A method for simultaneous detection of multiple drug resistance genes triple probe

Abstract

The application discloses a kind of synchronous detection multiple drug resistance gene triple probe method. Design and synthesis respectively for polymyxin drug resistance gene mcr-1, carbon penicillin resistance gene bla NDM‑1 , tigecycline drug resistance gene tet (X4) conservative region three pairs of specific primers and three fluorescently labeled probes, each probe is labeled with different fluorescent reporter group and corresponding fluorescent quenching group;Extract the genomic DNA of the sample to be tested;Prepare triple fluorescent quantitative detection reaction system, carry out amplification reaction, and synchronously collect the signals of three different fluorescent reporter groups;Detect whether drug resistance gene exists and copy number.This application realizes single tube synchronous detection of three types of drug resistance genes, compared with traditional single nucleic acid detection method, detection efficiency is improved, while reducing reagent consumption consumption, reduce detection cost;At the same time, it can detect whether drug resistance gene exists and abundance level.

Description

Technical Field

[0001] This application relates to the field of microbial detection technology, and in particular to a triple probe method for simultaneous detection of multiple drug resistance genes. Background Technology

[0002] Antimicrobial drugs are a key means of controlling bacterial diseases and ensuring profitability in livestock and poultry farming. However, their long-term, widespread, and irrational use has become a core factor driving the evolution and spread of bacterial resistance. The continued spread of bacterial resistance, especially the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains, not only seriously restricts the healthy and sustainable development of the livestock and poultry farming industry, but also enables the cross-species transmission of resistance genes through the food chain, environmental contact, and occupational exposure, posing a severe challenge to human clinical anti-infective treatment and becoming a major issue that urgently needs to be addressed in the global public health field.

[0003] Escherichia coli, a common commensal and opportunistic pathogen in the pig's intestines, is characterized by rapid reproduction, strong environmental adaptability, and high efficiency in horizontal gene transfer, making it an important vector for the storage, transmission, and spread of drug-resistant genes. In pig production, porcine pathogenic E. coli can cause various diseases in piglets, such as yellow-white scours, edema disease, and septicemia, leading to increased mortality and decreased growth performance, resulting in significant economic losses. Simultaneously, drug-resistant genes carried by porcine E. coli can be transmitted to humans through meat product contamination and environmental emissions, forming cross-resistance with clinically isolated pathogenic E. coli, significantly reducing the therapeutic effect of clinical antimicrobial drugs and posing a potential threat to human public health and safety.

[0004] In recent years, three types of mobile resistance genes mediating resistance to "last-line antibiotics" have rapidly spread globally, among which bla NDM-1 (Carbapenem resistance gene bla) NDM-1 Due to their high pathogenicity and transmissibility, mcr-1 (polymyxin resistance gene mcr-1) and tet(X4) (tigecycline resistance gene tet(X4)) have become core drug resistance threats of common concern in the veterinary and clinical fields.

[0005] Currently, in porcine Escherichia coli, mcr-1 and blavin are present. NDM-1- Co-detection of the tet(X4) gene is becoming increasingly common, and multidrug-resistant strains continue to spread throughout the entire chain of breeding, slaughtering, and sales. This not only increases the difficulty of controlling infections in pig herds but also threatens the health of workers and consumers through occupational exposure and meat product consumption. Traditional methods for detecting drug resistance mainly include drug susceptibility testing and conventional PCR. Drug susceptibility testing is cumbersome and time-consuming, requiring 18-24 hours, and cannot directly detect drug resistance genes. While conventional PCR can achieve rapid screening of drug resistance genes, it suffers from insufficient specificity, inability to simultaneously detect multiple genes, and difficulty in accurate quantification, making it difficult to meet the needs of rapid screening in breeding farms, large-scale epidemiological monitoring, and precise clinical diagnosis.

[0006] Quantitative real-time PCR (qPCR) technology, especially the TaqMan probe method, boasts advantages such as high specificity, high sensitivity, good reproducibility, accurate quantification, and multiplex detection. It can simultaneously identify multiple target genes in a single reaction system, significantly improving detection efficiency and accuracy. It has become the mainstream technology for rapid detection and quantitative analysis of drug resistance genes and is widely used in fields such as microbial detection and epidemiological surveys. Currently, although domestic and international researchers have established methods targeting mcr-1, blavin, and other drug resistance genes... NDM-1 While qPCR detection methods for the single tet(X4) gene are available, systematic research on triple TaqMan qPCR simultaneous detection technology for these three core drug resistance genes in porcine Escherichia coli is still lacking, making it difficult to meet the practical needs of simultaneous screening and quantitative analysis of multiple genes. Summary of the Invention

[0007] To solve at least one of the above-mentioned technical problems, a method capable of simultaneously detecting mcr-1 and blavin has been developed. NDM-1 This application provides a triple probe method for the simultaneous detection of multiple drug resistance genes, including tet(X4) and tet(X4).

[0008] On the one hand, this application provides a triple probe method for simultaneous detection of multiple drug resistance genes, including polymyxin resistance gene mcr-1 and carbapenem resistance gene bla. NDM-1 And the tigecycline resistance gene tet(X4), characterized in that, Includes the following steps: S1. Design and synthesize drugs targeting the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla, respectively. NDM-1 Three pairs of specific primers and three fluorescently labeled probes were used to target the conserved region of the tigecycline resistance gene tet(X4). Each probe was labeled with a different fluorescent reporter group at its 5' end and a corresponding fluorescent quencher group at its 3' end. S2. Extract genomic DNA from the sample to be tested; S3. Prepare a triple fluorescence quantitative detection reaction system, wherein the reaction system comprises a premixed solution, three pairs of specific primers, three fluorescently labeled probes, genomic DNA of the sample to be tested, and nuclease-free water; S4. Place the prepared reaction system in a fluorescence quantitative amplification instrument for amplification reaction, and simultaneously collect signals of three different fluorescent reporter groups during the amplification process; S5. Determine the presence of the three types of drug resistance genes in the sample based on the amplification curve and the cycle threshold, and calculate the copy number of the three types of drug resistance genes in combination with the standard curve.

