Liquid-phase chip method capable of simultaneously detecting 17 drug-resistant genes in fresh milk

Abstract

The invention relates to the technical field of biology, in particular to a liquid chip method capable of simultaneously detecting 17 drug-resistant genes in fresh milk. The invention provides a nucleic acid composition for simultaneously and rapidly detecting 17 drug-resistant genes in fresh milk. The nucleic acid composition comprises 17 primers and probe sequences. According to the high-throughput detection method provided by the invention, the common drug-resistant genes in the raw and fresh milk can be quickly locked, a reference is provided for timely prevention and control of the risk of continuous propagation of the drug-resistant genes along a production chain, and an effective clinical medication guidance is provided for diagnosis and treatment of cow mastitis. The method has the advantages of being small in required sample size, rapid, high in sensitivity, wide in linear range, good in repeatability, low in comprehensive detection cost and the like.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a liquid chip method capable of simultaneously detecting 17 drug-resistant genes in raw milk. Background Art

[0002] Due to the need for disease prevention and control in the dairy farming process, the use of antibiotics is still an important means to promote healthy dairy farming, and improper use of antibiotics can lead to drug resistance in dairy cow-derived bacteria. As a result, the risk of bacterial resistance factors entering raw milk along the dairy production chain increases, and may then spread along the industry chain, directly or indirectly harming consumers.

[0003] Raw milk can carry multiple drug-resistant genes at the same time. Rapid and efficient detection of these possible drug-resistant genes can not only provide a reference for timely prevention and control of the risk of continued spread of drug-resistant genes along the production chain, but also provide guidance for timely and accurate clinical medication for bovine mastitis. Liquid chip technology is a microsphere technology established by combining the application of fluid mechanics, optics and digital signal processing, which can achieve high-throughput detection of multiple targets in a sample. It is characterized by rapidity, high throughput, good specificity and high sensitivity. xMAP TM The system is a multiplexed microsphere-based suspension array platform that can analyze and report up to 500 different reactions in a single reaction well, reducing time, labor and costs.

[0004] This study firstly comprehensively identified 17 drug-resistant genes that are prevalent and harmful in raw milk through Meta-analysis and Delphi method, covering 9 types of antibiotics commonly used in clinical practice. The detection target sequences were determined through homology analysis, and then the corresponding specific detection primers and probes were designed. xMAP TM The liquid chip detection method was established on the platform, which can detect these 17 common drug-resistant genes simultaneously in one reaction well. It has good specificity and high sensitivity, which greatly improves the detection efficiency of drug-resistant genes. This method can provide technical support for the research and application related to the risk transmission and prevention of drug-resistant genes in raw milk.

[0005] The Luminex xMAP system uses 5.6 μm polystyrene microspheres that are internally dyed with two spectrally distinct fluorescent dyes. Precise amounts of each of these fluorescent dyes are used to create an array of 100 different microsphere sets with specific spectral addresses. Each microsphere set can have a different reactant on its surface. Because the microsphere sets can be distinguished by their spectral address, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. A third fluorescent dye coupled to a reporter molecule quantifies the biomolecular interactions occurring on the microsphere surface. When the microspheres are passed through 100 TM When two independent lasers are used in the analyzer, they are interrogated individually in a fast-flowing fluid stream. A 635nm, 10mW red diode laser excites two fluorescent dyes contained in the microspheres, and a 532nm, 13mW yttrium aluminum garnet (YAG) laser excites a reporter fluorescent dye (R-phycoerythrin, Alexa 532, or Cy3) bound to the surface of the microspheres. High-speed digital signal processing sorts the microspheres according to their spectral address and quantifies the reactions on the surface. Thousands of microspheres can be examined per second, resulting in an analytical system that can analyze and report up to 100 different reactions in a single reaction vessel in just a few seconds per sample.

[0006] With the completion of the sequencing of the human genome, a wealth of information now exists to advance our understanding of genetic variation and its relationship to disease and disease treatment. As the identification of disease-causing mutations increases, the number of DNA tests to be performed in molecular laboratories has also increased, highlighting the need for rapid, accurate, and cost-effective high-throughput nucleic acid detection methods. The Luminex xMAP system is a multiplexed microsphere-based suspension array platform that is capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. In recent years, xMAP technology has been increasingly used for multiplexed high-throughput nucleic acid detection in research, commercial, and diagnostic molecular laboratory settings. The platform has been documented to meet the current and future needs of molecular laboratories. With the commercialization of numerous test kits, nucleic acid detection using xMAP technology can be easily implemented into laboratory workflows and can be automated, reducing hands-on time. As this technology matures, it will continue to improve and be incorporated into new tests. It is expected that more xMAP-based nucleic acid detection applications will be available in the future, and xMAP technology will be more widely used for quantitative nucleic acid detection and gene expression analysis. The use of a liquid phase chip platform to establish this rapid, high-throughput multidrug-resistant gene detection method can provide a new method option for laboratory testing of raw milk quality and safety, and better ensure product quality and safety and people's food safety. Summary of the invention

[0007] The object of the present invention is to provide a liquid phase chip method capable of simultaneously detecting 17 drug-resistant genes in raw milk.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a nucleic acid composition for simultaneously and rapidly detecting 17 drug-resistant genes in raw milk, comprising the following primer and probe sequences:

[0010] Table 217 Primer and probe sequences of drug resistance genes

[0011]

[0012]

[0013] As shown in SEQ ID NOs: 1 to 51.

[0014] Preferably, the 5' end of the downstream primer of the nucleic acid composition is connected to biotin, and the 5' end of the probe is connected to NH2-C 12 .

