Vancomycin-resistant gene multiplex detection system based on real-time fluorescent PCR

Abstract

This invention discloses a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR, aiming to establish a TaqMan probe-based multiplex real-time fluorescence PCR detection method for evaluating five common vancomycin resistance gene subtypes in clinical isolates. vanA、vanB、vanC、vanD and vanM The presence of [a substance] is used to clarify the drug resistance mechanism of the strain and guide precise clinical medication. The detection method established in this invention has superior performance, wherein... vanA and vanM The limit of detection is 10 2 copies / µL, vanB、vanC and vanD 10 3 Copies / µL. No specific amplification was observed in non-target strains, vancomycin-sensitive Enterococcus faecalis, and template-free controls. The coefficients of variation of Ct values ​​for each target strain were <1.0% both intra- and inter-batch. With NGS results as a reference, the overall concordance of this system among 108 clinical isolates was 100.00% (108 / 108).

Description

Technical Field

[0001] This invention belongs to the field of medical testing and molecular diagnostic technology, specifically relating to a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR. Background Technology

[0002] Vancomycin-resistant Enterococci ( van Vancomycin-resistant enterococci (VRE) are a significant pathogen associated with hospital-acquired infections, often exhibiting acquired resistance. Listed as a priority pathogen for new antibiotic development by the World Health Organization, VRE primarily affects high-risk populations such as those in intensive care, organ transplant recipients, and those with prolonged hospital stays. It can cause bacteremia, complicated urinary tract infections, and postoperative infections, significantly increasing the difficulty of anti-infective treatment and the burden of hospital infection control. In recent years, VRE-related infections, particularly vancomycin-resistant Enterococci, have become increasingly prevalent. Enterococcus faecium The detection rate of VREfm in southern China continues to rise, further exacerbating the clinical burden and becoming a public health issue that urgently needs attention.

[0003] VREs carry drug resistance genes that exhibit significant heterogeneity, among which... vanA and vanB This is the most representative acquired drug resistance genotype. vanC This is often related to the inherent low-level glycopeptide resistance background of some enterococci. vanD and vanM Although relatively rare, it still has some supplementary significance in the identification of drug resistance mechanisms and the interpretation of results. Moreover, the drug resistance phenotypes, clinical medication and prognosis corresponding to different vancomycin resistance gene subtypes vary greatly.

[0004] Therefore, relevant molecular testing should not only determine the presence of drug resistance genes, but also cover as many representative major drug resistance mechanisms as possible to guide precision medicine in clinical practice. Globally, genomic studies based on publicly available global databases show that... vanA The detection rate was approximately 40.9%. vanB The detection rate was approximately 5.8%. Meanwhile, a Dutch study on screening hospitalized patients found... vanC-1 and vanC-2 / 3 The detection rates were 7.2% and 7.8%, respectively. vanD Although accounting for only 0.7%, its unique acquired drug resistance mechanism suggests a potential risk of transmission. Overall, globally prevalent... vanA、vanB、vanC and vanD, And China vanA The main one, of which vanMThis is a subtype specific to China, with an overall prevalence rate ranging from 16% to 40%, exhibiting significant regional variations. Considering clinical diagnosis and treatment, hospital infection control, and health economics, simultaneous testing is necessary. vanA、vanB、 vanC、vanD and vanM (The accession numbers of the nucleotide sequences in the GenBank database are: CP111713, KT003982, NC_020995, NG_048362, NZ_CP038997.) These five subtypes are currently the optimal combination.

[0005] In recent years, detection methods for drug resistance mechanism typing have emerged continuously, but the reported methods still suffer from problems such as insufficient target coverage and limited stability in multi-target detection. Therefore, it is necessary to develop a method with more comprehensive target coverage. 、 A more stable detection method is urgently needed in clinical practice. Therefore, this study aims to construct a five-fold real-time fluorescence PCR detection method based on TaqMan probes for the detection of [viruses / samples] in clinical isolates. vanA、vanB、vanC、vanD and vanM Rapid detection and resistance mechanism typing of five vancomycin resistance genotypes were developed. The sensitivity, specificity, reproducibility, and consistency with next-generation sequencing (NGS) results were systematically evaluated, which is expected to provide a reference for clinical laboratories to identify related resistance mechanisms and for precision medicine in clinical practice. Summary of the Invention

[0006] Purpose of the invention: To overcome the shortcomings of existing technologies, this invention provides a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR, for use in... vanA、vanB、vanC、vanD and vanM Rapid detection and drug resistance mechanism typing of five target genes. Methodological evaluation results showed that the system performed well in terms of sensitivity, specificity, repeatability, and clinical consistency, possessing stable analytical performance and clinical application potential. The establishment of this method provides a feasible solution for the simultaneous identification of multiple vancomycin resistance genes on a conventional real-time fluorescence PCR platform, and can provide technical support for early clinical assessment of drug resistance mechanisms, precise anti-infective therapy, and hospital infection surveillance.

