A multiplex fluorescent primer probe composition, kit and detection method for detecting fluoroquinolone resistance genes

Abstract

The application discloses a multiplex fluorescence primer probe composition, a kit and a detection method for detecting fluoroquinolone drug resistance genes, and belongs to the technical field of biology. The multiplex fluorescence primer probe composition for detecting fluoroquinolone drug resistance genes comprises a primer pair and a specific probe for detecting plasmid-mediated fluoroquinolone drug resistance genes, and the plasmid-mediated fluoroquinolone drug resistance genes are selected from one or more of aac(6')-Ib-cr, qnrA, qnrB, qnrVC, oqxA and oqxB. The multiplex fluorescence primer probe composition, the kit and the detection method for detecting fluoroquinolone drug resistance genes provided by the application provide a rapid, comprehensive and low-cost PMQR gene detection scheme, meet the urgent needs of drug resistance monitoring in a breeding site, and solve the problems of incomplete target coverage, complicated operation and insufficient sensitivity in the prior art.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to a multiplex fluorescent primer-probe composition, kit, and detection method for detecting fluoroquinolone resistance genes. Background Technology

[0002] Fluoroquinolones (FQs) are a class of synthetic broad-spectrum antibacterial drugs with advantages such as broad antibacterial spectrum, strong antibacterial activity, and few adverse reactions, and are widely used in clinical medicine and aquaculture. However, with their widespread use, drug resistance to fluoroquinolones has become increasingly prominent, especially in aquaculture, where excessive or improper use can lead to the emergence of drug-resistant strains. Plasmid-mediated fluoroquinolone resistance genes (PMQRs) are one of the main mechanisms of resistance transmission. These resistance genes spread horizontally among different bacteria via plasmids, leading to decreased efficacy of fluoroquinolone drugs and serving as an early warning indicator of resistance spread in aquaculture environments. Unlike chromosome-mediated resistance (which usually spreads slowly through mutation accumulation), the presence of PMQR genes indicates an increased risk of resistance transmission within a region, and has important early warning value for guiding rational drug use and delaying the development of resistance.

[0003] Currently, the aquaculture industry lacks effective methods for screening for drug resistance genes. Traditional drug susceptibility testing methods are time-consuming (usually requiring 3-5 days) and have low sensitivity (difficult to detect low-abundance resistance), making them unsuitable for rapid detection. Drug resistance gene detection methods mainly include conventional PCR, sequencing, and quantitative real-time PCR. Existing conventional PCR + electrophoresis requires opening the container after PCR, a cumbersome procedure prone to contamination; sequencing is costly and time-consuming, unsuitable for rapid on-site screening; and existing quantitative real-time PCR products mostly detect only 1-3 PMQR genes such as qnrS, resulting in high false negative rates and an inability to comprehensively assess drug resistance risk.

[0004] Therefore, there is an urgent need to develop a method that can achieve comprehensive coverage and accurate and rapid detection of plasmid-mediated fluoroquinolone resistance genes in aquatic animal samples. This method should address the problems of long detection cycles and low sensitivity in traditional drug sensitivity testing, while also achieving efficient and accurate screening of resistance genes. This would provide a scientific basis for the aquaculture industry, guide aquaculture production, and improve product quality and safety. Summary of the Invention

[0005] The purpose of this invention is to provide a multiplex fluorescent primer-probe composition, kit, and detection method for detecting fluoroquinolone resistance genes, thereby overcoming at least one of the above-mentioned defects in the prior art.

[0006] To achieve this objective, the present invention adopts the following technical solution: The present invention provides a multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes, comprising: primer pairs and specific probes for detecting plasmid-mediated fluoroquinolone resistance genes, wherein the plasmid-mediated fluoroquinolone resistance genes are selected from one or more of aac(6')-Ib-cr, qnrA, qnrB, qnrVC, oqxA, and oqxB.

