reactions performed in two different PCR media (CPM and MIS). CPM samples were tested in 850 μL sample volumes 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, and MIS samples were tested in 400 μL sample volumes 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 composition of targets present in each sample tested is listed in Table 7. [Figure 4]Provides a graphical representation of the CRAB multiplex assay workflow in cases of suspected hospital-acquired bacterial pneumonia (HABP), ventilator-associated bacterial pneumonia (VABP), or bloodstream infection (BSI) caused by CRAB. [Figure 5A] A complete assay diagram (Figure 5A) is shown, as well as data for CRAB spiked into either sample diluent (CPM) clean system or pooled bronchoalveolar lavage (BAL) and / or sputum (SPU) specimens (Figure 5B and Figure 5C). [Figure 5B] Figure 5B shows assay performance using A. baumannii titration controls spiked into various concentrations of negative BAL matrix. [Figure 5C] Figure 5C shows the assay performance using A. baumannii titration controls spiked into various concentrations of negative SPU matrix. [Figure 6] A complete assay workflow diagram for preliminary testing is shown, using negative whole blood (WB) incubated in commercially available blood culture bottles as the negative matrix and commercially available A. baumannii titration controls spiked to three final CFU / mL concentrations (1 x 10, 1 x 10, 1 x 10). Data for CRAB assay performance are further presented in Table 8. DETAILED DESCRIPTION OF THE INVENTION

[0014] 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 A. baumannii). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors (e.g., MgCl and / or KCl) for optimal activity of the polymerase enzyme.

[0015] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, but also a modified oligonucleotide that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which a further "nucleotide" can be attached by a template-dependent DNA polymerase that establishes a 3' to 5' phosphodiester bond, thereby using a deoxynucleoside triphosphate and releasing pyrophosphate. Thus, there is no fundamental difference between a "primer," an "oligonucleotide," or a "probe," except perhaps for their intended function.

[0016] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0017] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0018] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme catalyzes the formation of primer extension products complementary to the template and does not irreversibly denature when exposed to high temperatures for the time required to cause denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in 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. Nevertheless, non-thermostable polymerases can also be used in polymerase chain reaction (PCR) assays if the enzyme is replenished.

[0019] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0020] The term "extension" or "elongation" when used with respect to nucleic acids refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, such as a polymerase, which typically adds a nucleotide to the 3' end of the nucleic acid.

[0021] The term "identical" or percent "identity" in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides when compared or aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool," J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search," Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server," Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Res. 7:649-656, each of which is incorporated herein by reference.

[0022] "Modified nucleotide" in the context of oligonucleotides refers to an alteration in which at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide that provides desired properties to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2'-0-methylribo-U, 2'-0-methylribo-C, N4-ethyl-dC, N6-methyl-dA, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, modified nucleotide substitutions modify the melting temperature (Tm) of oligonucleotides compared to the melting temperature of corresponding unmodified oligonucleotides. To further explain, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer-dimer formation, etc.), increase the yield of intended target amplicons, etc. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is incorporated herein by reference.

[0023] A "variant" of a given oligonucleotide may contain one or more nucleotide additions, deletions, or substitutions, such as one or more nucleotide additions, deletions, or substitutions at the 5' and / or 3' ends of the respective sequences of the oligonucleotide. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modifications but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide," thereby resulting in a "modified nucleotide." A natural base in a "nucleotide" may also be replaced, for example, by 7-desazapurine, thereby similarly resulting in a "modified nucleotide." The terms "modified nucleotide" and "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analogue") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogues").

[0024] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules, such as those encoding the nucleic acid sequences of the gyrB gene or the blaOXA and blaNDM genes, can be designed using computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, CO). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, appropriate size amplification products for ease of detection (e.g., by electrophoresis), similar melting temperatures of the members of the pair of primers, and the length of each primer (i.e., primers must be long enough to anneal and initiate synthesis with sequence specificity, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 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).

