COMPOSITION, METHOD, AND SYSTEM FOR A RAPID, REAL-TIME PENTAPLEX PCR ASSAY FOR MAJOR BETA-LACTAMASE GENES

Abstract

Provided herein are a composition, system, and kit for the rea-time pentaplex amplification and detection of DNA from at least one of beta-lactamase genes Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48).

Claims

1. A composition for real-time amplification of DNA from at least one of beta-lactamase genes Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48), the composition comprising at least one of: a PCR primer pair with the sequence of SEQ. ID Nos 1-2; a PCR primer pair with the sequence of SEQ. ID Nos 4-5; a PCR primer pair with the sequence of SEQ. ID Nos 7-8; a PCR primer pair with the sequence of SEQ. ID Nos 10-11; and a PCR primer pair with the sequence of SEQ. ID Nos 13-14.

2. A composition for real-time detection of amplified DNA from at least one of beta-lactamase genes Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48), the composition comprising at least one of: a probe with the sequence of SEQ. ID No 3; a probe with the sequence of SEQ. ID No 6; a probe with the sequence of SEQ. ID No 9; a probe with the sequence of SEQ. ID No 12; and a probe with the sequence of SEQ. ID No 15.

3. The composition of claim 2 wherein the probe comprises a detectable label.

4. The composition of claim 3 wherein the detectable label is a fluorophore.

5. The composition of claim 4 wherein the detectable label is selected based on emission spectra of each fluorophore.

6. The composition of claim 4 further comprising a quencher specific to the fluorophore.

7. A method for detecting at least one of beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 in a blood sample, the method comprising: obtaining a blood sample from a subject; extracting bacterial cells from the blood sample; providing at least one PCR primer pair comprising the sequence of at least one of SEQ ID No. 1-2, SEQ ID No. 4-5, SEQ ID No. 7-8, SEQ ID No. 10-11, and SEQ ID No. 13-14; providing at least one probe comprising a detectable label and comprising the sequence of at least one of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12 and SEQ ID No. 15; and amplifying the bacterial DNA in a PCR reaction in the presence of the primer pairs and probes.

8. The method of claim 7 wherein the bacterial cells, the five primer pairs, and the five probes are combined in a single-tube pentaplex reaction.

9. The method of claim 7 further comprising reading the detectable label of the probe.

10. The method of claim 9 wherein the bacterial cells are isolated directly from blood according to U.S. Pat. No. 10,457,975.

11. The method of claim 10 wherein the subject is a human patient.

12. The method of claim 11 wherein the human patient suffers from septicemia.

13. A system for detecting at least one of beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 in a blood sample, the system comprising: at least one PCR primer pair comprising the sequence of at least one of SEQ ID No. 1-2, SEQ ID No. 4-5, SEQ ID No. 7-8, SEQ ID No. 10-11, and SEQ ID No. 13-14; and at least one probe comprising the sequence of at least one of at least one of SEQ ID No 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12 and SEQ ID No. 15.

14. The system of claim 13 wherein the probe comprises a detectable label.

15. The system of claim 14 wherein the detectable label comprises a fluorophore and wherein the probe further comprises a quencher.

16. The system of claim 15 further comprising a pentaplex reaction comprising the five primer pairs and five probes.

17. The system of claim 16 further comprising PCR reaction reagents and/or laboratory apparatus.

18. A kit for detecting at least one of beta-lactamase genes KPC, NDM, CTX, CMY, and OXA-48 in a blood sample, the kit comprising: at least one primer pair having the sequence of at least one of SEQ ID No. 1-2; SEQ ID No. 4-5, SEQ ID No. 7-8, SEQ ID No. 10-11; and SEQ ID No. 13-14; and at least one probe having the sequence of at least one of SEQ ID No. 3, SEQ ID No. 6, SEQ ID No. 9, SEQ ID No. 12, and SEQ ID No. 15.