[0007] Preferably, the primer pair and specific probe for detecting the aac(6')-Ib-cr gene comprises: an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a specific probe as shown in SEQ ID NO.3; the primer pair and specific probe for detecting the qnrA gene comprises: an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a specific probe as shown in SEQ ID NO.6; the primer pair and specific probe for detecting the qnrB gene comprises: an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a specific probe as shown in SEQ ID NO.9; the primer pair and specific probe for detecting the qnrVC gene comprises: an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a specific probe as shown in SEQ ID NO.12; and the primer pair and specific probe for detecting the oqxA gene comprises: an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, a downstream primer as shown in SEQ ID NO.15, and a specific probe as shown in SEQ ID NO.16. The primer pair and specific probe for detecting the oqxB gene, shown in NO.14 (downstream primer) and SEQ ID NO.15 (specific probe), comprise: an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a specific probe as shown in SEQ ID NO.18.

[0008] Preferably, it further comprises a primer pair and a specific probe for detecting an internal standard, the primer pair and specific probe for detecting an internal standard comprising: an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a specific probe as shown in SEQ ID NO.21, wherein the internal standard is pUC18.

[0009] Preferably, the 5' end of the specific probe with the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21 is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group. The fluorescent reporter group is selected from FAM, HEX, ROX, and CY5, and the fluorescent quencher group is selected from MGB, BHQ1, and BHQ2.

[0010] The present invention also provides a multiplex fluorescent kit for detecting fluoroquinolone resistance genes, comprising the above-described multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes.

[0011] Preferably, it further comprises: qPCR premix, positive control, and negative control, wherein the qPCR premix contains hot-start Taq enzyme, dNTPs, and Mg²⁺. + The positive control was a mixed DNA template containing aac(6')-Ib-cr, qnrA, qnrB, oqxA, oqxB, qnrVC and pUC18 gene fragments, and the negative control was ribonuclease-free water.

[0012] Preferably, the primer-probe composition is dispensed into reaction tube A and reaction tube B. Reaction tube A contains at least one of the following: primer pair and specific probe for detecting the aac(6')-Ib-cr gene; primer pair and specific probe for detecting the qnrB gene; primer pair and specific probe for detecting the oqxA gene; and primer pair and specific probe for detecting the qnrVC gene. Reaction tube B contains a primer pair and specific probe for detecting pUC18. Reaction tube B also contains at least one of the following: primer pair and specific probe for detecting the qnrA gene; and primer pair and specific probe for detecting the oqxB gene.

[0013] The present invention also provides a multiplex fluorescence detection method for detecting fluoroquinolone resistance genes, comprising the following steps: S1: using the above-mentioned multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes or the above-mentioned multiplex fluorescent kit for detecting fluoroquinolone resistance genes to perform multiplex fluorescent PCR amplification on the nucleic acid of the sample to be tested; S2: determining the detection status of fluoroquinolone resistance genes in the nucleic acid of the sample to be tested based on the Ct value of PCR amplification, and verifying the detection effectiveness in conjunction with the amplification results of the internal standard.

[0014] Preferably, in step S1, PCR amplification is performed in reaction tube A and reaction tube B. In reaction tube A, the FAM channel detects the aac(6')-Ib-cr gene, the HEX channel detects the qnrB gene, the ROX channel detects the oqxA gene, and the CY5 channel detects the qnrVC gene; in reaction tube B, the HEX channel detects the qnrA gene, the ROX channel detects the oqxB gene, and the CY5 channel detects the internal standard.

[0015] Preferably, the judgment rule for step S2 is as follows: using a Ct value of 40 as the threshold, Ct < 40 is judged as positive, and Ct ≥ 40 or not detected is judged as negative. If the Ct value of the CY5 channel in reaction tube B is ≥ 40 or not detected, the test result is invalid. If the Ct value of the CY5 channel in reaction tube B is < 40, the test result is valid. Under the valid condition: if the FAM channel of reaction tube A is positive, the aac(6')-Ib-cr gene is judged to be positive; if the HEX channel of reaction tube A is positive, the qnrB gene is judged to be positive. If the ROX channel of reaction tube A is positive, the oqxA gene is considered positive; if the CY5 channel of reaction tube A is positive, the qnrVC gene is considered positive; if the HEX channel of reaction tube B is positive, the qnrA gene is considered positive; and if the ROX channel of reaction tube B is positive, the oqxB gene is considered positive. Multiple gene positivity is allowed simultaneously. If the Ct values ​​of the FAM, HEX, ROX, and CY5 channels of reaction tube A and the HEX and ROX channels of reaction tube B are all negative, the result is considered negative.