[0025] In addition to a set of primers, the method may use one or more probes to detect the presence or absence of the ACB complex and carbapenem resistance mechanisms. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that, by design or selection, contains a specific nucleotide sequence that allows it to specifically (i.e., preferentially) hybridize to a "target nucleic acid," in this example, the Abi gyrB (target) nucleic acid, and / or the blaOXA-23-like, blaOXA-24-like, and bla-OXA-58-like alleles of OXA carbapenemase, and the blaNDM carbapenemase (target) nucleic acid, under a defined, predetermined stringency. The "probe" may also be referred to as a "detection probe," which means that it detects the target nucleic acid.

[0026] In some embodiments, the probes described can be labeled with at least one fluorescent label. In one embodiment, the probes can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor fluorescent moiety, such as a quencher.

[0027] The design of oligonucleotides used as probes can be performed similarly to the design of primers. In embodiments, a single probe or a pair of probes may be used for detection of amplification products. Depending on the embodiment, the probe(s) used may contain at least one label and / or at least one quencher moiety. Like primers, probes typically have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is compromised during synthesis. Oligonucleotide probes are generally 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0028] The constructs may include vectors each containing one of the primer and probe nucleic acid molecules (e.g., SEQ ID NOS: 1-15). The constructs may be used, for example, as a control template nucleic acid molecule. Suitable vectors for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. Target nucleic acid molecules may be obtained, for example, by chemical synthesis, direct gene cloning, or PCR amplification.

[0029] Constructs suitable for use in the present methods typically contain, in addition to the target nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-15), a sequence 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, host cell choice, replication efficiency, selectability, inducibility, and ease of recovery.

[0030] Constructs containing target nucleic acid molecules can be propagated in host cells. As used herein, the term host cell is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant, and animal cells. Prokaryotic hosts can include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris; mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells; insect cells; and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. Constructs can be introduced into host cells using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into host cells.In addition, naked DNA can be directly delivered into cells (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0031] polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). In some embodiments, useful primers include oligonucleotides that can act as initiation points for nucleic acid synthesis within the described target gene and target allele nucleic acid sequences (e.g., SEQ ID NOS: 1, 2, 4, 5, 7, 8, 10, 11, and 13, 14). Primers can be purified from restriction digests by conventional methods or produced synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, but primers can also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method for denaturing double-stranded nucleic acids is by heating.

[0032] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating nucleic acid strands involves heating the nucleic acid until it is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid will depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on reaction characteristics such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0033] If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the nucleic acid molecule. The annealing temperature is typically about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes polymerase activity, i.e., a temperature sufficient for extension of the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but not so high as to denature the extension products from their complementary templates (e.g., the temperature for extension is generally in the range of about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). The extension time can be from about 10 seconds to about 5 minutes (eg, from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0034] PCR assays can use target gene and / or allele nucleic acids such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified and can be a minor fraction of a complex mixture, such as the target nucleic acid contained in a biological sample. Target nucleic acid molecules can be extracted from biological samples by routine techniques, such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, including plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0035] Oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl, 0.001% (w / v) gelatin, 0.5–1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150–320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0036] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The strand separation, annealing, and extension steps can be repeated as many times as necessary to generate the desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficiently for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or even 100 times.

[0037] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are positioned within a certain distance from each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule containing a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0038] In one example, an oligonucleotide probe may contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent, that dissipates the transferred energy in a form other than light.When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, such that the fluorescence 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, for example, by the 5' to 3' nuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched.Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785.A commonly used donor-acceptor pair includes the FAM-TAMRA pair.Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, MI).

[0039] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to an amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0040] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor fluorescence emission in a specific range), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, a fiber optic light source, or other high-intensity light source appropriately filtered for excitation of the desired range.

[0041] As used herein with respect to a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, "corresponding" refers to an acceptor fluorescent moiety having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety, thereby allowing efficient non-radiative energy transfer between them.