[0007] Technical Solution: To achieve the above objectives, on one hand, the present invention provides a real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes, wherein the real-time fluorescent PCR primer and probe composition is for detecting... vanA、vanB、 vanC、vanD and vanM Primer pairs and probes for five vancomycin resistance genotypes; among them, The vanAThe primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 1~2. vanA The probe for the gene is the nucleotide sequence shown in SEQ ID No. 3; The vanB The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 4-5. vanB The probe for the gene is the nucleotide sequence shown in SEQ ID No. 6; The vanC The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 7-8. vanC The probe for the gene is the nucleotide sequence shown in SEQ ID No. 9; The vanD The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 10-11. vanD The probe for the gene is the nucleotide sequence shown in SEQ ID No. 12; The vanM The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 13-14. vanM The probe for the gene is the nucleotide sequence shown in SEQ ID No. 15.

[0008] Optionally, in one embodiment of the present invention, the... vanA、vanB、vanC、vanD and vanM The final concentrations of primer pairs and probes for the five vancomycin resistance genotypes were independently 0.1–1 μmol / L, with 0.2 μmol / L being preferred.

[0009] Optionally, in one embodiment of the present invention, the probe contains a fluorescent group and a quenching group. The fluorescent groups include FAM, VIC, and Cy5; The quenching group includes BHQ1.

[0010] Optionally, in one embodiment of the present invention, the probe is a TaqMan probe.

[0011] On the other hand, the present invention also provides a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR, comprising a real-time fluorescence PCR primer and probe composition for detecting vancomycin resistance genes as described in any one of the above.

[0012] Optionally, in one embodiment of the present invention, the vancomycin resistance gene multiplex detection system may be in the form of a kit; The kit also includes a premix and quality control materials.

[0013] On the other hand, the present invention also provides a method for preparing a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR as described in any one of the above claims. The preparation method includes: synthesizing primer pairs and probes, constructing positive control samples, and then placing them in a premixed solution to obtain the vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR.

[0014] On the other hand, the present invention also provides a non-diagnostic or therapeutic method for detecting vancomycin resistance genes, the method comprising: extracting nucleic acid from a sample, performing PCR detection using the real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes described in any one of the above and / or the vancomycin resistance gene multiplex detection system based on real-time fluorescent PCR described in any one of the above, and obtaining the result.

[0015] Optionally, in one embodiment of the present invention, the amplification program for the PCR detection includes: 25℃ for 2 min, 95℃ for 20 s; followed by 45 cycles: 95℃ for 15 s, 60℃ for 35 s; and finally 38℃ for 5 s.

[0016] On the other hand, the present invention also provides the application of any of the above-described real-time fluorescence PCR primer and probe compositions for detecting vancomycin resistance genes and / or any of the above-described real-time fluorescence PCR-based multiplex detection systems for vancomycin resistance genes, or any of the methods described in any of the above-described methods, in the detection of vancomycin resistance genes in non-diagnostic or therapeutic purposes.

[0017] Beneficial effects: The vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR provided by this invention can detect vancomycin resistance genes. vanA、vanB、vanC、vanD and vanM Five common vancomycin resistance genotypes were detected simultaneously.

[0018] The results showed that vanA and vanM The limit of detection is 10 2 copies / µL, vanB、vanC and vanD 10 3 Copies / µL. No specific amplification was observed in non-target strains, vancomycin-sensitive Enterococcus faecalis, and template-free controls. The coefficients of variation of Ct values ​​for each target strain were <1.0% both intra- and inter-batch. With NGS results as a reference, the overall concordance of this system among 108 clinical isolates was 100.00% (108 / 108).

[0019] The method of this invention exhibits good analytical performance, demonstrating stability in sensitivity, specificity, and repeatability, and maintains high consistency with NGS genotyping results in the detection of clinical isolates. These results indicate that, under the condition of limited fluorescence channels in conventional real-time PCR platforms, it is feasible to conduct stable and interpretable genotyping detection targeting multi-target drug resistance mechanisms, providing clinical laboratories with a drug resistance mechanism identification strategy that is closer to practical application scenarios. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 for vanA、vanB、vanC、vanD and vanM Gel electrophoresis images of five candidate primer pairs for each of the following: A: vanA B: vanB C: vanC D: vanD E: vanM .

[0022] Figure 2 This diagram illustrates the workflow and principle of multiplex real-time fluorescence PCR detection of vancomycin resistance genotypes. A: Detection process; B: TaqMan probe detection principle; C: Representative amplification curve.

[0023] Figure 3 This diagram illustrates the construction and identification of standard recombinant plasmids. A: The process of constructing standard recombinant plasmids; B: Agarose gel electrophoresis results of the PCR amplification products of the standard recombinant plasmids.

[0024] Figure 4 This diagram illustrates the localization and amplification regions of each target within the vancomycin resistance gene cluster. A: vanA Type gene cluster structure and amplification region design; BE: respectively vanB、vanC、vanD and vanM Representative structure of a gene cluster. Arrows indicate the direction of gene transcription; red lines represent the gene cluster skeleton, colored arrows represent the core structural genes of the corresponding subtype, and gray arrows represent other related genes in the same cluster.

[0025] Figure 5 This is a schematic diagram illustrating the sensitivity evaluation of a multiplex real-time fluorescence PCR detection system. AE represent... vanA、vanB、 vanC、vanD and vanM The amplification curve.