[0016] Preferably, the amplification program in step S1 is as follows: pre-denaturation at 95℃ for 1-2 min, denaturation at 95℃ for 5-15 s, annealing at 60℃ for 30-60 s, 40-45 cycles, with fluorescence signal collected at the end of each cycle.

[0017] The present invention also provides the use of the above-described multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes or the above-described multiplex fluorescent kit for detecting fluoroquinolone resistance genes in the detection of fluoroquinolone resistance genes or in the preparation of products for detecting fluoroquinolone resistance genes.

[0018] The beneficial effects of this invention are as follows: 1. Provide a rapid, comprehensive, and low-cost PMQR gene detection solution to meet the urgent need for drug resistance monitoring in aquaculture sites and solve problems such as incomplete target coverage, cumbersome operation, and insufficient sensitivity in existing technologies.

[0019] 2. The targeted PMQR gene is located on a mobile plasmid and can be horizontally transferred between different bacterial species, serving as a "sentinel indicator" of drug resistance spread. Monitoring changes in the prevalence of the PMQR gene can provide early warning of the risk of fluoroquinolone resistance in a region, offering greater early warning value than simply detecting chromosomal mutations.

[0020] 3. For the first time, six major PMQR genes were combined with internal standards to cover more than 80% of PMQR positive samples in aquaculture environments, solving the problems of single target and high false negative rate of existing products.

[0021] 4. Through the optimized allocation of 4 fluorescence channels, 6 targets + internal standard can be detected in 2 tubes, reducing reagent consumption by more than 50% compared with traditional singleton PCR.

[0022] 5. This invention uses the pUC18 plasmid as an exogenous internal standard, which can effectively monitor nucleic acid extraction quality and PCR inhibition, avoid false negatives, and ensure the reliability of results. In contrast, protocols without an internal standard cannot identify false negative results caused by sample extraction failures.

[0023] 6. The reagents can be packaged into lyophilized pellets, which can be transported and stored at room temperature and are convenient to use.

[0024] 7. It has no cross-reactivity with common aquatic pathogens and fish genomes, and no cross-reactivity with the aac(6')-Ib gene, exhibiting high specificity.

[0025] 8. The lowest detection limit can reach 100 copies / reaction, with high sensitivity.

[0026] 9. Intra-batch CV < 1%, inter-batch CV < 2%, indicating good repeatability.

[0027] 10. This invention uses fluorescent probes as the detection method, eliminating the need for opening the lid after PCR, thus preventing contamination problems. Attached Figure Description

[0028] Figure 1 These are amplification curves of each target and internal standard in reaction tube A and reaction tube B of this invention.

[0029] Figure 2 This is a graph showing the sensitivity test results of the aac(6')-Ib-cr target of this invention.

[0030] Figure 3 This is a graph showing the sensitivity test results of the qnrB target of this invention.

[0031] Figure 4 This is a graph showing the sensitivity test results of the oqxA target of this invention.

[0032] Figure 5 This is a graph showing the sensitivity test results of the qnrVC target of this invention.

[0033] Figure 6 This is a graph showing the sensitivity test results of the qnrA target of this invention.

[0034] Figure 7 This is a graph showing the sensitivity test results of the oqxB target of this invention.

[0035] Figure 8 This is a graph showing the sensitivity test results of the internal standard pUC18 in this invention.

[0036] Figure 9 The concentration of this invention is 10. 3 10 5 10 7 Figure showing the specificity detection results of copy / reaction of the aac(6')-Ib plasmid and positive and negative controls. Detailed Implementation

[0037] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0038] This embodiment provides a multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes, comprising: primer pairs and specific probes for detecting plasmid-mediated fluoroquinolone resistance genes, and primer pairs and specific probes for detecting internal standards. The plasmid-mediated fluoroquinolone resistance genes include aac(6')-Ib-cr, qnrA, qnrB, qnrVC, oqxA, and oqxB.