19. The kit of claim 18 further comprising PCR reaction reagents.

20. The kit of claim 19 further comprising instructions for use.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent application as filed contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0023] FIG. 1 is a table depicting isolates used to validate the assay and resistance genes present in each isolate in accordance with the present invention;

[0024] FIG. 2 is a table depicting Pentaplex primer and probe sequences;

[0025] FIG. 3 is a table depicting r2 values and sensitivities of each gene from culture in singleplex and pentaplex, or pentaplex from blood in accordance with the present invention;

[0026] FIG. 4(a) is a set of graphs depicting sensitivity of an individual target assay for the presence of KPC from the strain 0112 in accordance with the present invention;

[0027] FIG. 4(b) is a set of graphs depicting sensitivity of an individual target assay for the presence of NDM from the strain 0119 in accordance with the present invention;

[0028] FIG. 4(c) is a set of graphs depicting sensitivity of an individual target assay for the presence of CTX from the strain 0119 in accordance with the present invention;

[0029] FIG. 4(d) is a set of graphs depicting sensitivity of an individual target assay for the presence of CMY from the strain 0119 in accordance with the present invention;

[0030] FIG. 4(e) is a set of graphs depicting sensitivity of an individual target assay for the presence of OXA-48 from the strain 0160 in accordance with the present invention;

[0031] FIG. 5(a) is a set of graphs depicting sensitivity of the pentaplex assay from bacterial culture for the presence of KPC from the strain 0112 in accordance with the present invention;

[0032] FIG. 5(b) is a set of graphs depicting sensitivity of the pentaplex assay from bacterial culture for the presence NDM from the strain 0119 in accordance with the present invention;

[0033] FIG. 5(c) is a set of graphs depicting sensitivity of the pentaplex assay from bacterial culture for the presence CTX from the strain 0119 in accordance with the present invention;

[0034] FIG. 5(d) is a set of graphs depicting sensitivity of the pentaplex assay from bacterial culture for the presence CMY from the strain 0119 in accordance with the present invention;

[0035] FIG. 5(e) is a set of graphs depicting sensitivity of the pentaplex assay for the presence of OXA-48 from the strain 0160 in accordance with the present invention;

[0036] FIG. 6 (a) is a set of graphs depicting sensitivity of pentaplex assay in bacteria derived from blood for the presence of KPC from the strain 0112 in accordance with the present invention;

[0037] FIG. 6 (b) is a set of graphs depicting sensitivity of pentaplex assay in bacteria derived from blood for the presence of NDM from the strain 0119 in accordance with the present invention;

[0038] FIG. 6 (c) is a set of graphs depicting sensitivity of pentaplex assay in bacteria derived from blood for the presence of CTX the strain 0119 in accordance with the present invention;

[0039] FIG. 6 (d) is a set of graphs depicting sensitivity of pentaplex assay in bacteria derived from blood for the presence of CMY the strain 0119 in accordance with the present invention;

[0040] FIG. 6 (e) is a set of graphs depicting sensitivity of pentaplex assay in bacteria derived from blood for the presence of OXA-48 from the strain 0160 in accordance with the present invention;

[0041] FIG. 7 is an amplification plot depicting simultaneous amplification of all five beta-lactamase gene targets in the pentaplex format in accordance with the present invention;

DETAILED DESCRIPTION OF THE INVENTION

[0042] Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0043] Furthermore, the described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, structures, materials, or operations that are known in the art are not shown or described in detail to avoid obscuring aspects of the invention.

[0044] Provided herein are primers and probes which can identify all subtypes of Klebsiella pneumoniae carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48).

[0045] Therefore, it is possible according to this invention to identify in real time the major beta-lactamase genes present in bacteria isolated directly from blood. This quantitative PCR assay was able to concurrently identify the five most common beta-lactamase genes (KPC, NDM, CTX, CMY, and OXA-48) in a single-tube reaction format with no loss of sensitivity due to multiplexing. Consequently, this assay is capable of rapid, precise identification of the major beta-lactamase genes present in bacteria taken directly from blood, requiring no blood culturing. These results can be available in under 2 hours from blood having bacterial concentrations down to 4 c.f.u. ml-.sup.1.

[0046] In the United States, most carbapenem-resistant blood infections are caused by organisms carrying either KPC or NDM; these organisms also commonly carry multiple ESBL, AmpC, or oxacillinase (OXA) genes. The most common ESBL gene present in resistant pathogens in the United States is Cefotaximase-Munich (CTX), while the most common AmpC gene is CMY. Oxacillinase-48 is a less common enzyme but is present in many antibiotic-resistant isolates and is capable of hydrolysing carbapenems and other commonly used antibiotics.