[0042] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) highly efficient Förster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of emission as far as possible into the red portion of the visible spectrum (>600 nm), and (d) a shift of emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its excitation maximum near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0043] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include, depending on the donor fluorescent moiety used, LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium). Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0044] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Typically, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to a specific nucleotide base and for attaching the fluorescent moiety to the linker arm.

[0045] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-aminophosphoramidites available from ABI (Foster City, CA) or Glen Research (Sterling, VA)) to produce, for example, LC Red 640-labeled oligonucleotides. Linkers frequently used to couple donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, MA)), amide-linkers (derived from fluorescein-NHS-esters, e.g., CX-Fluorescein-CPG from BioGenex (San Ramon, CA)), or 3'-amino-CPG, which requires coupling of the fluorescein-NHS-ester after oligonucleotide synthesis.

[0046] Detection of target genes and alleles in CRAB The present disclosure provides a method for detecting the presence or absence of the A. baumannii gyrB gene, blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like alleles of the OXA carbapenemase gene, and the blaNDM-like NDM carbapenemase gene in a biological or non-biological sample. The provided method avoids problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step involving amplifying a portion of a target nucleic acid molecule from the sample using multiple pairs of target primers, and a FRET detection step. The multiple cycling steps are preferably performed in a thermal cycler. The target primers and probes can be used to perform the method for detecting the presence of the 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.

[0047] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and therefore the presence or absence of CRAB. TaqMan® technology utilizes, for example, a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is subsequently degraded during the extension phase by, for example, the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of CRAB in a sample.

[0048] Molecular beacons combined with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and fluorescent labels are typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows secondary structure formation (e.g., a hairpin). As a result of the formation of the secondary structure within the probe, both fluorescent moieties are spatially close together when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted, separating the fluorescent moieties from each other so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0049] Another common format of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize close to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, such as fluorescein, is excited at 470 nm by the LightCycler® instrument's light source. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, such as LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the LightCycler® instrument's optical detection system. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules. When amplification of the target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable FRET-based signal between the members of the probe pair.

[0050] Generally, the presence of FRET indicates the presence of target sequence in sample, and the absence of FRET indicates the absence of target sequence in sample.However, insufficient specimen collection, delayed transport, improper transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shaft) are all conditions that can affect the success and / or accuracy of test results.Using the method disclosed herein, for example, the detection of FRET within 45 cycle steps indicates CRAB infection.

[0051] The representative biological sample that can be used in the implementation of this method includes, but is not limited to, skin swab, nasal swab, wound swab, blood culture, skin and soft tissue infection.The collection and preservation method of biological sample is known to those skilled in the art.Biological sample can be processed (for example, by nucleic acid extraction method and / or kit known in the art) to release target gene nucleic acid, or in some cases, biological sample can be directly contacted with PCR reaction components and suitable oligonucleotide.

[0052] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA duplex separates into single strands. The melting temperature of DNA depends primarily on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature of the probe from the amplification product can confirm the presence or absence of the target sequence in the sample.

[0053] Control samples can be cycled during each thermal cycler run as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the listed target gene) using, for example, control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample using the same primers and probes used to detect the intended target. Such controls are indicators of the success or failure of the amplification, hybridization, and / or FRET reaction. Each thermal cycler run can also include a negative control, for example, lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false-positive signals. Thus, control reactions can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, and the ability of probes to hybridize with sequence specificity and undergo FRET.

[0054] In one embodiment, the method includes a step of avoiding contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase to reduce or eliminate contamination between one thermal cycler run and the next are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313.

[0055] The method can be implemented using conventional PCR methods combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0056] The LightCycler® can be operated using a PC workstation and utilizes the Windows® NT operating system. Signals from samples are acquired as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The generated data can be saved for further analysis.