[0039] The primer pair and specific probe used to detect the aac(6')-Ib-cr gene include: an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a specific probe as shown in SEQ ID NO.3; The primer pairs and specific probes used to detect the qnrA gene include: an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a specific probe as shown in SEQ ID NO.6; The primer pairs and specific probes used to detect the qnrB gene include: an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a specific probe as shown in SEQ ID NO.9; The primer pairs and specific probes used to detect the qnrVC gene include: an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a specific probe as shown in SEQ ID NO.12; The primer pair and specific probe used to detect the oqxA gene include: an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a specific probe as shown in SEQ ID NO.15; The primer pair and specific probe used to detect the oqxB gene include: an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a specific probe as shown in SEQ ID NO.18; The primer pair and specific probe used for detecting the internal standard include: an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a specific probe as shown in SEQ ID NO.21; The internal standard in this embodiment is pUC18.

[0040] The nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21 have a fluorescent reporter group at the 5' end and a fluorescent quencher group at the 3' end. The fluorescent reporter group is selected from FAM, HEX, ROX, and CY5, and the fluorescent quencher group is selected from MGB, BHQ1, and BHQ2.

[0041] This embodiment also provides a multiplex fluorescent kit for detecting fluoroquinolone resistance genes, comprising the above-described multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes.

[0042] It also includes: qPCR premix, positive control, and negative control. The qPCR premix contains hot-start Taq enzyme, dNTPs, and Mg²⁺. + The positive control was a mixed DNA template containing aac(6')-Ib-cr, qnrA, qnrB, oqxA, oqxB, qnrVC and pUC18 gene fragments, and the negative control was ribonuclease-free water.

[0043] In this embodiment, the primer and probe composition is dispensed into reaction tube A and reaction tube B. Reaction tube A contains primer pairs and specific probes for detecting the aac(6')-Ib-cr gene, primer pairs and specific probes for detecting the qnrB gene, primer pairs and specific probes for detecting the oqxA gene, and primer pairs and specific probes for detecting the qnrVC gene. Reaction tube B contains primer pairs and specific probes for detecting pUC18, primer pairs and specific probes for detecting the qnrA gene, and primer pairs and specific probes for detecting the oqxB gene.

[0044] The kit may be a lyophilized formulation containing lyophilized reaction reagents, which contain the primer-probe composition and qPCR premix before lyophilization.

[0045] This embodiment also provides a multiplex fluorescence detection method for detecting fluoroquinolone resistance genes, including the following steps: S1: Multiplex fluorescent PCR amplification of the nucleic acid in the test sample was performed using the aforementioned multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes or the aforementioned multiplex fluorescent kit for detecting fluoroquinolone resistance genes. PCR amplification was performed in reaction tubes A and B. In reaction tube A, the FAM channel detected the aac(6')-Ib-cr gene, the HEX channel detected the qnrB gene, the ROX channel detected the oqxA gene, and the CY5 channel detected the qnrVC gene; in reaction tube B, the HEX channel detected the qnrA gene, the ROX channel detected the oqxB gene, and the CY5 channel detected the internal standard. The amplification program was: 95℃ pre-denaturation for 1-2 min, 95℃ denaturation for 5-15 s, 60℃ annealing for 30-60 s, for 40-45 cycles. Fluorescence signals were collected at the end of each cycle.

[0046] S2: Determine the detection status of fluoroquinolone resistance genes in the nucleic acid of the sample based on the Ct value of PCR amplification, and verify the detection effectiveness in combination with the amplification results of the internal standard.

[0047] The judgment rule for step S2 is as follows: A Ct value of 40 is used as the threshold; Ct < 40 is judged as positive, and Ct ≥ 40 or not detected is judged as negative. If the Ct value of the CY5 channel in reaction tube B is ≥ 40 or not detected, the test result is invalid; if the Ct value of the CY5 channel in reaction tube B is < 40, the test result is valid. Under valid conditions: if the FAM channel in reaction tube A is positive, the aac(6')-Ib-cr gene is judged to be positive; if the HEX channel in reaction tube A is positive, the qnrB gene is judged to be positive. If the ROX channel of reaction tube A is positive, the oqxA gene is considered positive; if the CY5 channel of reaction tube A is positive, the qnrVC gene is considered positive; if the HEX channel of reaction tube B is positive, the qnrA gene is considered positive; and if the ROX channel of reaction tube B is positive, the oqxB gene is considered positive. Multiple gene positivity is allowed simultaneously. If the Ct values ​​of the FAM, HEX, ROX, and CY5 channels of reaction tube A and the HEX and ROX channels of reaction tube B are all negative, the result is considered negative.