[0026] Figure 6 This is a schematic diagram illustrating the specificity evaluation of a multiplex real-time fluorescence PCR detection system. AE represent... vanA、vanB、 vanC、vanD and vanM The test results. Kp It is Klebsiella pneumoniae. Pa It is Pseudomonas aeruginosa. E. coli It is Escherichia coli. Ab Acinetobacter baumannii, Efm It is Enterococcus faecalis. Sa It is Staphylococcus aureus. Sh Staphylococcus aureus Shl It is a hemolytic staphylococcus. Sp It is a pyogenic streptococcus. Ec It is Enterobacter cloacae.

[0027] Figure 7 This diagram illustrates the genotyping results of a multiplex real-time fluorescence PCR detection system in 108 clinical isolates and its consistency analysis with NGS results. A: Genotype distribution. BC: Overview of amplification curves for clinical isolates. The horizontal axis represents cycle number, and the vertical axis represents ΔRn; different colors represent different targets, with black representing the negative control. D: Comparison of qPCR and NGS consistency on a strain-by-strain basis. TP, TN, FP, and FN represent true positive, true negative, false positive, and false negative, respectively. E: Confusion matrix of qPCR and NGS at each target level.

[0028] Figure 8 This is a schematic diagram illustrating the diagnostic efficacy of multiplex real-time fluorescence PCR for detecting vancomycin resistance gene subtypes using NGS as a reference method. Detailed Implementation

[0029] Comprehensive coverage of the detection target is one of the core considerations of this invention. At least nine types of vancomycin resistance mechanisms have been reported to date, among which... vanA、vanB、vanC、vanD and vanM This constitutes the most representative genotype combination in clinical testing. vanA and vanB Acquired drug resistance is the main mechanism, but its prevalence is not constant. National surveillance data from Denmark from 2015 to 2022 shows that... vanA The [type] was dominant from 2015 to 2019, but after 2020... vanB The significant increase in genotypes suggests that mainstream genotypes exhibit dynamic evolution, and single-target detection may be insufficient to adapt to epidemiological changes. Meanwhile, vanM As an important subtype unique to my country, its detection rate reached 64.3% (out of 70 strains) in a study conducted in nine hospitals in Shanghai, which is higher than... vanA 35.7%; further research in Hangzhou found that 56 strains vanMOf the positive bacteria, 55 strains remained phenosensitive to vancomycin and teicoplanin, suggesting that relying solely on phenotypic screening or testing alone is insufficient. vanA / vanB This could lead to a significant proportion of drug-resistant strains being missed. Furthermore, vanC The inclusion of this test in the study is helpful in distinguishing between inherent resistance and acquired high-level resistance, given the background of low-level vancomycin resistance in Enterococci. vanD Although relatively rare, vanA / vanB Reports of detection have emerged in clinically suspected samples that were PCR negative. Therefore, [the following is missing from the original text]. vanA、vanB、vanC、vanD and vanM Placing them within the same testing system is not simply about expanding the testing scope, but rather about taking into account the identification of typical acquired drug resistance, the differentiation of inherent drug resistance backgrounds, and the coverage of rare mechanisms, thereby improving the comprehensiveness of the testing and the completeness of the result interpretation, and better serving clinical medication guidance, drug resistance monitoring, and hospital infection control.

[0030] The appropriate selection of the technology platform laid the foundation for the feasibility of this invention. TaqMan probe-based real-time fluorescence PCR technology is relatively mature. Based on dual specificity recognition of primers and probes, it can simultaneously complete fluorescence signal acquisition and result interpretation during amplification. Compared to detection methods that rely on amplification endpoint products or terminal signals, it has higher specificity and more stable interpretation capabilities, helping to reduce non-specific amplification and cross-reaction interference. This technology system is well-developed; primer and probe design, synthesis and labeling, enzyme reaction systems, and supporting buffer systems are all mature, with high methodological repeatability and standardization, providing reliable support for the construction and performance evaluation of multiplex detection systems. From a clinical application economic perspective, although the initial investment in specific probes makes the cost per detection slightly higher than ordinary amplification methods, it has good overall application economy in scenarios with large sample volumes and high degree of process standardization. More importantly, most hospitals, especially secondary and higher-level medical institutions, are now equipped with molecular detection laboratories and real-time fluorescence detection equipment; a four-channel real-time fluorescence PCR instrument is one of the most basic and commonly used configurations in clinical laboratories. The research method of this invention relies on the conventional real-time fluorescence PCR platform represented by ABI 7500. The detection system is based on four-channel fluorescence signal recognition and has strong compatibility at the instrument level. It can be applied without purchasing additional high-end equipment or reconstructing the detection platform, which provides a realistic basis for its promotion and transformation in clinical laboratories.

[0031] Therefore, this invention aims to establish a TaqMan probe-based multiplex real-time fluorescence PCR detection method for evaluating five common vancomycin resistance genotypes in clinical isolates. vanA、vanB、vanC、vanD and vanM To determine the presence of the virus in the bacteria, we can clarify the drug resistance mechanism of the strains and guide precise clinical medication.

[0032] The method of this invention includes: collecting 108 strains of vancomycin-resistant Enterococcus faecalis clinically isolated from two tertiary general hospitals in Shanghai and Guangdong between January 2022 and December 2024. Primers and probes were designed based on GenBank reference sequences, and a standard recombinant plasmid containing five vancomycin resistance genotypes was constructed. A multiplex real-time fluorescent PCR detection system was established, and its sensitivity, specificity, repeatability, and consistency with the results were evaluated.