[0057] As an alternative to FRET, amplification products can be detected using double-stranded DNA-binding dyes, such as fluorescent DNA-binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). Upon interaction with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescent signal after excitation with light of a suitable wavelength. Double-stranded DNA-binding dyes, such as nucleic acid intercalating dyes, can also be used. When using double-stranded DNA-binding dyes, a melting curve analysis is usually performed to confirm the presence of amplification products.

[0058] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0059] Manufactured Products / Kits Embodiments of the present disclosure further provide articles of manufacture or kits for detecting the A. baumannii gyrB gene, blaOXA-23-like, blaOXA-24-like, and blaOXA-58-like alleles of OXA carbapenemase genes, and blaNDM-like NDM carbapenemase genes (i.e., genes and alleles involved in CRAB). The articles of manufacture may include primers and probes used to detect CRAB, along with suitable packaging materials. Representative primers and probes for detecting CRAB are capable of hybridizing to target nucleic acid molecules. Furthermore, the kits may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as solid supports, buffers, enzymes, and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to target nucleic acid molecules are provided.

[0060] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or the probes provided with the kit may be labeled. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0061] The article of manufacture may also include a package insert or packaging label that describes instructions for using the target primers and probes to detect CRAB in a sample. The article of manufacture may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents for preventing contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0062] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. [Example]

[0063] 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 is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0064] Example 1 Example 1 provides, in part, primer and probe sequences for use in accordance with the present disclosure. Table 1 provides primers and probes used in multiplex PCR assays for the detection of ACB complex and carbapenem resistance mechanisms.

[0065] [Table 1]

[0066] Example 2 Example 2 partially describes PCR experimental conditions according to the present disclosure. Real-time PCR detection of target genes was performed using either the cobas® 4800 system or the cobas® 6800 / 8800 system platform (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of amplification reagents are shown in Table 2.

[0067] [Table 2]

[0068] [Table 3]

[0069] The pre-PCR program included incubations at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. The three-temperature incubations combined the beneficial effects of suppressing the formation of RNA secondary structures at higher temperatures, thereby resulting in more efficient transcription, while allowing for transcription of even slightly mismatched target sequences (e.g., genetic variants of an organism) at lower temperatures. PCR cycling was divided into two runs, each using a single-stage setup (combined annealing and extension). The first five cycles at 55°C increased inclusiveness by preamplifying the slightly mismatched target sequences, while the second run (45 cycles) increased specificity by using an annealing / extension temperature of 58°C.

[0070] Example 3 Example 3 partially demonstrates the performance of a multiplex PCR assay for the detection of CRAB according to the present disclosure. A prototype multiplex PCR assay targeting the most common carbapenemases (blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM-like) found in ACB complex species and CRAB using the primer and probe sequences listed in Table 1. The most common carbapenemases found in CRAB include the previously described OXA enzymes and new deli-metallo-β-lactamase (blaNDM) enzymes (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, assay performance 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 assay linearity and dynamic range are shown in Table 4. The multiplex assay was performed in 10 2 ~10 6 It is efficient and linear in terms of copies / reaction, and no significant interference from human genomic DNA is observed.

[0071] [Table 4]

[0072] Example 4 Example 4 partially illustrates 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 involved real-time PCR detection of target genes using the cobas® 6800 / 8800 system, which has five distinct detection channels. Channel 1, not listed in Table 5, was left empty but could be utilized for the detection of additional targets (e.g., novel resistance mechanisms). Channel 2 was used to detect the primary CRAB-specific resistance mechanism (blaOXA-23 / 24 / 58-like). Channel 3 was used to detect the conserved region of the ACBgyrB gene and identify the presence of Acinetobacter spp. (also utilized to distinguish pathogens from commensals for LRTI specimens). Channel 4 was used to detect emerging CRAB resistance mechanisms (blaNDM-like) and should be correlated with channel 3 to indicate CRAB versus other carbapenem-resistant organisms (CROs). Channel 5 was used to detect the generic internal control (GIC), which is a control for sample preparation and PCR amplification used to distinguish between valid and invalid samples.