[0048] This embodiment also provides the application of the above-described multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes or the above-described multiplex fluorescent kit for detecting fluoroquinolone resistance genes in the detection of fluoroquinolone resistance genes or in the preparation of products for detecting fluoroquinolone resistance genes.

[0049] I. Target gene and internal standard combination The following six plasmid-mediated fluoroquinolone resistance genes and one exogenous internal standard are included, as shown in Table 1: Table 1. Target Gene and Internal Standard Combinations

[0050] II. Two-pipe allocation scheme This invention rationally distributes 6 targets and 1 exogenous internal standard into 2 reaction tubes, with each tube distinguished by 4 fluorescence channels, as shown in Table 2: Table 2 Target and Internal Standard Allocation Table

[0051] III. Primer and Probe Sequences The primer-probe composition of this embodiment contains the sequences shown in Table 3: Table 3 Primer-Probe Composition Sequence Listing

[0052] IV. Kit Components and Formulation 4.1 Components of Reaction Tube A (20 μL per reaction): Contains a primer and probe composition for the detection of aac(6')-Ib-cr, qnrB, oqxA, and qnrVC. See Table 4 for details. Table 4 Composition of Reaction Tube A

[0053] 4.2 Components of reaction tube B (20 μL per reaction): Contains a primer and probe composition for detecting qnrA, oqxB, and the internal standard pUC18. See Table 5: Table 5 Composition of reaction tube B

[0054] Note: 1) The 4× qPCR premix contains hot-start Taq enzyme, dNTPs, and Mg²⁺. + and buffer solutions, etc.

[0055] 2) The total volume of the reaction system is 20 μL, which is 15 μL of reaction solution + 5 μL of template.

[0056] 3) This reagent can also be prepared as lyophilized beads, aliquoted into PCR tubes, and sealed in aluminum foil bags for storage at room temperature. When using, simply add the appropriate number of lyophilized beads directly to the template.

[0057] 4.3 Positive / Negative Controls This kit also includes one tube of positive control and one tube of negative control: 1) Positive control: A mixed DNA template containing gene fragments from aac(6')-Ib-cr, qnrA, qnrB, oqxA, oqxB, qnrVC, and pUC18. The primary function of the positive control is to demonstrate that the entire PCR reaction system, from primers, enzymes, buffers to the thermal cycling procedure, is functioning correctly in the current experiment.

[0058] 2) Negative control: RNase-Free Water. The negative control is a sample known to be free of any target nucleic acid to be detected (in this invention, it refers to the six drug resistance genes aac(6')-Ib-cr, qnrA, qnrB, oqxA, oqxB, and qnrVC), used to monitor whether the entire detection process is subject to non-specific contamination or interference.

[0059] V. Testing Methods (a) Sample preprocessing 1. Fish tissue: Take an appropriate amount of fish gills or intestines and other tissue samples into a homogenization bag, weigh them, add 3-6 times the amount of physiological saline and grind them thoroughly into a homogenate. Take 100 μL of the homogenate supernatant (avoiding unground tissue fragments) for extraction.

[0060] 2. Bacterial solution: Extract directly from 100 μL of bacterial solution.

[0061] (II) Nucleic acid extraction from samples Sample extraction using the magnetic bead automated nucleic acid extraction reagent: Add 100 μL of the above-prepared sample and 20 μL of internal control solution to the sample well of the reagent strip. Place the sample-added reagent strip into the nucleic acid extractor for nucleic acid extraction. The instrument automatically performs sample lysis, nucleic acid binding with magnetic beads, washing of the magnetic bead-nucleic acid conjugate, and nucleic acid elution, finally obtaining pure nucleic acid.

[0062] (III) Preparation of PCR reaction solution Prepare reaction tubes A and B according to the required number of reactions, referring to the kit formulation tables in 4.1 and 4.2, and aliquot them into single PCR tubes or 8-tube strips at 15 μL / tube. Alternatively, use lyophilized reagent beads directly.

[0063] (iv) Template addition Using a micropipette, add 5 μL of the corresponding sample nucleic acid or negative / positive control to reaction tube A and reaction tube B respectively, and immediately cap the tubes tightly, mix thoroughly and centrifuge briefly.

[0064] Note: If using freeze-dried balls, the template addition amount is 20 μL.