[0033] Evaluation and verification have shown that the detection method established in this invention has superior performance, wherein... vanA and vanM The limit of detection is 10 2 copies / µL, vanB、vanC and vanD 10 3 Copies / µL. No specific amplification was observed in non-target strains, vancomycin-sensitive Enterococcus faecalis, and template-free controls. The coefficients of variation of Ct values ​​for each target strain were <1.0% both intra- and inter-batch. With NGS results as a reference, the overall concordance of this system among 108 clinical isolates was 100.00% (108 / 108).

[0034] The multiplex real-time fluorescence PCR detection method established in this invention has good sensitivity, specificity and repeatability, and high consistency with NGS results. The detection performance can meet the requirements of clinical application and can be used for rapid detection and resistance mechanism typing of five common vancomycin resistance gene subtypes, providing a reference for clinical laboratories to carry out related resistance mechanism identification and clinical precision medicine.

[0035] The present invention will be further described below with reference to the accompanying drawings and embodiments. The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the present invention as described in detail in the claims.

[0036] Example 1: Strain Collection and Identification From January 2022 to December 2024, a total of 108 vancomycin-resistant Enterococcus faecium clinical isolates were obtained from routine clinical specimens submitted by two tertiary general hospitals in Shanghai and Guangdong Province. The primary isolates were inoculated onto blood agar plates and incubated at 37°C for 24 hours. Single colonies were then picked for subculturing and purification for subsequent experiments.

[0037] Bacterial species identification was performed using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS), and drug resistance phenotypes were confirmed using the VITEK 2 COMPACT automated microbial identification and drug susceptibility testing system. Confirmed strains were prepared into cryopreserved bacterial suspensions with a final concentration of 30% sterile glycerol and stored at −80°C for later use. To avoid duplicate inclusion, only the first isolate from each patient was included; for repeated isolates from the same patient during the same hospitalization, only the first isolate was retained.

[0038] Example 2: Design and Synthesis of Primers and Probes Using reference sequences of relevant drug resistance genes in the GenBank database as templates (accession numbers: CP111713, KT003982, NC_020995, NG_048362, NZ_CP038997), respectively targeting vanA、vanB、vanC、vanD and vanM Primer design. Primer design was performed using Primer Premier 5 and Oligo 7 software. Five pairs of candidate primers were initially designed for each target gene (specific sequences are shown in Table 1). After screening with NCBI BLAST to exclude potential homology with non-target sequences, preliminary experiments were performed using conventional PCR for verification.

[0039] Table 1 Candidate primer sequences

[0040]

[0041]

[0042] The amplified products were separated by 1% agarose gel electrophoresis, and the results were observed using a Tanon 1600 fully automated gel imaging system. Optimal primer pairs were selected based on factors such as band uniformity, consistency of theoretical fragment length, and the presence of non-specific amplification or primer dimers.

[0043] right vanA、vanB、vanC、vanD and vanM The designed candidate primers were amplified and analyzed by gel electrophoresis, such as Figure 1 As shown vanA、vanB、vanC、vanD and vanM Gel electrophoresis images of five candidate primer pairs for each group. The results showed that the amplification effects of different candidate primer pairs varied. After screening, vanA 4th primer pair, vanB First pair of primers, vanC 5th primer pair, vanDThe first pair of primers and vanM The first primer pair showed good amplification results, with the amplified band size consistent with expectations, clear bands, and no obvious non-specific bands. The remaining candidate primers exhibited weak bands, interference from stray bands, or poor resolution. After comprehensive evaluation, the aforementioned primers were ultimately selected for the subsequent detection system construction. Based on this, the corresponding probes and detection combinations for each target were further determined, with amplified product lengths ranging from 143 to 340 bp. A dual-reaction tube detection mode was established based on the fluorescence channel configuration: reaction tube 1 detection... vanB , vanC and vanM Detection in reaction tube 2 vanA and vanD The sequences of each primer and probe, as well as the lengths of the amplification products, are shown in Table 2.

[0044] After determining the primers, gene-specific TaqMan probes were designed based on the same reference sequence, with all probes located inside the corresponding amplification fragment.

[0045] Based on the fluorescence channel configuration, a dual-reaction tube detection mode is established: reaction tube 1 detection. vanB (FAM channel) vanC (VIC channel) and vanM (Cy5 channel), detection in reaction tube 2 vanA (FAM channel) and vanD (VIC channel). All probes used BHQ1 as the quencher. Primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The lyophilized products were dissolved in nuclease-free water to prepare stock and working solutions, which were stored at −20℃ protected from light. The final concentration of each primer and probe was 0.2 μmol / L. Specific sequences and amplified fragment lengths are shown in Table 2.

[0046] Table 2 Primer and probe sequences

[0047] Example 3 Construction of standard plasmids According to the GenBank database vanA、vanB、vanC、vanD and vanM The target sequence was designed and synthesized into a fragment, which was synthesized by Sangon Biotech (Shanghai) Co., Ltd., and then cloned into the pUC57 vector through homologous recombination to construct a recombinant plasmid.