[0009] By adopting the above technical solution, simultaneous detection of three types of drug resistance genes—polymyxins, carbapenems, and tigecycline—can be achieved in a single tube. Compared with traditional single-tube nucleic acid detection methods, this method improves detection efficiency while reducing reagent and consumable consumption and lowering detection costs. The probe-based quantitative fluorescence detection technology completes amplification and signal acquisition entirely in a closed tube, eliminating the need for subsequent electrophoresis and effectively avoiding cross-contamination. It can detect both the presence and abundance levels of drug resistance genes.

[0010] Optionally, the nucleotide sequences of the three pairs of specific primers and the three fluorescently labeled probes are as follows: HEX-CACATCGTGCGGCACATCGACGGCGT-BHQ1ROX-TCCAGGCGGTATCGACCACCAGCAC-BHQ2FAM-ACCTTGATGAGCAGCCATTAGCCGT-BHQ1 Upstream primer targeting the polymyxin resistance gene mcr-1: 5'-GATGGCGTGACCAATTTTAGCAATG-3'; Downstream primer targeting the polymyxin resistance gene mcr-1: 5'-CCAAGCGATCCAGCGTATCC-3'; Probe targeting the polymyxin resistance gene mcr-1: 5'-HEX-CACATCGTGCGGCACATCGACGGCGT-BHQ1-3'; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 5'-GGCAGCACACTTCCTATCTC-3'; Targeting carbapenem resistance genes bla NDM-1 Downstream primer: 5'-GCTTGATCCAGTTGAGGATCTG-3'; Targeting carbapenem resistance genes bla NDM-1 Probe: 5'-ROX-TCCAGGCGGTATCGACCACCAGCAC-BHQ2-3'; Upstream primer targeting the tigecycline resistance gene tet(X4): 5'-GCACCATTATTATTAGGATTCGCAA-3'; Downstream primer targeting the tigecycline resistance gene tet(X4): 5'-CACGGAAGTTGAAGAAACAGGTA-3'; Probe targeting the tigecycline resistance gene tet(X4): 5'-FAM-ACCTTGATGAGCAGCCATTAGCCGGT-BHQ1-3'.

[0011] By employing the above technical solution, all three primer pairs are designed to target highly conserved regions of the three types of drug resistance genes, effectively avoiding false negatives caused by gene sequence variations. Furthermore, the primer sequences are not homologous to the endogenous genome of *E. coli*, specifically amplifying only the target drug resistance genes without cross-reactivity, resulting in high detection specificity. The three probes are labeled with different fluorescent reporter groups and corresponding quencher groups, with non-overlapping fluorescence emission spectra. They can independently identify the three types of target genes through different fluorescence channels in the same reaction system, truly achieving single-tube triple detection without fluorescence signal crosstalk, thus improving accuracy. The primer and probe sequence combination has been optimized, amplifying the target fragment length to a suitable level, resulting in high DNA polymerase extension efficiency, effectively improving detection sensitivity and ensuring stable detection of low copy number target genes, with amplification performance approaching that of single-detection.

[0012] Optionally, in S3, the volume percentage of each component in the triple fluorescence quantitative detection reaction system is: Premixed solution: 50%; Genomic DNA template to be tested: 2%; Upstream primers targeting the polymyxin resistance gene mcr-1: 1-4%; Downstream primers targeting the polymyxin resistance gene mcr-1: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Downstream primers: 1-4%; Upstream primers targeting the tigecycline resistance gene tet(X4): 1-4%; Downstream primers targeting the tigecycline resistance gene tet(X4): 1-4%; Probes targeting the polymyxin resistance gene mcr-1: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Probe density: 1-4%; Probes targeting the tigecycline resistance gene tet(X4): 1-4%; Nuclease-free water: Balance.

[0013] By adopting the above technical solution, the volume percentage matching range of primers and probes is limited, which can effectively alleviate the competitive inhibition effect when multiple primers and probes coexist in the same system, and ensure that the three types of target genes can be amplified efficiently and synchronously in the same reaction, without the situation where the amplification advantage of a single gene excessively suppresses other genes.

[0014] Optionally, in S3, the volume percentage of each component in the triple fluorescence quantitative detection reaction system is: Premixed solution: 50%; Genomic DNA template to be tested: 2%; Upstream primer targeting the polymyxin resistance gene mcr-1: 3%; Downstream primers targeting the polymyxin resistance gene mcr-1: 3%; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 3%; Targeting carbapenem resistance genes bla NDM-1 Downstream primers: 3%; Upstream primer targeting the tigecycline resistance gene tet(X4): 3%; Downstream primers targeting the tigecycline resistance gene tet(X4): 3%; Probes targeting the polymyxin resistance gene mcr-1: 4%; Targeting carbapenem resistance genes bla NDM-1 Probes: 4%; Probe targeting the tigecycline resistance gene tet(X4): 1%; Nuclease-free water: Balance.