[0065] (v) PCR amplification 1) Place the centrifuged reaction tubes sequentially into the PCR reaction chamber of the instrument and tighten the chamber lid. A and B reaction tubes of the same sample must be placed adjacent to each other. The amplification reaction program settings are shown in Table 6: Table 6 Amplification Reaction Procedure

[0066] 2) After the program finishes running (approximately 1 hour), remove the PCR thin-walled reaction tube (closed tube) and place it in a sealed bag. Seal the bag tightly and treat it as a source of contamination. Amplification results are as follows: Figure 1 As shown.

[0067] (vi) Interpretation of results The detection status of each drug resistance gene was determined based on the Ct values ​​provided by the instrument, as shown in Table 7: Table 7. Detection of each drug resistance gene

[0068] VI. Key Experimental Data Refer to section V, Detection Methods, for nucleic acid extraction and PCR amplification experiments: 1. Sensitivity Verification Experimental method: Positive plasmids containing 6 drug resistance genes were diluted to 10⁻⁶. 5 104 10 3 10 2 10 1 Copy / reaction (internal standard pUC18 concentration was 10) 5 (Copy / reaction), each concentration was repeated 3 times.

[0069] Experimental data: Detection data (average Ct) for each target and internal standard are shown in Table 8: Table 8 Detection data for each target and internal standard

[0070] Conclusion: The sensitivity experiment results are as follows Figure 2-8 As shown. The overall sensitivity of the system can reach 100 copies / reaction.

[0071] 2. Repeatability verification Experimental method: Use a medium concentration of positive control (10 4 (Copy / Reaction), test 3 different batches (inter-batch repeatability), 3 reaction tubes per batch (intra-batch repeatability), and calculate the repeatability of Ct values ​​for each target and internal standard.

[0072] Experimental data: Detection data for each target are shown in Table 9. Table 9 Detection data for each target

[0073] Conclusion: Intra-batch CV < 1%, inter-batch CV < 2%, indicating good reproducibility.

[0074] 3. Specificity verification 3.1 Validation of cross-reactivity of homologous genes Experimental methods: The aac(6')-Ib gene, which has the highest homology with aac(6')-Ib-cr, was selected for cross-reactivity validation. Concentrations of 10-1 were measured. 3 10 5 10 7 Copy / reaction of aac(6')-Ib plasmid and positive and negative controls.

[0075] Experimental data: The results of cross-reactivity verification of homologous genes are shown in Table 10: Table 10 Results of Homologous Gene Cross-Reactivity Detection

[0076] Conclusion: Specificity test results are as follows Figure 9 As shown, the reagent of this invention can specifically detect the aac(6')-Ib-cr gene and has no cross-reactivity with the aac(6')-Ib gene, which has a homology of over 95%, even at 10 7The fact that no nonspecific amplification occurs even at high concentrations of copy / reaction indicates that the present invention has good specificity.

[0077] 3.2 Verification of cross-reactivity of nucleic acids in common aquatic pathogens Experimental methods: Ten strains of fish isolated from fish samples and confirmed by sequencing to be free of the six drug resistance genes of this invention were selected for cross-reaction verification, along with three healthy fish tissue samples.

[0078] Experimental data: The detection data are shown in Table 11: Table 11 Results of cross-reactivity detection of nucleic acids in common aquatic pathogens

[0079] Note: "+" indicates positive, and "-" indicates negative.

[0080] Conclusion: No cross-reactivity with common aquatic pathogens, 100% specificity.

[0081] 4. Clinical sample validation Experimental methods: Thirty-two strains isolated from fish samples were collected and detected using the reagents of this invention and sequencing, and the concordance rate was calculated.

[0082] Experimental data: The detection of drug resistance genes in 32 bacterial strains is shown in Table 12: Table 12 Results of drug resistance gene detection in 32 bacterial strains

[0083] The number of positive samples detected for each target and the statistical results of the sequencing are shown in Table 13. Table 13 Statistical table of the number of positive samples detected for each target and the concordance rate with sequencing

[0084] Conclusion: 32 clinical samples were validated, and the consistency rate with the sequencing results was 100%.