[0048] Transformation of recombinant plasmids to E. coliDH5α competent cells were amplified, and plasmid DNA was extracted using the SanPrep column-based plasmid DNA mini-extraction kit (Sangon Biotech). Conventional PCR amplification was performed using primers for each target gene. The amplified products were verified by agarose gel electrophoresis and then sequenced. Successful construction was defined as sequencing results that matched the corresponding GenBank reference sequences. The identified recombinant plasmids were used as positive standards for establishing the detection system and evaluating analytical performance such as sensitivity and repeatability.

[0049] After determining the plasmid DNA concentration, it was converted to copy number using the following formula: copies / μL = (6.02 × 10⁻⁶) / μL. 23 ×C×10 -9 ) / (L×660), where C is the plasmid concentration (ng / μL), L is the total plasmid length (bp, including vector and insert fragment), and 660 is the average molecular weight per bp (g / mol·bp). This study vanA、vanB、vanC、vanD and vanM Positive controls were all prepared using recombinant plasmids containing the corresponding target gene. Due to limitations in sample availability, no samples carrying the gene were obtained. vanC or vanD Clinically positive isolates, therefore vanC and vanD Only the performance evaluation was completed.

[0050] like Figure 3 The diagram shows the construction and identification of a standard recombinant plasmid. Figure 3 A shows the insertion of pUC57 (2677 bp) as a carrier. vanA、vanB、vanC、vanD and vanM The target fragment was used to construct a standard recombinant plasmid; the key elements of the vector and the relevant restriction endonuclease sites are marked in the figure. Figure 3 B shows the agarose gel electrophoresis results of the standard recombinant plasmid PCR amplification products. Each target gene showed a single specific band, and the band size was consistent with expectations. M represents the relative molecular mass of the DNA.

[0051] Example 4: Nucleic acid extraction from clinical samples After resuscitation and purification of the VREfm strain stored at −80℃, genomic DNA was extracted from fresh bacterial cells. DNA extraction was performed on 108 isolates using the Ezup column-based bacterial genomic DNA extraction kit (Sangon Biotech) according to the manufacturer's instructions. DNA concentration and purity were determined using micro-spectrophotometry. Samples with an A260 / A280 ratio of 1.8–2.0 and an A260 / A230 ratio > 2.0 were used for subsequent amplification. The extracted DNA was not uniformly diluted and was directly used as template for real-time fluorescence PCR detection. 2 μL was added to each reaction, and the remaining sample was stored at −20℃ for later use.

[0052] Example 5: TaqMan fluorescent PCR reaction system A multiplex TaqMan real-time fluorescence PCR detection method was established using the Takara Probe qPCR Mix MultiPlus Multiplex Probe Quantitative PCR Kit (RR393A), and amplification and fluorescence signal acquisition were performed on an ABI 7500 real-time fluorescence quantitative PCR instrument.

[0053] The reaction was carried out in a 96-well plate, with a total reaction volume of 25 μL, prepared according to the kit instructions. Since the system contained ROX passive reference dye, the Passive Reference was set to ROX in the ABI 7500 software. Detection was performed using a dual-tube mode: reaction tube 1 was used for detection. vanB、vanC and vanM Detection in reaction tube 2 vanA and vanD Add 2 μL of template DNA to each well, and bring the remaining volume to 25 μL with reaction solution and nuclease-free water. A template-free control (NTC) is included in each experiment.

[0054] The amplification program was as follows: 25℃ for 2 min, 95℃ for 20 s; followed by 45 cycles: 95℃ for 15 s, 60℃ for 35 s; and finally 38℃ for 5 s. Fluorescence signals were acquired at the 60℃ stage and detected through the FAM, VIC, and Cy5 channels. The detection procedure and representative amplification curves are shown below. Figure 1 .

[0055] like Figure 2 The diagram shows the process and principle of multiplex real-time fluorescence PCR detection of vancomycin resistance gene subtypes. Figure 2 A illustrates the detection process of the present invention, which specifically involves purifying and identifying clinical isolates and confirming vancomycin resistance phenotypes, followed by enrichment culture and extraction of genomic DNA, and then using the extracted DNA as a template for multiplex real-time fluorescent PCR detection.

[0056] Figure 2 B illustrates the TaqMan probe detection principle, in which the 5′ end of the probe is labeled with a reporter group (R) and the 3′ end is labeled with a quencher group (Q). After the probe specifically binds to the target sequence, it is cleaved by the 5′→3′ exonuclease activity of Taq DNA polymerase during amplification, separating the reporter group from the quencher group and releasing a fluorescent signal.

[0057] Figure 2 C shows a representative amplification curve, in which vanA、vanB、vanC、vanD and vanM All curves exhibited a typical S-shaped amplification curve. The horizontal axis represents the cycle number, and the vertical axis represents ΔRn, indicating the change in normalized fluorescence signal after correction by the passive reference dye.