[0015] By employing the above technical solution, the amplification curves of the three target genes exhibit clear inflection points, high fluorescence signal intensity, and stable cycle thresholds. There is no non-specific amplification or primer dimer interference, resulting in optimal overall amplification efficiency. The amplification efficiency of different genes is balanced, avoiding the phenomenon of high-efficiency genes suppressing low-efficiency genes, ensuring that the limits of detection for all three gene types are at a high level. The coefficients of variation for cycle thresholds within and between groups are both below 4%, indicating high consistency in detection results.

[0016] Optionally, in S4, the amplification reaction conditions are: 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 59℃ annealing extension and fluorescence signal acquisition for 30s, for a total of 45 cycles.

[0017] By adopting the above technical solution and employing a two-step amplification procedure, annealing and extension are combined into a single step, simplifying the reaction process. The entire quantitative fluorescence amplification process can be completed within one hour, significantly increasing the sample detection throughput per unit time. The annealing and extension temperatures are simultaneously adapted to the annealing temperature requirements of all three primer pairs, ensuring that each primer pair can bind to the template DNA efficiently and specifically; it avoids both excessively high temperatures leading to decreased primer binding efficiency and excessively low temperatures causing non-specific amplification. Fluorescence signals are collected simultaneously during the annealing and extension stages, synchronized with the process of TaqMan probe cleavage and fluorescence release, ensuring precise signal acquisition timing and high reliability of quantitative results. A 45-cycle amplification system can cover target gene amplification down to the single-copy level, fully guaranteeing the detection rate of low-abundance clinical and environmental samples and effectively avoiding false negative results.

[0018] Optionally, the method for establishing the standard curve includes: Construct separate gene packs containing the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla. NDM-1 Recombinant plasmid standard for the target fragment of the tigecycline resistance gene tet(X4); The concentration of the recombinant plasmid standard was determined, converted to copy number, and then serially diluted 10-fold to obtain standard solutions with different copy numbers. Using standard solutions with different copy numbers as templates, amplification reactions were carried out according to the triple fluorescence quantitative detection reaction system and conditions described above, and standard curves for three types of drug resistance genes were plotted based on the amplification results.

[0019] By employing the above technical solution and using recombinant plasmids as standards, the sequences are accurate, the properties are stable, and they are easy to preserve for long periods and prepare in batches with minimal batch-to-batch variation, ensuring the reproducibility and traceability of the standard curves. A series of standard solutions of varying concentrations were prepared through 10-fold serial dilutions, covering a wide range of copy number concentrations. The resulting standard curves for the three gene classes exhibited high linear correlation coefficients, good linearity, and high quantitative accuracy. Based on the standard curves established using this method, the three types of drug resistance genes in the test samples can be quantified, and the initial copy number of the target gene in the sample can be calculated. This not only serves as a qualitative screening tool but also provides quantitative data support for the abundance analysis of drug resistance genes and the assessment of transmission risks.

[0020] Optionally, the result judgment criteria are as follows: if a certain fluorescence channel shows a typical S-type amplification curve and the cycle threshold is ≤35, then the drug resistance gene corresponding to that channel is determined to be positive; if a certain fluorescence channel has no amplification curve or the cycle threshold is >35, then the drug resistance gene corresponding to that channel is determined to be negative.

[0021] By adopting the above technical solution and using the dual criteria of amplification curve morphology and cycle threshold, false positive results caused by non-specific amplification and instrument background noise can be effectively eliminated, resulting in highly reliable test results.

[0022] Secondly, this application provides a triple probe method fluorescence quantitative detection kit for the simultaneous detection of polymyxin, carbapenems, and tigecycline resistance gene tet(X4) in porcine Escherichia coli; the kit contains: three pairs of specific primers and three fluorescently labeled probes, premixed solution, nuclease-free water, positive control and negative control.

[0023] By adopting the above technical solution, operators are no longer required to prepare single-component reagents; simply adding the nucleic acid template to be tested is sufficient to initiate the detection, significantly reducing operational errors and improving the stability of test results. The kit format facilitates storage, transportation, and standardized management. Positive and negative controls are included to avoid false positives and false negatives, ensuring the reliability of test results.

[0024] Optionally, the positive controls are those containing the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla, respectively. NDM-1 A mixture of recombinant plasmids containing the target fragment of the tigecycline resistance gene tet(X4); the negative control is nuclease-free water.

[0025] Thirdly, this application provides the above-mentioned detection kit for polymyxin resistance gene mcr-1 and carbapenem resistance gene blavin. NDM-1 And its application in the detection of the tigecycline resistance gene tet(X4).

[0026] By employing the above technical solution, the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla can be rapidly detected. NDM-1 Screening and quantification of the tigecycline resistance gene tet(X4); efficient detection of bacterial resistance in livestock and poultry farming and precise use of antimicrobial drugs.

[0027] In summary, this invention enables simultaneous single-tube detection of three classes of drug resistance genes: polymyxins, carbapenems, and tigecyclines. Compared to traditional single-nucleic acid detection methods, it improves detection efficiency while reducing reagent and material consumption and lowering detection costs. Employing probe-based quantitative fluorescence detection technology, amplification and signal acquisition are completed entirely in a closed tube, eliminating the need for subsequent electrophoresis and effectively avoiding cross-contamination. Furthermore, it can detect both the presence and abundance levels of drug resistance genes. Attached Figure Description

[0028] Figure 1 Examples of the present application include mcr-1 and bla. NDM-1、PCR identification results of tet(X4), where M: DL2000 DNA Marker; 1-3: tet(X4) gene fragment; 5-6: bla NDM-1 Gene fragments; 8-9: mcr-1 gene fragments; 4, 7, 10: negative controls.

[0029] Figure 2 This is the standard curve for the drug resistance gene mcr-1 in the embodiments of this application.

[0030] Figure 3 The drug resistance gene bla is an example of the embodiment of this application. NDM-1 The standard curve.