[0085] This invention is the first to monitor fluoroquinolone resistance in farmed fish, screening out combinations of six PMQR genes plus exogenous internal standards. Specific sequences for each target ensure no cross-reactivity. The six targets and one exogenous internal standard are rationally allocated to two tubes, each separated by four fluorescence channels. The pUC18 plasmid is used as the internal standard to monitor PCR inhibition. Furthermore, the reagent components can be packaged into lyophilized beads for room temperature transport and use, and are easily adapted to automated platforms.

[0086] This invention has the following beneficial effects: 1. Focusing on the risk of drug resistance transmission, this invention provides early warning value: The PMQR gene targeted in this invention is located on a mobile plasmid and can be horizontally transferred between different bacterial species, serving as a "sentinel indicator" of drug resistance spread. Monitoring changes in the prevalence of the PMQR gene can provide early warning of the risk of fluoroquinolone resistance in a region, offering greater early warning value than simply detecting chromosomal mutations.

[0087] 2. Comprehensive target combination: For the first time, six major PMQR genes are combined with internal standards, covering more than 80% of PMQR positive samples in the breeding environment, solving the problems of single target and high false negative rate of existing products.

[0088] 3. Low-cost two-tube design: Through optimized allocation of 4 fluorescence channels, 6 targets + internal standard can be detected in 2 tubes, reducing reagent consumption by more than 50% compared with traditional singleton PCR.

[0089] 4. Exogenous internal standard quality control: pUC18 plasmid is used as an exogenous internal standard, which can effectively monitor the quality of nucleic acid extraction and PCR inhibition, avoid false negatives, and ensure the reliability of results.

[0090] 5. Easy to operate and stable at room temperature: The reagent can be packaged into lyophilized pellets, which can be transported and stored at room temperature and are convenient to use.

[0091] 6. High specificity: No cross-reaction with common aquatic pathogens and fish genomes, and no cross-reaction with the aac(6')-Ib gene.

[0092] 7. High sensitivity: The lowest detection limit can reach 100 copies / reaction.

[0093] 8. Good repeatability: intra-batch CV <1%, inter-batch CV <2%.

[0094] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes, characterized in that, Include: Primer pairs and specific probes for detecting plasmid-mediated fluoroquinolone resistance genes; The plasmid-mediated fluoroquinolone resistance gene is selected from one or more of aac(6')-Ib-cr, qnrA, qnrB, qnrVC, oqxA, and oqxB; The primer pair and specific probe used to detect the aac(6')-Ib-cr gene include: an upstream primer as shown in SEQ ID NO.1, a downstream primer as shown in SEQ ID NO.2, and a specific probe as shown in SEQ ID NO.3; The primer pairs and specific probes used to detect the qnrA gene include: an upstream primer as shown in SEQ ID NO.4, a downstream primer as shown in SEQ ID NO.5, and a specific probe as shown in SEQ ID NO.6; The primer pairs and specific probes used to detect the qnrB gene include: an upstream primer as shown in SEQ ID NO.7, a downstream primer as shown in SEQ ID NO.8, and a specific probe as shown in SEQ ID NO.9; The primer pairs and specific probes used to detect the qnrVC gene include: an upstream primer as shown in SEQ ID NO.10, a downstream primer as shown in SEQ ID NO.11, and a specific probe as shown in SEQ ID NO.12; The primer pair and specific probe used to detect the oqxA gene include: an upstream primer as shown in SEQ ID NO.13, a downstream primer as shown in SEQ ID NO.14, and a specific probe as shown in SEQ ID NO.15; The primer pairs and specific probes used to detect the oqxB gene include: an upstream primer as shown in SEQ ID NO.16, a downstream primer as shown in SEQ ID NO.17, and a specific probe as shown in SEQ ID NO.

18.

2. The multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes according to claim 1, characterized in that: It also includes primer pairs and specific probes for detecting internal standards; The primer pair and specific probe for detecting the internal standard include: an upstream primer as shown in SEQ ID NO.19, a downstream primer as shown in SEQ ID NO.20, and a specific probe as shown in SEQ ID NO.

21.

3. The multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes according to claim 2, characterized in that: The nucleotide sequences shown in SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.9, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.18, and SEQ ID NO.21 have a fluorescent reporter group labeled at the 5' end and a fluorescent quencher group labeled at the 3' end. The fluorescent reporter group is selected from one of FAM, HEX, ROX, and CY5, and the fluorescent quencher group is selected from one of MGB, BHQ1, and BHQ2.