[0073] [Table 5] *Detection of an ACB gene in channel 3 indicates the presence of an ACB or CRAB result if the CRAB resistance mechanism is also detected in channels 2 and / or 4. **Detection of CRAB with NDM must be correlated between channel 3 and channel 4.

[0074] In silico analysis was performed and predicted 100% reactivity for clinically relevant ACB species and known blaOXA-23-like, blaOXA-24-like, blaOXA-58-like, and blaNDM-like variants available in the National Center for Biotechnology Information (NCBI) database. The predicted (in silico) reactivities are shown in Table 6. Assay inclusiveness for ACB species (n=15) and exclusiveness for other bacterial species commonly found in respiratory samples (n=18) were also 10. 4 and 10 7 This was confirmed based on wet-lab testing in a clean system in CFU / mL (Figure 1A and Figure 1B).

[0075] [Table 6]

[0076] Referring to Figures 2A and 2B, additional assay interpretation is required to distinguish pathogens versus commensals for LRTI specimen types using the Ct threshold for channel 3 (gyrB) or the ΔCt threshold calculated between the Ct for channel 3 (gyrB) and channel 5 (internal control). LRTI specimens can also be a challenging sample type for bacterial DNA recovery due to LRTI specimen characteristics such as heterogeneity and viscosity. With this in mind, as shown in Figure 3 and Table 7, the CRAB assay was tested with multiple sample diluents, such as microbial inactivation solution (MIS) and cobas® PCR medium (CPM), but no significant differences were observed. Therefore, the optimal sample transfer method for the LRTI specimen workflow was identified using the cobas® uniswab with CPM dilution tubes loaded directly into the workflow.

[0077] [Table 7]

[0078] Referring to FIG. 4, a 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-acquired bacterial pneumonia (VABP). In a subsequent step 102, a primary sample is obtained from the subject identified in step 101. The sample may 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 a subsequent step 103, a portion of the primary sample from step 102 is transferred to a secondary tube. In this example, the portion of the primary sample was transferred using a swab or another similar tool. Alternatively, or in addition, method 100 may include a step 104 of identifying a subject suspected of having a bloodstream infection (BSI). Following step 104, the method may include a step 105 of obtaining a primary sample from the subject in step 104 in a blood culture bottle. Step 105 may 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 of a positive blood culture bottle result, the next step 106 of method 100 involves 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 resulting from one or both of steps 103 and 106 is loaded into a high-throughput PCR system and processed therein. An example of a suitable high-throughput PCR system includes ROCHE's cobas® X800 (e.g., 4800, 5800, 6800, and 8800) series of instruments. In the next step 108, a result is determined based on the information derived from step 107. In one embodiment, the result is a qualitative assay result. Possible results include the sample being positive for CRAB, being negative for CRAB, and the result being indeterminate.

[0079] Referring to Figures 5A-C, to verify the performance of the CRAB assay, negative and spiked BAL and sputum samples were screened with a titered Acinetobacter baumannii control or CRAB strain using the cobas® uniswab transfer method shown in Figure 5A. With continued reference to Figure 5A, a method 200 for detecting CRAB according to this example includes a first step 201 of preparing a titration control containing a known concentration (e.g., CFU / mL) of Acinetobacter baumannii. In this example, a commercially available titration control of Acinetobacter baumannii was used. In a subsequent step 203, a portion of the spiked sample from step 201 is transferred to a secondary tube. In one embodiment, a portion of the spiked sample can be transferred with a swab (as in this example) or another similar tool. Alternatively or additionally, method 200 includes a step 204 of preparing a pooled matrix sample. A 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 of (i.e., negative for) Acinetobacter species to provide a clean background for testing. After step 204, a spiked pooled sample is prepared by diluting the titration control from step 201 with the product of step 204. In this example, dilutions were prepared at a ratio of 1 part titration control to 20 parts of either pooled BAL samples or pooled sputum samples. Method 200 can further include step 206, transferring a portion of the primary sample from step 205 to a secondary tube. Next in method 200, in step 207, the secondary tubes resulting from one or both of steps 203 and 206 are loaded into a high-throughput PCR system and processed using the high-throughput PCR system. The high throughput PCR system used in this example was a cobas® 6800 instrument manufactured by ROCHE. In the next step 208, a result is determined based on the information derived from step 207. In this example, the result is a qualitative assay result.Possible outcomes include the sample being positive for CRAB, being negative for CRAB, and the result being indeterminate.