[0031] Figure 4 This is the standard curve for the drug resistance gene tet(X4) in the embodiments of this application.

[0032] Figure 5 This application provides an example of triple real-time quantitative PCR detection of mcr-1 and blavin. NDM-1 The standard curve of tet(X4).

[0033] Figure 6 For the specific detection results of the embodiments of this application, 1-2: DNA of mcr-1; 3-4: DNA of tet(X); 5-6: DNA of bla NDM-1 7: DNA; 8-9: DNA without the three drug resistance genes; 10: Negative control.

[0034] Figure 7 For the embodiment tet(X4) of this application, 10 0 -10 9 Sensitivity test for concentration of copies / μL, where 1-8:10 0 -10 9 Amplification curves of plasmids at copies / μL concentration; 9-10: negative controls.

[0035] Figure 8 For the embodiments of this application, bla NDM-1 10 0 -10 9 Sensitivity test for concentration of copies / μL, where 1-8:10 0 -10 9 Amplification curves of plasmids at copies / μL concentration; 9-10: negative controls.

[0036] Figure 9 For the 10 of the mcr-1 in the embodiment of this application 2 -10 9 Sensitivity test for concentration of copies / μL, where 1-8:100 -10 9 Amplification curves of plasmids at copies / μL concentration; 9-10: negative controls.

[0037] Figure 10 The results of the repeatability test in this application (tet(X4), FAM channel), where 1-3: 10 7 copies / μL; 10 6 copies / μL; 10 5 Amplification curves of plasmids at a concentration of copies / μL.

[0038] Figure 11 The repeatability test results of the embodiments of this application (bla) NDM-1 (ROX channel), where 1-3:10 7 copies / μL; 10 6 copies / μL; 10 5 Amplification curves of plasmids at a concentration of copies / μL.

[0039] Figure 12 The results of the repeatability test (mcr-1, VIC channel) for the embodiments of this application are shown, where 1-3: 10 7 copies / μL; 10 6 copies / μL; 10 5 Amplification curves of plasmids at a concentration of copies / μL. Detailed Implementation

[0040] The present application will be further described in detail below with reference to the accompanying drawings and embodiments.

[0041] Unless otherwise specified, the main components and instruments involved in the following embodiments of this application are all purchased from commercially available products.

[0042] Carrying mcr-1, bla NDM-1 The strain containing the tet(X4) single gene was isolated by our project group, and the blank control strains (Escherichia coli ATCC25922) containing the three target genes were purchased from the China Veterinary Microbial Culture Collection Center.

[0043] The real-time quantitative PCR instrument (model: Mastercycler® ep realplex) is manufactured by Eppendorf GmbH, Germany.

[0044] The real-time quantitative PCR instrument (model: QuantStudio™ 1 Plus) is manufactured by Applied Biosystems (ABI), a brand under Thermo Fisher Scientific.

[0045] The high-speed centrifuge (model: centrifuge5804R) is manufactured by Eppendorf GmbH in Germany.

[0046] The constant temperature incubator (model: BPX-82) is manufactured by Shanghai Yiheng Scientific Instruments Co., Ltd.

[0047] The temperature-controlled oscillator (model: TH2-98A) is manufactured by Shanghai Yiheng Scientific Instruments Co., Ltd.

[0048] The ultra-clean workbench (model: SW-CJ-1FD) is manufactured by Suzhou Purification Equipment Co., Ltd.

[0049] The electronic analytical balance (model: ARZ140) is manufactured by Ohaus Corporation of the United States.

[0050] The vertical pressure steam sterilizer (model: YXQ-LS-50SII) is manufactured by Shanghai Boxun Medical Bio-Instrument Co., Ltd.

[0051] The nucleic acid extractor (model: Lab-Aid820) was manufactured by Xiamen Baiweixin Biotechnology Co., Ltd.

[0052] The gel imaging system (model: GelDocXR+) is manufactured by Bio-Rad Laboratories, Inc.

[0053] The LED digital display heated metal bath (model: HB120-S) is manufactured by Beijing Dalong Instrument Co., Ltd.

[0054] The ice maker (model: SIMF140) is manufactured by SANYO Corporation of Japan.

[0055] The PCR instrument (model: Thermo-5020) was manufactured by Thermo Fisher Scientific.

[0056] The electrophoresis apparatus (model: DYY-6C) was manufactured by Beijing Liuyi Biotechnology Co., Ltd.

[0057] The pipettes were purchased from Dalong Medical Equipment Co., Ltd. and Eppendorf China Co., Ltd.

[0058] The -20℃ refrigerator (model: M1-L213C) is manufactured by Guangdong Midea Kitchen Appliances Manufacturing Co., Ltd.

[0059] The -80℃ refrigerator (model: DW-86L416G) is manufactured by Haier Biomedical Co., Ltd.

[0060] pMD19-T vector was purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0061] Pro Taq HS Premixed Probe qPCR Kit II, nuclease-free water, and 0.1 ml qPCR 96-well plates (with full skirt) were all purchased from Guangzhou Ruizhen Biotechnology Co., Ltd.

[0062] Primers and TaqMan fluorescent probes were synthesized by Guangzhou Ruizhen Biotechnology Co., Ltd.

[0063] Plasmid extraction kit and bacterial genomic DNA extraction kit were purchased from Kangwei Century Biotechnology Co., Ltd.

[0064] The gel recovery kit and plasmid DNA extraction kit were purchased from Tiangen Biotech Co., Ltd.