[0058] like Figure 4 The diagram shows the location and amplification regions of each target in the vancomycin resistance gene cluster. Figure 4 A shows vanA Type gene cluster structure and amplification region design. The above shows... vanA The typical composition and arrangement order of type gene clusters are shown below. vanA The location of the amplified region within the reference sequence. Red indicates primer binding sites, blue indicates probe binding sites and the core recognition region, and arrows indicate the direction and extent of amplification. Figure 4 BE are respectively vanB , vanC , vanD and vanM Representative structure of a gene cluster. Arrows indicate the direction of gene transcription; red lines represent the gene cluster skeleton, colored arrows represent the core structural genes of the corresponding subtype, and gray arrows represent other related genes in the same cluster.

[0059] Example 6 Sensitivity Experiment use vanA、vanB、vanC、vanD and vanM Standard recombinant plasmids were serially diluted 10-fold (10 6 ~10 1 (copies / μL) to evaluate the sensitivity of the multiplex real-time fluorescence PCR detection system.

[0060] The specific method is as follows: using standard recombinant plasmids of each target gene as templates, prepare 10-fold serial dilutions. 6 ~10 1 Copies / μL series concentrations. Add 2 μL of the corresponding concentration template for each reaction, and simultaneously set NTC. Set 3 replicates for each concentration, and perform detection according to the conditions described in Section 2.5.

[0061] Results interpretation combines amplification curve morphology and Ct value: a typical S-shaped amplification curve with Ct < 36 is considered positive; Ct 36-38 is considered suspicious and requires retesting for confirmation; Ct > 38 or no Ct value is considered negative. The limit of detection (LOD) is defined as the lowest concentration detected in all three replicates. If NTC shows a reproducible typical amplification curve, the batch is considered to be at risk of contamination, the result is invalid, and retesting is required; if only a non-reproducible late atypical signal appears, it is not considered a positive result, but should be verified in conjunction with the curve morphology.

[0062] The results showed that typical amplification curves could be obtained for each target in the corresponding fluorescence channel, and the Ct value gradually increased with decreasing template concentration. Figure 4 The lowest detection limit (LOD) is determined by the detection of the lowest concentration standard in all three replicates. This system... vanA and vanM The LOD is 10 2 copies / μL, for vanB、vanC and vanD The LOD is 10 3 copies / μL. No amplification signal was observed in either the blank negative control or the negative control.

[0063] like Figure 5 The diagram shows a sensitivity evaluation of a multiplex real-time fluorescence PCR detection system. vanA、vanB、 vanC、vanD and vanM The standard recombinant plasmid was used as a template and amplified after being serially diluted 10-fold. As the template concentration decreased, the amplification curve gradually shifted to the right, and the Ct value increased; no typical amplification curve was observed in the negative control. Figure 5 AE are respectively vanA、vanB、 vanC, vanD and vanM The amplification curve.

[0064] Example 7 Repeatability Experiment Using 10 6 ~10 1 Standard recombinant plasmids were serially diluted 10-fold (copies / μL) to evaluate the reproducibility of the multiplex real-time fluorescence PCR detection system.

[0065] The specific method is as follows: using standard recombinant plasmids of each target gene as templates, prepare 10-fold serial dilutions. 6 ~10 1 The concentrations were set in copies / μL, and the NTC was set. The reaction system and amplification conditions described in Example 5 were used for detection, and the Ct values ​​for each target were recorded. Repeatability evaluation included intra-batch repeatability and inter-batch repeatability. Intra-batch repeatability involved three parallel detections of each concentration template in the same experiment; inter-batch repeatability involved three detections at different times and in different experimental batches. Repeatability was evaluated using the coefficient of variation (CV), calculated as: CV(%) = SD / Mean × 100%. Where Mean is the mean of Ct, and SD is the standard deviation of Ct. CV ≤ 5% indicates good repeatability.

[0066] The results showed that each target could be stably amplified at each concentration gradient with minimal fluctuations in Ct values. The coefficient of variation (CV) of Ct values ​​in intra-batch and inter-batch repeated detections was <1.0%, indicating that the detection system had good repeatability.

[0067] Example 8 Specificity Experiment To evaluate the specificity of the multiplex real-time fluorescence PCR detection system, genomic DNA of common clinical pathogens was selected as non-target templates, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus aureus, Staphylococcus pyogenes, and Streptococcus pyogenes. Vancomycin-sensitive Enterococcus faecalis (VSEfm), blank negative control (NTC), and positive controls for each target were also set up.

[0068] The specific method is as follows: Genomic DNA of common clinical pathogens was selected as non-target templates, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, Staphylococcus hominis, Staphylococcus hemolyticus, and Streptococcus pyogenes, with one strain of each strain. Simultaneously, DNA from a clinical isolate of vancomycin-susceptible Enterococcus faecium (VSEfm) was selected as a negative control, and deionized water was used as the NTC. Each template was detected according to the reaction system and amplification conditions described in Section 2.5, with 2 μL of DNA template added to each well. Specificity was evaluated by whether typical amplification curves and specific fluorescence signals appeared in each fluorescence channel. The judgment criteria were: no specific amplification was observed in all non-target templates and negative controls, and no amplification signal was observed in the NTC.

[0069] The results showed that typical amplification curves were obtained in the corresponding fluorescence channels for each target positive control; no positive amplification signals were observed in the FAM, VIC, and Cy5 channels for all non-target strains, VSEfm, and NTC, indicating that the detection system has good specificity. Figure 6 .