[0080] 5B and 5C, results from performing method 200 according to this example are shown for both spiked pooled BAL samples (FIG. 5B) or spiked pooled sputum samples (FIG. 5C). Notably, differences in Ct for results measured in the first channel (gyrB) compared to results measured in the second channel (internal control) were observed for pooled BAL and sputum samples, respectively, at all concentrations of Acinetobacter baumannii tested.

[0081] With reference to Figure 6 for the BC sample type, the complete CRAB assay workflow (Method 300) was performed on a negative VERSATREK (商標) Blood culture bottle media and dipotassium ethylenediaminetetraacetic acid (K2-EDTA) whole blood (WB) were tested, spiked with controls containing pure titered cultures of A. baumannii. This automated prototype assay can be employed to screen ACB / CRAB from LRTI and BSI specimens.

[0082] In particular, method 300 includes a first step 301 of collecting a whole blood sample from a subject in a collection tube containing EDTA. In a 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 is transferred to a 40 mL VERSATREK (商標)The primary sample was then transferred to a blood culture bottle. The next step 303 involves 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 step 304. If a negative blood culture bottle results, the next step 305 of method 300 involves transferring a portion of the sample from the processed blood culture bottle to a first tube containing dilution media. In this example, 0.1 mL of negative or spiked blood culture was transferred to the first tube containing 1 mL of CPM. The next step 306 of method 300 involves transferring a portion of the material from the first tube to a secondary tube. In this example, at least 0.6 mL of material from the first tube was transferred to the secondary tube.

[0083] Continuing with reference to FIG. 6, the method 300 for detecting CRAB according to this example includes step 308, which involves preparing a titration control containing a known concentration (e.g., CFU / mL) of Acinetobacter baumannii. In this example, a commercially available titration control for Acinetobacter baumannii was used. In the next step 309, a spiked matrix sample is prepared by diluting the titration control from step 308 with a portion of the negative whole blood sample obtained from step 304. In this example, a mixture was prepared by transferring 0.1 mL of 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 embodiment, the portion of the spiked sample can be transferred with a swab or another similar tool. In this example, at least 0.6 mL of material from the first tube in step 309 was transferred to the secondary tube in step 310.

[0084] In the next step 307, the secondary tubes resulting from one or both of steps 306 and 310 are loaded into and processed in a high-throughput PCR system. The high-throughput PCR system used in this example was a cobas® 6800 instrument manufactured by ROCHE. In the next step 311, a result is determined based on the information derived from step 307. In one embodiment, the result is a qualitative assay result. Possible results include the sample being positive for CRAB, being negative for CRAB, and the result being indeterminate. The performance of the CRAB assay for negative and spiked blood culture samples in CPM (BC-WB:CPM) is shown in Table 8.