[0065] Escherichia coli competent cells (DH5α) were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0066] Anhydrous ethanol and other test reagents were purchased from Shanghai Hushi Laboratory Equipment Co., Ltd. Specific Implementation The following embodiments of this application use porcine Escherichia coli as an example, as detailed below.

[0068] Step 1: Establishment of a singleton quantitative PCR typing method First, primers and probes were designed and synthesized. As shown in Table 1, based on the mcr-1 and bla of Escherichia coli in GenBank... NDM-1 Primers and specific probes were designed corresponding to the tet(X4) gene sequence.

[0069] Table 1 Primer and probe sequences and target fragment sizes for real-time PCR Preparation of plasmid standards for real-time PCR Extraction of bacterial genomic DNA: Bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech Co., Ltd.

[0070] PCR amplification of the target gene: Conventional PCR amplification was performed using three different quantitative PCR primers, with mcr-1, blavin, and bla primers respectively. NDM-1 Using the DNA of the tet(X4) gene as a template, as shown in Table 2, amplification was performed in a 25 µL reaction system. Positive strains were used as positive controls, and ultrapure water was used as a negative control. Electrophoresis of the amplified products was performed using a 2% agarose gel.

[0071] Table 2 PCR amplification reaction system for Escherichia coli in pigs Amplification reaction conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles from denaturation to extension, and a final extension at 72℃ for 10 min. Electrophoresis of the amplified products was performed on a 2% agarose gel, and images were taken using a UV gel imaging system.

[0072] PCR product recovery and purification: The PCR amplification products were subjected to 2% agarose gel electrophoresis to analyze the amplification results. Each target band was cut out and purified by gel extraction using a gel extraction kit purchased from Tiangen Biotech Co., Ltd. The operation steps were as described in the kit instructions.

[0073] Ligation vector: The reaction system was prepared using Novizan's 5 min TA / Blunt-Zero Cloning Kit, as shown in Table 3. Table 3 Connection Reaction System Transformation of competent cells: Thaw DH5α competent cells frozen at -80℃ on ice. When just completely thawed, quickly add the ligation product and gently mix. Incubate on ice for 30 min, heat shock at 42℃ for 90 s, immediately place on ice for 1 min, add 700 μL of LB liquid medium, and incubate at 37℃ with shaking at 200 rpm for 60 min. Centrifuge at 5000 rpm for 2 min, discard the supernatant, add 200 μL of LB medium, mix thoroughly by pipetting, and spread on LB agar plates containing X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and Amp. Incubate overnight at 37℃ until single colonies appear.

[0074] Extraction of recombinant plasmids: Pick a single colony from the plate and inoculate it into LB liquid medium containing 25 μg / mL ampicillin. Incubate at 37°C with shaking until the bacteria become turbid. Extract the recombinant plasmid using the Novizan plasmid mini-prep kit, following the kit instructions. Store the DNA product at -20°C to prevent DNA degradation.

[0075] Screening and identification of recombinant plasmids: After single colonies were cultured from the culture plate, a single colony with neat edges and smooth morphology was picked using a 10 μL pipette tip that had been sterilized by high temperature and placed in a 1.5 mL EP tube. 1 mL of LB liquid medium containing ampicillin and kanamycin was added, and the culture was incubated at 37℃ and 180 rpm / min for 6 h on an air shaker. Using the cultured bacterial culture as a template, conventional PCR amplification was performed using three types of fluorescent quantitative PCR primers. The reaction system and procedure were the same as in 3.3.1.2. After the reaction, 7 μL of the PCR product was electrophoresed on a 2% agarose gel to detect whether the amplified DNA fragment matched the expected result. The recombinant plasmids that were correctly identified by PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing comparison to verify whether the recombinant plasmids were successfully constructed.

[0076] Preparation of recombinant plasmid standards: After the recombinant plasmids that have been successfully sequenced and verified are measured using an AA7003 UV spectrophotometer, their concentrations are converted into the corresponding copy numbers, as shown in the following formula.

[0077] Plasmid copy number (copies / μL) = The plasmids were then serially diluted in 10-fold increments, and the diluted plasmids were used as standards to establish standard curves.

[0078] Three types of genotyping quantitative PCR primers and probes were used, with mcr-1, blavin, and bla... NDM-1 Using the recombinant plasmid tet(X4) as a template, the reaction systems and conditions for the three genotyping quantitative PCR methods were optimized according to the instructions of the ProTaqHS premixed probe qPCR kit II. The reaction systems and conditions for the three methods were made to have the same reaction system and conditions, which facilitated the establishment of the triple quantitative PCR genotyping method. The final reaction system and reaction conditions are shown in Table 4.

[0079] Table 4 mcr-1, bla NDM-1 tet(X4) real-time PCR system Reaction conditions: pre-denaturation at 95℃ for 30s, denaturation at 95℃ for 5s, fluorescence signal collection at 59℃ for 30s, 45 cycles.

[0080] Establishment of a standard curve for singleton quantitative PCR: Following the established singleton quantitative PCR reaction conditions for the three drug resistance genes, a final concentration of 10 was selected. 2 -10 9 A plasmid mixture of copies / μL was used as a positive template, and Nuclease-free Water was used as a negative control. Amplification curves and standard curves were plotted.