[0047] FIG. 1 (Table 1) depicts isolates used to validate assay and resistance genes present in each isolate according to the present invention. The isolates listed in Table 1 were obtained from the Centre for Disease Control Antibiotic Resistance Isolate Bank Enterobacterales Carbapenemase Diversity Panel and were used to develop and validate this assay. Stock cultures of each isolate were grown on Luria broth (LB) agar plates containing 8 μg ml-.sup.1 imipenem, then incubated at 37° C. for 24 h. Plates were stored at 4° C.

[0048] A “+” indicates the presence of a beta-lactamase gene in the given isolate. Beta-lactamase target genes were present in at least one strain of bacteria, with CMY present in seven strains, KPC and NDM in six strains, CTX in two strains and OXA-48 in one strain.

[0049] FIG. 2 (Table 2) depicts the novel primer and probe sequences used to test for each of KPC, NDM, CTX, CMY, and OXA-48 genes according to the present invention. _F indicates a forward primer; _R indicates a reverse primer; _Pro indicates a probe; BHQ1 indicates Black Hole Quencher 1; BHQ2 indicates Black Hole Quencher 2; and IBRQ, indicates Iowa Black R Quencher. The novel primer and probe sequences provided herein yield pentaplex assay sensitivities at least substantially comparable to individual assays performed for each beta-lactamase gene.

[0050] FIG. 3 (Table 3) indicates the r.sup.2 values and sensitivities of each beta lactamase gene assay from culture in singleplex and pentaplex, or pentaplex from blood according to the present invention. The bacteria-blood separation technology removes interfering elements from the blood, which allows the pentaplex qPCR assay to detect low levels of bacterial DNA targets typically present in blood samples from patients with early bacteremias. Additionally, this assay returns results within the desired 1 to 3 h range for septicemia identification and is effective in a single-tube format without losing sensitivity due to multiplexing, a common problem for many multiplex PCR assays. Using the results from this assay in conjunction with existing methods for bacterial identification and antibiotic susceptibility may help inform increasingly effective treatment options for patients presenting with septicemia.

[0051] FIGS. 4 (a), (b), (c), (d), and (e) depict a standard curve and an amplification plot for the sensitivities of individual target assays according to the present invention. Serial tenfold dilutions of CRE DNA containing the desired target genes were tested in a singleplex reaction to determine the detection limit for each of: (a) KPC, from strain 0112; (b) NDM, strain 0119; (c) CTX, strain 0119; (d) CMY, strain 0119; and (e) OXA-48, strain 0160. Individual runs with different DNA concentrations provided Ct values which were plotted against the log of the concentration of DNA to obtain the standard curve. DNA quantity was adjusted to 481 μg ml-.sup.1 for each starting concentration. The assays demonstrated similar sensitivities.

[0052] FIGS. 5 (a), (b), (c), (d), and (e) depict a standard curve and an amplification plot for sensitivities of the pentaplex assay for target genes from bacteria in culture according to the present invention. Serial tenfold dilutions of CRE DNA containing the desired target gene(s) were tested in a single tube format as a pentaplex reaction. Target genes were: (a) KPC, from strain 0112; (b) NDM, strain 0119; (c) CTX, strain 0119; (d) CMY, strain 0119; and (e) OXA-48, strain 0160. Individual runs with different DNA concentrations provided Ct values which were plotted against the log of the concentration of DNA to obtain the standard curve. DNA quantity was adjusted to 481 μg ml-.sup.1 for each starting concentration. The pentaplex assay yielded sensitivities substantially comparable to individual assays for beta-lactamase DNA.

[0053] FIG. 6 depicts sensitivities of pentaplex assay for target gene DNA in bacteria derived from blood according to the present invention. Serial tenfold dilutions of CRE DNA containing the desired target gene(s) were tested in a single tube format as a pentaplex reaction. Targeted genes were: (a) KPC, from strain 0112; (b) NDM, strain 0119; (c) CTX, strain 0119; (d) CMY, strain 0119; and (e) OXA-48, strain 0160. Individual runs with different DNA concentrations provided Ct values which were plotted against the log of the concentration of DNA to obtain the standard curve. DNA quantity was adjusted to 481 μg ml-.sup.1 for each starting concentration. The pentaplex assay yielded sensitivities substantially comparable to individual assays for target gene DNA.