4. A multiplex fluorescent reagent kit for detecting fluoroquinolone resistance genes, characterized in that: The multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes as described in any one of claims 1-3.

5. The multiplex fluorescent reagent kit for detecting fluoroquinolone resistance genes according to claim 4, characterized in that, Also includes: qPCR premix, positive control, and negative control; The qPCR premix contains hot-start Taq enzyme, dNTPs, and Mg²⁺. + and buffer solution; The positive control is a mixed DNA template containing the gene fragments aac(6')-Ib-cr, qnrA, qnrB, oqxA, oqxB, qnrVC and pUC18; The negative control was ribonuclease-free water; The primer-probe composition was dispensed into reaction tube A and reaction tube B; The reaction tube A contains at least one of the following: primer pair and specific probe for detecting the aac(6')-Ib-cr gene, primer pair and specific probe for detecting the qnrB gene, primer pair and specific probe for detecting the oqxA gene, and primer pair and specific probe for detecting the qnrVC gene. The reaction tube B contains primer pairs and specific probes for detecting pUC18; The reaction tube B also contains at least one of a primer pair and a specific probe for detecting the qnrA gene, and a primer pair and a specific probe for detecting the oqxB gene.

6. A multiplex fluorescence detection method for detecting fluoroquinolone resistance genes, characterized in that, Includes the following steps: S1: Perform multiplex fluorescent PCR amplification on the nucleic acid of the sample to be tested using the multiplex fluorescent primer and probe composition for detecting fluoroquinolone resistance genes as described in any one of claims 1-3 or the multiplex fluorescent kit for detecting fluoroquinolone resistance genes as described in any one of claims 4-5. S2: Determine the detection status of fluoroquinolone resistance genes in the nucleic acid of the sample based on the Ct value of PCR amplification, and verify the detection effectiveness in combination with the amplification results of the internal standard.

7. The multiplex fluorescence detection method for detecting fluoroquinolone resistance genes according to claim 6, characterized in that: In step S1, the PCR amplification is performed in reaction tube A and reaction tube B. In reaction tube A, the FAM channel detects the aac(6')-Ib-cr gene, the HEX channel detects the qnrB gene, the ROX channel detects the oqxA gene, and the CY5 channel detects the qnrVC gene; in reaction tube B, the HEX channel detects the qnrA gene, the ROX channel detects the oqxB gene, and the CY5 channel detects the internal standard. The determination rule for step S2 is as follows: Using a Ct value of 40 as the threshold, Ct < 40 is considered positive, and Ct ≥ 40 or not detected is considered negative; If the Ct value of the CY5 channel in reaction tube B is ≥40 or not detected, the detection result is invalid. If the Ct value of channel CY5 in reaction tube B is < 40, the detection result is valid. Under valid conditions: If the FAM channel of reaction tube A is positive, then the aac(6')-Ib-cr gene is considered positive. If the HEX channel of reaction tube A is positive, then the qnrB gene is determined to be positive. If the ROX channel of reaction tube A is positive, then the oqxA gene is determined to be positive; If the CY5 channel in reaction tube A is positive, then the qnrVC gene is considered positive. If the HEX channel of reaction tube B is positive, then the qnrA gene is determined to be positive. If the ROX channel in reaction tube B is positive, then the oqxB gene is considered positive. Multiple gene positivity is allowed simultaneously; If the Ct values ​​of the FAM, HEX, ROX, and CY5 channels of reaction tube A and the HEX and ROX channels of reaction tube B are all negative, then it is determined to be negative.

8. The multiplex fluorescence detection method for detecting fluoroquinolone resistance genes according to claim 6, characterized in that, The amplification procedure for step S1 is as follows: Pre-denaturation at 95℃ for 1-2 min; Denaturation at 95℃ for 5-15 seconds, annealing at 60℃ for 30-60 seconds, 40-45 cycles; Fluorescence signals are collected at the end of each cycle.

9. The use of the multiplex fluorescent primer-probe composition for detecting fluoroquinolone resistance genes as described in any one of claims 1-3 or the multiplex fluorescent kit for detecting fluoroquinolone resistance genes as described in any one of claims 4-5 in the detection of fluoroquinolone resistance genes or in the preparation of products for detecting fluoroquinolone resistance genes.

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