[0081] Step 2: Establishment of a triple real-time PCR typing method for porcine Escherichia coli Optimization of the triplet real-time PCR reaction system: With both primer and probe concentrations at 10 μM, 0.2-0.8 μM primers and 0.2-0.8 μM probes were added to a 20 μL system for cross-reaction. The optimal amounts of primers and probes were screened using a matrix method, and the concentrations of primers and probes with the lowest Ct value and highest fluorescence intensity were considered the optimal concentrations. Three recombinant plasmids with a concentration of 10... 2 Using standard plasmids (copies / μL) as templates, equal volumes were mixed and subjected to triplet quantitative PCR. The reaction system and procedure were set according to the ProTaq HS Premixed Probe qPCR Kit II instructions. The total reaction volume was 20 μL, including 10 μL of 2×ProTaq HS Probe Premix II, 0.2-0.8 μL of primers at 10 μM concentration for three drug resistance genes, 0.2-0.8 μL of probes at 10 μM concentration, and 0.4 μL of template sample. The remaining volume was made up with nuclease-free water. Reaction conditions: pre-denaturation at 95℃ for 30 s, denaturation at 95℃ for 5 s, fluorescence signal collection at 59℃ for 30 s, for 45 cycles. The optimal triplet quantitative PCR reaction system was obtained based on the results of each reaction.

[0082] Establishment of triple fluorescence quantitative PCR standard curve: Take 10 concentration gradients of recombinant plasmid standards respectively. 1 copies / μL-10 10 Using copies / μL as template, the reaction was carried out according to the established triplet quantitative PCR reaction conditions. The standard copy number of the recombinant plasmid for each well was set on the instrument. After the PCR reaction, a standard curve was generated using software.

[0083] Triple quantitative PCR specificity assay: Following the optimized triple quantitative PCR system, recombinant plasmid DNA was added as a model. The specificity of the assay was verified by using a plate with Nuclease-free Water as a negative control and the corresponding drug resistance gene standard plasmid as a positive control.

[0084] Triple real-time PCR sensitivity test: 10 μL of recombinant plasmid standard was added. 1 copies / μL-10 8 Eight plasmid concentrations (copies / μL) were used as reaction templates, and triplet quantitative PCR was performed under established conditions. The lowest copy number of the standard plasmid that could amplify the curve is the sensitivity of the triplet quantitative PCR system.

[0085] Triple real-time PCR repeatability test: 10 recombinant plasmids were selected respectively. 5 10 6 10 7 Using standard plasmids at a concentration of copies / μL as templates, intra- and inter-group replicate experiments were performed according to the established triplet quantitative PCR system. Three parallel controls were set up for each concentration for inter-group replicate experiments. Simultaneously, each concentration was amplified three times consecutively for intra-group replicate experiments. The coefficient of variation (Ct) values ​​for intra- and inter-group replication were calculated using software to evaluate the repeatability of the triplet quantitative PCR system.

[0086] Step 3: Clinical sample testing using triple TaqMan qPCR for porcine E. coli. Porcine Escherichia coli isolated from farms, slaughterhouses, and farmers' markets of different sizes in Guizhou Province were collected and detected using an optimized triple real-time PCR method. The results were compared with those of conventional PCR to determine the concordance rate between the newly developed method and conventional PCR.

[0087] Recombinant plasmid PCR identification results: Based on the analysis of the tet(X4) gene and bla... NDM-1 PCR amplification of the gene and the mcr-1 gene fragment, the results are as follows: Figure 1 The results showed that the target fragment sizes were consistent with the expected product lengths of 156bp, 151bp, and 144bp.

[0088] Preparation of plasmid standards: The concentration of recombinant plasmids was determined using a UV spectrophotometer. The average value was obtained from three measurements. The concentrations of mcr-1 and blavin were also measured. NDM-1 The concentrations of the recombinant plasmids tet(X4) were 83.3 ng / μL, 90.7 ng / μL, and 139.9 ng / μL, respectively, with A260 / 280 values ​​of 1.91, 1.99, and 1.87, respectively. Substituting the concentrations of the recombinant plasmids into the formula, the copy numbers were calculated to be 4.34 × 10⁻⁶. 10 copies / μL, 6.74×10 10 copies / μL, 4.01×10 10 Copies / μL. The three standard plasmids were diluted to the same concentration of 4.01 × 10⁻⁶. 10 Copies / μL, diluted 10-fold, up to 4.01×10⁻⁶. 0 Used as a template for subsequent reactions.

[0089] Establishment of a standard curve for singleton quantitative PCR: (e.g.) Figures 2-4 As shown, with mcr-1, bla NDM-1 4.01 × 10⁴ tet(X⁴) 1 -4.01×10 8These eight different dilutions of recombinant plasmids were used to establish a standard curve for the standard. Figure 1-2 ~4). mcr-1, bla NDM-1 The correlation coefficients (R²) of the three standard curves for tet(X4) were 0.9986, 0.9976, and 0.9992, respectively. The results showed that the standard for singleton quantitative PCR was within the range of 4.01 × 10⁻⁶. 7 copies / μL - 2.62 × 10 2 Within the dilution range of copies / μL, a good linear relationship was observed.

[0090] Establishment of a standard curve for triple quantitative PCR: using the drug resistance genes mcr-1 and blavin from porcine Escherichia coli. NDM-1 Using tet(X4) genomic DNA as a template, triplet real-time PCR was performed. Primers (0.2-0.8 μM) and probes (0.2-0.8 μL) at 10 μM concentrations for the three drug resistance genes were optimized. The optimal reaction system is shown in Table 5. Table 5 Optimized System for Triple Real-Time PCR The standard with a known copy number was serially diluted to a concentration of 10-1. 1 copies / μL-10 10 Ten concentration gradients of copies / μL were calculated, and the results showed that all three standard curves exhibited good linearity. For example... Figure 5 As shown, the detection of mcr-1 and bla NDM-1 The R² values ​​of the three standard curves, tet(X4), are 0.9958, 0.9973, and 0.9985, respectively, all above 0.99.