[0054] FIG. 7 depicts an amplification plot showing simultaneous amplification of all five beta-lactamase gene targets in the pentaplex format. Red represents KPC (strain 0112); green represents CMY (strain 0119); blue represents NDM (strain 0119); yellow represents CTX (strain0119); grey represents OXA-48 (strain 0160). As illustrated, the compositions and methods of the invention herein identify the major beta-lactamase genes present in bacteria isolated directly from blood.

[0055] This quantitative PCR assay was able to concurrently amplify and identify the five most common beta-lactamase genes (KPC, NDM, CTX, CMY, and OXA-48) in a single-tube reaction format with no loss of sensitivity due to multiplexing. The assay provided herein can be completed in under 2 hours using DNA from bacteria taken directly from blood, requiring no blood culturing, and identifies the most common beta-lactamase genes present in bacteria that cause bloodstream infections in the United States. Detection limits are as low as four genome copies, i.e. 4 c.f.u. ml-.sup.1, per blood sample.

[0056] The information provided by this assay may improve patient care and outcomes by allowing for early CRE detection and characterization. In combination with other technologies such as tests for vancomycin resistance, this assay may promote the utilization of antibiotics which can successfully treat multi-drug resistant infections, improving the current bleak survival rates of bacteremias caused by these organisms.

EXAMPLES

Example 1: DNA Preparation

[0057] A single colony from a stock plate of each isolate was used to inoculate 10 ml LB containing 8 μg ml-.sup.1 imipenem at 37° C. in a non-shaking incubator. Bacteria were allowed to grow for 12 hours to an OD reading of 1, roughly 2×10.sup.9 c.f.u. ml-.sup.1. Total genomic DNA was extracted from each isolate from 1 ml of culture suspension using the DNeasy UltraClean Microbial Kit (Qiagen). DNA concentration was measured using an ND-1000 spectrophotometer (Nanodrop Technologies). The DNA concentration before dilution averaged 4800 μg ml-.sup.1.

Example 2: Primer and Probe Design and Testing

[0058] Quantitative PCR assays for each beta-lactamase gene using 5′-hydrolysis Taqman probes were designed using Clustal Omega and software available from Integrated DNA Technologies. Fluorophores were chosen based on emission spectra of each fluorophore. Eighty sequences for each beta-lactamase gene subtype were obtained from NCBI and aligned using the Clustal Omega software. The BLASTN programme at the National Centre for Biotechnology Information (NCBI) and the Clustal Omega software confirmed that the PCR target regions were specific to each gene and covered the most common single-nucleotide polymorphism subtypes for each gene (i.e. the PCR target regions for KPC target KPC-2, KPC-3, etc.), but did not target other genes such as CTX. Blind tests were conducted using a random numbering system of the target DNA from each isolate listed in Table 1 (FIG. 1). These tests were used to test sensitivity and detect false positives, false negatives, and ensure accurate identification of each target gene. No false positives or false negatives were found, and each target was accurately identified each time.

[0059] The assay indicated similar sensitivity, regardless of the isolate from which the DNA was obtained. Replicates for each gene were repeated 3-6 times. Difficulties commonly faced when designing multiplex PCR assays, such as differing primer annealing temperatures and inter-region differences in GC content were overcome by careful design of novel primers and analysis of the surrounding regions of the primers and probes. The primer and probe sequences used are listed in Table 2 (FIG. 2). Primers and probes were purchased from Integrated DNA Technologies.

Example 3: PCR Cycling Conditions

[0060] Quantitative PCR assays were performed on a QuantStudio five using Integrated DNA Technology's PrimeTime Gene Expression Master Mix. Individual assay conditions were 500 nM forward primer, 500 nM reverse primer, 250 nM probe, 50 ng target DNA, and HPLC-grade H.sup.2O for a final assay volume of 20 pl. Pentaplex reaction conditions were the same for each target gene, except no water was added. The final sample volume for pentaplex reactions was 25.2 μl. Thermal cycling conditions were 5 min at 95° C., followed by 40 cycles of 5 seconds at 95° C. and 30 seconds at 70° C. and a post-read cycle of 30 seconds at 60° C. A positive signal was determined by QuantStudio five threshold software before cycle 22. Cycle 22 was chosen because it was the latest cycle that the positive control samples amplified the target gene. The baseline and threshold settings used were the standard default values on the QuantStudio five platform.