[0070] like Figure 6 The diagram illustrates the specificity evaluation of a multiplex real-time fluorescence PCR detection system. Standard recombinant plasmids for each target gene were used as positive controls, genomic DNA from common clinical pathogens was used as a non-target template, and a negative control was included. Only the positive control showed a typical amplification curve; no specific amplification was observed in the non-target template or the negative control. Figure 6 AE respectively show the relevant vanA, vanB, vanC, vanD and vanM The results of the system-specific tests showed no specific amplification, indicating that the system has good specificity. Kp It is Klebsiella pneumoniae. Pa It is Pseudomonas aeruginosa. E. coli It is Escherichia coli. Ab Acinetobacter baumannii, Efm It is Enterococcus faecalis. Sa It is Staphylococcus aureus. Sh Staphylococcus aureus Shl It is a hemolytic staphylococcus.Sp It is a pyogenic streptococcus. Ec It is Enterobacter cloacae.

[0071] Example 9: Detection of clinical isolates and evaluation of concordance with NGS To evaluate the performance of the multiplex real-time fluorescence PCR detection system in clinical isolates, genomic DNA from 108 VREfm clinical isolates was used as a template for detection, and NGS results were used as a reference method for consistency analysis.

[0072] The specific method is as follows: using genomic DNA extracted from 108 VREfm clinical isolates as templates, simultaneous detection... vanA, vanB, vanC, vanD and vanM The reaction system, amplification conditions, and result judgment criteria are the same as described in Examples 5 and 6, respectively. Based on the detected values ​​of each sample... van Gene combinations were genotyped, and consistency was evaluated using next-generation sequencing (NGS) results as a reference method. Raw sequencing data underwent quality control and de novo assembly using SPAdes. Resistance gene retrieval was performed using BLASTn based on the CARD database. van Genotype determination; the criteria for a positive result are alignment coverage ≥95% and sequence consistency ≥99%. For samples inconsistent with NGS results, further sequence verification is performed.

[0073] The consistency evaluation included both target gene and sample genotype levels. At the target gene level, a 2×2 contingency table was constructed using NGS as a reference to calculate sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. 95% confidence intervals were calculated using the Clopper-Pearson exact method. When the NGS positive count for a target was 0, the relevant indicator was considered Not Applicable (NA). At the sample genotype level, the consistency was compared with the results detected by NGS. van Complete consistency of gene combinations was considered acceptable. Overall consistency was calculated and evaluated using Cohen's kappa coefficient. Statistical analysis was performed in R software.

[0074] like Figure 7 The diagram shows the typing results of the multiplex real-time fluorescence PCR detection system in 108 clinical isolates and its consistency analysis with NGS results. Among them, Figure 7 A shows the genotype distribution. Figure 7 BC shows an overview of the amplification curves of clinical isolates. The horizontal axis represents the cycle number, and the vertical axis represents ΔRn; different colors represent different targets, and black represents the negative control. Figure 7D shows the strain-by-strain consistency comparison between qPCR and NGS. TP, TN, FP, and FN represent true positives, true negatives, false positives, and false negatives, respectively. E: Confusion matrix of qPCR and NGS at each target level. vanA and vanM The test results were consistent with those of NGS. vanB, vanC and vanD No positive results were detected in either method.

[0075] like Figure 7 The NGS results shown indicate that, among the 108 isolates, vanA 95 single-positive strains vanM One single positive strain. vanA and vanM Eight strains were double-positive, and four others did not detect the aforementioned target genes. Based on target count, NGS detected a total of [number missing] [unclear - likely related to NGS detection]. vanA 103 positive strains vanM Nine positive strains, vanB, vanC and vanD No positive results were detected. Multiplex real-time fluorescence PCR results showed that... vanA 103 strains tested positive. vanB, vanC and vanD No positive results were detected in any of them. vanM Nine strains tested positive. Among them, vanA and vanM The detection results were consistent with those of NGS. vanB, vanC and vanD No positive results were detected in either method.

[0076] The consensus was based on complete consistency of vancomycin resistance gene combinations at the sample level, with 108 samples showing consistency, resulting in an overall consistency of 100.00% (108 / 108). Further consistency analysis using sample genotype as a categorical variable yielded a Cohen's kappa value of 0.921 (95% CI: 0.814–1.000), indicating high consistency between the two methods in sample genotyping. Regarding target identification... vanA and vanM Completely consistent with NGS results, see Figure 8 Since NGS did not detect it in the samples of this study. vanB , vanC or vanD For positive events, the sensitivity and positive predictive value of the relevant targets were not calculated and are denoted as NA. The 95% CI for each proportional category indicator was calculated using the Clopper-Pearson exact method.

[0077] like Figure 8The diagram illustrates the diagnostic efficacy of multiplex real-time fluorescence PCR for detecting vancomycin resistance gene subtypes using NGS as a reference method. NGS serves as the reference standard for determining whether the corresponding resistance gene is positive or negative. Sensitivity = TP / (TP+FN), specificity = TN / (TN+FP), positive predictive value = TP / (TP+FP), negative predictive value = TN / (TN+FN), accuracy = (TP+TN) / N, and consistency is evaluated using Cohen's kappa. Results are expressed as point estimates [95% CI], and the 95% CI for proportional indicators was calculated using the Clopper-Pearson exact method. If no NGS-positive sample is found for a target, the corresponding sensitivity, positive predictive value, and kappa value are denoted as NA. qPCR refers to real-time quantitative PCR; CI is the confidence interval; PPV is the positive predictive value; NPV is the negative predictive value; NA indicates not applicable. Related indicators are calculated based on the sample distribution of this study. vanB, vanC and vanD Due to the lack of NGS-positive clinical samples, some diagnostic efficacy indicators cannot be fully obtained.