[0091] Laboratory evaluation of triple real-time PCR typing method for porcine Escherichia coli Specificity test results of triple quantitative PCR method: mcr-1, blavin, and bla... NDM-1 Using DNA from three drug-resistant strains (tet(X4) and strains without the three drug resistance genes) as templates, the specificity of the strains was verified in laboratory evaluation, and the drug resistance genes mcr-1 and bla of *E. coli* were obtained. NDM-1 The Ct values ​​of tet(X4) were all less than 35, while the remaining Ct values ​​were greater than 35, indicating a negative result. Figure 6 As shown, it exhibits good specificity.

[0092] Sensitivity test results of triple fluorescence quantitative PCR method: Using optimized reaction conditions and system, 10 0 -10 9Sensitivity tests were performed in the laboratory to assess the concentration of copies / μL. Results are as follows: Figures 7-9 As shown, mcr-1, bla NDM-1 The limit of detection for tet(X4) is 10. 0 copies / μL.

[0093] Repeatability test results of triplet quantitative PCR method: 2.62×10 7 -2.62×10 3 Repeatability tests were performed in the laboratory evaluation using three gradients of copies / μL. The repeatability test results are as follows: Figures 10-12 As shown in Table 6, the curves overlap well and have a high degree of overlap. The CV values ​​are all below 4%, indicating good repeatability.

[0094] Table 6 Results of Within-Group and Between-Group Repeatability Tests Concordance rate detection of triple quantitative PCR method for porcine Escherichia coli: The triple quantitative PCR typing method established in this study was used to simultaneously detect porcine Escherichia coli samples from clinical porcine Escherichia coli in Guizhou Province, and the concordance rate was compared. The results are shown in Table 7. 64 strains of mcr-1 and blavin were detected by triple quantitative PCR. NDM-1 45 strains of bacterial strain and 12 strains of tet(X4) were detected, which is consistent with the positive detection rate obtained by ordinary PCR detection, with a concordance rate of 100%.

[0095] Table 7. Detection results of triple real-time PCR and conventional PCR This embodiment successfully established a method for simultaneously detecting the polymyxin resistance gene mcr-1mcr-1 (mcr-1) and the carbapenem resistance gene bla in porcine Escherichia coli. NDM-1 (bla) NDM-1 The triple probe method for quantitative fluorescence detection of the tigecycline resistance gene tet(X4) has been fully validated in terms of specificity, sensitivity, repeatability, and clinical samples. The detection method described in this application is rapid, accurate, and has high throughput, and can provide reliable technical support for the epidemiological monitoring and targeted control of multidrug-resistant Escherichia coli from livestock and poultry.

[0096] The specificity verification results show that the detection method of this application has good specificity for all three types of target genes. It only generates specific amplification signals for positive templates containing the corresponding target genes and has no cross-reaction with the Escherichia coli ATCC25922 standard strain that does not contain the target genes. The primers and probes designed for the detection method of this application are designed for conserved gene regions, which can effectively avoid non-specific amplification.

[0097] Sensitivity validation results show that the detection method of this application has excellent sensitivity, with the lowest detection limit for polymyxin resistance gene mcr-1mcr-1 and tigecycline resistance gene tet(X4) reaching 10. 2 Copy / μL, carbapenem resistance gene bla NDM-1 The lowest detection limit can reach 10 3 With a sensitivity of copies / μL, it can meet the needs of rapid screening for low-copy drug resistance genes, and its detection performance is superior to traditional conventional PCR methods.

[0098] Repeatability verification results show that the cyclic threshold variation coefficients of the detection method in this application are both less than 3% for both intra-group and inter-group detection. The method has strong stability, good repeatability, and high consistency of detection results, and is suitable for batch detection of large-scale samples.

[0099] Clinical sample testing results showed that the results obtained using the detection method described in this application had a 100% concordance rate with those obtained using the conventional PCR method, further confirming the accuracy of the detection method described in this application.

[0100] Compared with traditional drug susceptibility testing and conventional PCR methods, the detection method of this application can simultaneously perform qualitative and quantitative analysis of three types of drug resistance genes in a single reaction tube, reducing the total detection time to less than 1.5 hours. This overcomes the technical shortcomings of traditional methods, such as long detection time, low throughput, and inability to quantify. This detection method is not only suitable for detecting drug resistance genes at the strain level, but can also be extended to the rapid screening of drug resistance genes in complex matrices such as feces and environmental samples, meeting the needs of full-chain drug resistance monitoring.

[0101] The detection method proposed in this application can rapidly identify high-risk multidrug-resistant strains, track the transmission chain of drug-resistant genes, assist in the precise use of antimicrobial drugs in the breeding process, and reduce the risk of multidrug-resistant bacteria spreading.

[0102] The above are all preferred embodiments of this application, and are not intended to limit the scope of protection of this application. Therefore, all equivalent changes made in accordance with the structure, shape and principle of this application should be covered within the scope of protection of this application.

Claims

1. A triple probe method for simultaneous detection of multiple drug resistance genes, including polymyxin resistance gene mcr-1 and carbapenem resistance gene bla. NDM-1 And the tigecycline resistance gene tet(X4), characterized in that, Includes the following steps: S1. Design and synthesize drugs targeting the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla, respectively. NDM-1 Three pairs of specific primers and three fluorescently labeled probes were used to target the conserved region of the tigecycline resistance gene tet(X4). Each probe was labeled with a different fluorescent reporter group at its 5' end and a corresponding fluorescent quencher group at its 3' end. S2. Extract genomic DNA from the sample to be tested; S3. Prepare a triple fluorescence quantitative detection reaction system, wherein the reaction system comprises a premixed solution, three pairs of specific primers, three fluorescently labeled probes, genomic DNA of the sample to be tested, and nuclease-free water; S4. Place the prepared reaction system in a fluorescence quantitative amplification instrument for amplification reaction, and simultaneously collect signals of three different fluorescent reporter groups during the amplification process; S5. Determine the presence of the three types of drug resistance genes in the sample based on the amplification curve and the cycle threshold, and calculate the copy number of the three types of drug resistance genes in combination with the standard curve.

2. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 1, characterized in that, The nucleotide sequences of the three pairs of specific primers and the three fluorescently labeled probes are as follows: Upstream primer targeting the polymyxin resistance gene mcr-1: 5'-GATGGCGTGACCAATTTTAGCAATG-3'; Downstream primer targeting the polymyxin resistance gene mcr-1: 5'-CCAAGCGATCCAGCGTATCC-3'; Probe targeting the polymyxin resistance gene mcr-1: 5'-hexachlorofluorescein-CACATCGTGCGGCACATCGACGGCGT-black hole quencher 1-3'; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 5'-GGCAGCACACTTCCTATCTC-3'; Targeting carbapenem resistance genes bla NDM-1 Downstream primer: 5'-GCTTGATCCAGTTGAGGATCTG-3'; Targeting carbapenem resistance genes bla NDM-1 Probe: 5'-Rhodamine X-TCCAGGCGGTATCGACCACCAGCAC-Black Hole Quencher 2-3'; Upstream primer targeting the tigecycline resistance gene tet(X4): 5'-GCACCATTATTATTAGGATTCGCAA-3'; Downstream primer targeting the tigecycline resistance gene tet(X4): 5'-CACGGAAGTTGAAGAAACAGGTA-3'; Probe targeting the tigecycline resistance gene tet(X4): 5'-Carboxyfluorescein-ACCTTGATGAGCAGCCATTAGCCGGT-Blackhole Quencher 1-3'.

3. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 1, characterized in that, In S3, the volume percentage of each component in the triple fluorescence quantitative detection reaction system is: Premixed solution: 50%; Genomic DNA template to be tested: 2%; Upstream primers targeting the polymyxin resistance gene mcr-1: 1-4%; Downstream primers targeting the polymyxin resistance gene mcr-1: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Downstream primers: 1-4%; Upstream primers targeting the tigecycline resistance gene tet(X4): 1-4%; Downstream primers targeting the tigecycline resistance gene tet(X4): 1-4%; Probes targeting the polymyxin resistance gene mcr-1: 1-4%; Targeting carbapenem resistance genes bla NDM-1 Probe density: 1-4%; Probes targeting the tigecycline resistance gene tet(X4): 1-4%; Nuclease-free water: Balance.

4. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 3, characterized in that, In S3, the volume percentage of each component in the triple fluorescence quantitative detection reaction system is: Premixed solution: 50%; Genomic DNA template to be tested: 2%; Upstream primer targeting the polymyxin resistance gene mcr-1: 3%; Downstream primers targeting the polymyxin resistance gene mcr-1: 3%; Targeting carbapenem resistance genes bla NDM-1 Upstream primer: 3%; Targeting carbapenem resistance genes bla NDM-1 Downstream primers: 3%; Upstream primer targeting the tigecycline resistance gene tet(X4): 3%; Downstream primers targeting the tigecycline resistance gene tet(X4): 3%; Probes targeting the polymyxin resistance gene mcr-1: 4%; Targeting carbapenem resistance genes bla NDM-1 Probes: 4%; Probe targeting the tigecycline resistance gene tet(X4): 1%; Nuclease-free water: Balance.

5. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 1, characterized in that, In S4, the amplification reaction conditions are: 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 59℃ annealing extension and fluorescence signal acquisition for 30s, for a total of 45 cycles.

6. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 1, characterized in that, The method for establishing the standard curve includes: Construct separate gene packs containing the polymyxin resistance gene mcr-1 and the carbapenem resistance gene bla. NDM-1 Recombinant plasmid standard for the target fragment of the tigecycline resistance gene tet(X4); The concentration of the recombinant plasmid standard was determined, converted to copy number, and then serially diluted 10-fold to obtain standard solutions with different copy numbers. Using standard solutions with different copy numbers as templates, amplification reactions were carried out according to the triple fluorescence quantitative detection reaction system and conditions described above, and standard curves for three types of drug resistance genes were plotted based on the amplification results.

7. The triple probe method for simultaneous detection of multiple drug resistance genes according to claim 6, characterized in that, The criteria for judging the results are as follows: if a fluorescent channel shows a typical S-shaped amplification curve and the cycle threshold is ≤35, then the drug resistance gene corresponding to that channel is determined to be positive; if a fluorescent channel has no amplification curve or the cycle threshold is >35, then the drug resistance gene corresponding to that channel is determined to be negative.

8. A triple probe method fluorescence quantitative detection kit, characterized in that, This kit is used for the simultaneous detection of polymyxin, carbapenem, and tigecycline resistance genes tet(X4) in porcine Escherichia coli. The kit contains: three pairs of specific primers and three fluorescently labeled probes, premixed solution, nuclease-free water, positive control, and negative control.

9. The detection kit according to claim 8, characterized in that, The positive controls consisted of polymyxin resistance gene mcr-1 and carbapenem resistance gene bla, respectively. NDM-1 A mixture of recombinant plasmids containing the target fragment of the tigecycline resistance gene tet(X4); the negative control is nuclease-free water.

10. A detection kit according to claim 8 for detecting polymyxin resistance gene mcr-1 and carbapenem resistance gene bla NDM-1 And its application in the detection of the tigecycline resistance gene tet(X4).

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