[0085] [Table 8]

Claims

1. 1. A method for detecting Acinetobacter baumannii having a carbapenem resistance mechanism in a sample, comprising: - carrying out an 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 generate amplification product(s) if any of these target genes are present in the sample; - carrying out a hybridization step comprising contacting said 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; - detecting the presence or absence of said amplification product(s), wherein the presence of said amplification product(s) indicates the presence of Acinetobacter baumannii and / or carbapenem resistance mechanisms in said sample, and the absence of said amplification product(s) indicates the absence of Acinetobacter baumannii and / or carbapenem resistance mechanisms in said sample; and A method comprising:

2. the set of gyrB primers comprises a forward primer comprising the nucleic acid sequence of SEQ ID NO: 1 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 2, the detectable gyrB probe comprises the nucleic acid sequence of SEQ ID NO: 3, or a complement thereof, and / or the set of blaOXA-23-like primers comprises a forward primer comprising the nucleic acid sequence of SEQ ID NO: 4 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 5, the detectable blaOXA-23-like probe comprises the nucleic acid sequence of SEQ ID NO: 6, or a complement thereof, and / or the set of blaOXA-24-like primers comprises a forward primer comprising the nucleic acid sequence of SEQ ID NO: 7 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO:

8.

2. The method of claim 1, wherein the detectable blaOXA-24-like probe comprises the nucleic acid sequence of SEQ ID NO: 9, or a complement thereof, and / or the set of blaOXA-58-like primers comprises a forward primer comprising the nucleic acid sequence of SEQ ID NO: 10 and a reverse primer comprising the nucleic acid sequence of SEQ ID NO: 11, the detectable blaOXA-58-like probe comprises the nucleic acid sequence of SEQ ID NO: 12, or a complement thereof, and / or the set of blaNDM primers comprises a forward primer comprising the nucleic acid sequence of SEQ ID NO: 13 and a reverse primer comprising 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 a complement thereof.

3. The method of any one of claims 1 to 2, wherein the amplification step uses a polymerase enzyme with 5' to 3' nuclease activity.

4. - the hybridizing step comprises contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety; and the detecting 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, the presence or absence of fluorescence FRET being indicative of the presence or absence in the sample; The method according to any one of claims 1 to 3.

5. The method of claim 4 , wherein the donor fluorescent moiety and the corresponding acceptor fluorescent moiety are within 8 nucleotides of each other on the probe.

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

7. Detecting the presence or absence of said amplification product(s), - detecting the presence or absence of a gyrB amplification product in a first detection channel; - detecting the presence or absence of amplification product(s) of a blaOXA-23-like allele, a blaOXA-24-like allele, and / or a blaOXA-58-like allele in a second detection channel; - detecting the presence or absence of an amplification product of blaNDM in a third detection channel; The method of any one of claims 1 to 6, further comprising:

8. 8. The method of claim 7, wherein the detectable gyrB probe comprises a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety, 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 each comprise a second donor fluorescent moiety and a corresponding second acceptor fluorescent moiety, and the one or more detectable blaNDM probes comprise a third donor fluorescent moiety and a corresponding third acceptor fluorescent moiety.

9. 9. The method of 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 NOs: 1-15.

11. 1. A kit for detecting Acinetobacter baumannii and one or more nucleic acids of a carbapenem resistance mechanism, 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 blaOXA-23-like genes, and one or more detectable blaOXA-23-like probes specific for the detection of blaOXA-23-like gene amplification products; a set of blaOXA-24-like gene primers specific for the amplification of a blaOXA-24-like gene, and one or more detectable blaOXA-24-like probes specific for the detection of a blaOXA-24-like gene amplification product; a set of blaOXA-58-like gene primers specific for the amplification of blaOXA-58-like genes, and one or more detectable blaOXA-58-like probes specific for the detection of blaOXA-58-like gene amplification products; 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 blaNDM gene amplification products; Includes a kit.

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

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

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

15. 15. The kit of claim 11, wherein the detectable gyrB probe comprises a first donor fluorescent moiety and a corresponding first acceptor fluorescent moiety; 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 fluorescent moiety and a corresponding second acceptor fluorescent moiety; and the one or more detectable blaNDM probes comprises a third donor fluorescent moiety and a corresponding third acceptor fluorescent moiety.

16. 16. The kit of claim 15, wherein the first donor fluorescent moiety is HEX, the second donor fluorescent moiety is FAM, and the third donor fluorescent moiety is JA270.

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