[0078] In summary, this study established a method for detecting vancomycin-related drug resistance genotypes based on real-time fluorescence PCR, which can be used for... vanA, vanB, vanC, vanD and vanM Five vancomycin resistance genotypes were simultaneously detected. Results showed that this method exhibited good analytical performance, demonstrating stability in sensitivity, specificity, and repeatability, and maintained high consistency with NGS genotyping results in clinical isolate detection. These results indicate that, under the condition of limited fluorescence channels in conventional real-time PCR platforms, stable and interpretable genotyping detection for multi-target resistance mechanisms is feasible, providing clinical laboratories with a resistance mechanism identification strategy that is closer to practical applications.

[0079] From a clinical application perspective, the research method of this invention meets the comprehensive clinical needs for rapid identification of drug-resistant bacteria in terms of timeliness, standardization, and practicality. Compared to the conventional approach of gradually determining drug resistance based on culture and drug sensitivity results, this method can directly provide clear drug resistance genotyping results in a shorter time. This helps clinicians identify target drug resistance mechanisms earlier, appropriately advancing the determination of drug resistance and providing more proactive intervention opportunities for adjusting antimicrobial drugs, modifying empirical treatment, and stratifying patient management. The TaqMan probe method is based on specific amplification signals, resulting in relatively objective interpretation and standardized reporting. It is less affected by subjective experience and maintains good consistency across different batches, different testing personnel, and different time periods. Therefore, it is more suitable as a routine and continuous monitoring technique in clinical laboratories. For large-volume sample submissions, this method has a mature technical path, a clear testing process, and is easy to integrate into existing molecular testing workflows, with a relatively controllable average cost per sample. More importantly, this method can not only determine the presence of drug resistance genes but also provide relatively detailed genotyping information, helping clinicians to combine the drug resistance characteristics corresponding to different genotypes to make more targeted interpretations of the test results.

[0080] It should be noted that in this application, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0081] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes, characterized in that, The real-time fluorescent PCR primer and probe composition is for the separate detection of... vanA, vanB, vanC, vanD and vanM Primer pairs and probes for five vancomycin resistance genotypes; among them, The vanA The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 1~2. vanA The probe for the gene is the nucleotide sequence shown in SEQ ID No. 3; The vanB The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 4-5. vanB The probe for the gene is the nucleotide sequence shown in SEQ ID No. 6; The vanC The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 7-8. vanC The probe for the gene is the nucleotide sequence shown in SEQ ID No. 9; The vanD The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 10-11. vanD The probe for the gene is the nucleotide sequence shown in SEQ ID No. 12; The vanM The primer pairs for the gene are the nucleotide sequences shown in SEQ ID No. 13-14. vanM The probe for the gene is the nucleotide sequence shown in SEQ ID No.

15.

2. The real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes according to claim 1, characterized in that, The vanA, vanB, vanC, vanD and vanM The final concentrations of primer pairs and probes for the five vancomycin resistance genotypes were independently 0.1–1.0 μmol / L.

3. The real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes according to claim 1, characterized in that, The probe contains a fluorescent group and a quenching group. The fluorescent groups include FAM, VIC, and Cy5; The quenching group includes BHQ1.

4. The real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes according to claim 1, characterized in that, The probe is a TaqMan probe.

5. A vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR, characterized in that, Includes the real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes as described in any one of claims 1-4.

6. The vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR according to claim 5, characterized in that, The vancomycin resistance gene multiplex detection system includes kits. The kit also includes a premix and quality control materials.

7. A method for preparing a vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR as described in claim 5 or 6, characterized in that, The preparation method includes: synthesizing primer pairs and probes, constructing positive control samples, and then placing them into a premixed solution to obtain the vancomycin resistance gene multiplex detection system based on real-time fluorescence PCR.

8. A non-diagnostic or therapeutic method for detecting vancomycin resistance genes, characterized in that, The method for detecting vancomycin resistance genes includes: extracting nucleic acid from a sample, performing PCR detection using the real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes as described in any one of claims 1-4 and / or the vancomycin resistance gene multiplex detection system based on real-time fluorescent PCR as described in claim 5 or 6, and obtaining the results.

9. The method according to claim 8, characterized in that, The PCR amplification program includes: 25℃ for 2 min, 95℃ for 20 s; followed by 45 cycles: 95℃ for 15 s, 60℃ for 35 s; and finally 38℃ for 5 s.

10. The application of the real-time fluorescent PCR primer and probe composition for detecting vancomycin resistance genes according to any one of claims 1-4 and / or the vancomycin resistance gene multiplex detection system based on real-time fluorescent PCR according to claim 5 or 6, or any one of the methods described in claim 8 or 9, in the detection of vancomycin resistance genes in non-diagnostic or therapeutic purposes.

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