Example 4: Separating Bacteria from Blood and Bacterial DNA Isolation

[0061] Whole human blood was obtained from volunteers (BYU IRBNo.: X18-340) by venipuncture into EDTA tubes and spiked with a dilution of the bacterial cultures prepared as described above. The final concentration of bacteria in the spiked blood samples was about 10 c.f.u. ml-.sup.1. Bacteria were separated from human blood using the spinning disc method of U.S. Pat. No. 10,457,975 previously described by Buchanan et al. This spinning disc method utilizes novel separation technology that exploits the subtle size and density differences between erythrocytes and bacterial cells, which conventional centrifugation does not.

[0062] Following plasma collection, samples were added to microcentrifuge tubes and lysozyme (0.5 mg ml-.sup.1, Sigma-Aldrich, chicken egg white #L6876) was added to each tube, vortexed for 5 seconds, then incubated for 10 minutes at room temperature. One hundred microlitres each of 6 M guanidine HCl (Promega, #H5381) solution and 1% sodium dodecyl sulphate (SDS, USB #18220) solution was then added to each microcentrifuge tube and vortexed for 30 seconds. Following lysis, 200 μl of isopropanol and 25 μl of Spherotech magnetic beads (#SIM-05-10 h) were added to each microcentrifuge tube, which were vortexed for 30 seconds and placed on a magnet holder. Beads were allowed to aggregate on the side of the tube wall adjacent to the magnet for two minutes at which time the liquid was removed. Then 450 μl of Wash 1 solution (6 M guanidine isothiocyanate, 20 mM Tris-HCl, v/v isopropanol, balance water) was added to each microcentrifuge tube and the tubes were vortexed for 30 seconds before being placed back in the magnet holder. Beads were aggregated and after 2 minutes, Wash 1 solution was removed and Wash 2 solution (0.1 M NaCl, 10 mM Tris-HCl, 70% v/v ethanol, balance water) was added. The tubes were vortexed again for 30 seconds, placed back in the magnet holder, and beads allowed to aggregate for 2 minutes. Wash 2 solution was removed and the tubes were air-dried for 5 minutes. Fifteen microlitres of EDTA elution buffer solution (1 mM EDTA in water) was added and tubes were vortexed for 2 minutes to allow the DNA to detach from the beads for final resuspension in the elution buffer solution. The microcentrifuge tubes were placed back in the magnet holder for 1 minute, after which the solutions were placed in clean microcentrifuge tubes. DNA was then serially diluted for further analysis via qPCR. The bacterial separation and DNA extraction took approximately 25 minutes, while the time to prepare and run the qPCR was about an hour. Combined, this process required almost 1.5 hour from time of blood sample collection to reading the assay results.

Example 5: Pentaplex Detection Sensitivity in Bacteria Derived from Pure Culture and from Blood

[0063] Serial tenfold dilutions of whole genomic DNA from clinical CRE isolates were used for optimization of primer and probe sets. Many iterations were performed to arrive at a successful combination of the five individual assays into a single tube, pentaplex reaction. FIG. 5 shows the sensitivity of the pentaplex reaction using DNA from pure cultures. FIG. 6 shows the performance of the pentaplex reaction using DNA extracted from bacteria in blood. The sensitivities of the individual assays did not decrease when combined into the final single reaction, whether the DNA was from pure cultures (FIG. 5), or from bacteria present in blood (FIG. 6). To prove that the concurrent presence of all target genes in a pentaplex reaction did not decrease the sensitivities of any of the individual assays, all five genes were combined into a pentaplex reaction in a single tube. Gene targets were present in three different bacteria which were spiked into human blood. Following separation, extraction, and serial tenfold dilution, each target gene amplified in the pentaplex reaction at the same rate as when run in a single reaction. The assay's successful identification of all five targets concurrently also confirmed no fluorophore interference (FIG. 7). See Table 3 (FIG. 3) for r2 values and sensitivities of each gene in pentaplex with each gene target present.

[0064] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.