[0015] The present invention also provides a kit for simultaneously and rapidly detecting 17 drug-resistant genes in raw milk, comprising the nucleic acid composition and microspheres.

[0016] Preferably, it also includes multiple PCR Mix, MES solution, EDC solution, Tween 20 solution, SDS solution, TE solution and SAPE reporter solution.

[0017] The present invention also provides a method for simultaneously and rapidly detecting 17 drug-resistant genes in raw milk, comprising the following steps:

[0018] (1) using the sample DNA sequence as a template, performing multiplex PCR amplification using the upstream primer and the downstream primer to obtain multiplex PCR products;

[0019] (2) coupling the probe and the microspheres at a volume ratio of (1-5):50 to obtain a liquid chip working solution;

[0020] (3) The multiplex PCR product is hybridized with the liquid chip working solution in a volume ratio of (4-6):33, and then incubated with SAPE to detect the MFI value; the detection result is judged according to the MFI value; if the qualitative ratio result is ≥6, the result is positive.

[0021] Preferably, the total system of the multiplex PCR amplification is 50 μL, including 25 μL of multiplex PCR Mix, 0.5 μL of each upstream primer of each drug resistance gene, 0.5 μL of each downstream primer of each drug resistance gene, 5 μL of template DNA, and then ddH2O is added to 50 μL.

[0022] Preferably, the multiplex PCR amplification program is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 30 s; 35 cycles; and finally extension at 72°C for 10 min.

[0023] Preferably, the qualitative ratio result = MFI value of the test sample / MFI value of the blank control.

[0024] Preferably, except for the upstream primer of mecA, which is added in an amount of 0.1 to 0.4 μL, the concentration of the upstream primer of each of the other drug-resistant genes is 8 to 12 μmol / L;

[0025] Except for the downstream primer of mecA, which was added in an amount of 0.1 to 0.4 μL, the concentration of the downstream primer of each of the other drug-resistant genes was 8 to 12 μmol / L.

[0026] Preferably, the hybridization procedure in step (3) is 95°C, 5 min; 50°C, 15 min.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The high-throughput detection method provided by the present invention can quickly lock common drug-resistant genes in raw milk, provide a reference for timely blocking and controlling the risk of continuous spread of drug-resistant genes along the production chain, and provide effective clinical medication guidance for the diagnosis and treatment of bovine mastitis. The method has the advantages of small sample size, rapidity, high sensitivity, wide linear range, good repeatability, and low comprehensive detection cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0030] Figure 1 It is the specific detection result of 17 drug resistance genes;

[0031] Figure 2 The results of sensitivity testing of 17 drug-resistant genes;

[0032] Figure 3This is the common PCR gel imaging image of 17 drug resistance genes;

[0033] Figure 4 Temperature optimization results for 17 drug resistance genes;

[0034] Figure 5 Optimization results for the mecA reaction system. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Experimental Example 1

[0037] Methods:

[0038] 1. Drug resistance gene sequencing

[0039] Based on literature meta-analysis and metagenomic sequencing, this study comprehensively examined 7 indicators, including the proportion of veterinary antibiotic use, resistance gene prevalence, resistance gene loci, public health importance of corresponding antibiotics, public health hazard of corresponding resistant bacteria, resistance rate of human bacteria, and resistance rate of milk bacteria for 30 resistance genes of 9 types of antibiotics commonly used in clinical practice, and scored them. The semi-quantitative risk assessment method was used to rank the risks, and 17 resistance genes with the highest prevalence and greatest harm in raw milk were identified, which are:

[0040] β-lactam resistance genes blaSHV, blaNDM, mecA, blaZ; tetracyclines tetM, tetX, tetB; aminoglycosides ant(4')-Ia, aadA; quinolones qnrB, gyrA; macrolides ermA, ermB; sulfonamides Sul1; amide alcohols floR; lincosamides lnuA, peptides mcr-1. All the above 17 resistance genes were detected in milk samples provided by the Pathogenic Microorganism Monitoring Office of the China Animal Health and Epidemiology Center.

[0041] Table 3 Scoring of 17 drug resistance genes

[0042]

[0043]

[0044] 2. Determination of drug resistance gene sequence

[0045] About 30 sequences of each drug resistance gene in different species and strains were retrieved and downloaded from GenBank. The most conservative segment was selected as the region with high homology through analysis by bioinformatics software such as Laser gene, and used as the target sequence for designing multiple PCR primers. The corresponding NCBI sequence number is shown in Table 2. Using PrimerPlex primer design software, according to the primer and design principles, appropriate parameters were set to ensure that the melting temperature of the primer pair was 51-60°C and the size of the PCR amplification product was 100-200 bp, and the optimal set of 17 pairs of primers and probe sequences was determined, as shown in Table 2.

[0046] 3. Construction of drug resistance gene positive plasmid

[0047] The specific primers of 17 drug-resistant genes were used to amplify their target fragments by PCR method. The PCR reaction conditions were pre-denaturation at 95℃ for 10min; 35 cycles of amplification (denaturation at 95℃ for 30s; annealing at 56℃ for 30s; extension at 72℃ for 30s); and finally extension at 72℃ for 10min.

[0048] The drug-resistant gene reaction system was 12.5 μL of PCR Mix, 0.5 μL of upstream primer (10 μmol), 0.5 μL of biotin-modified downstream primer (10 μmol), 1 μL of template DNA, and water was added to make up to 25 μL.

[0049] 2% agarose gel electrophoresis was used to identify the PCR amplification product. The product was quickly purified and recovered according to the kit instructions, and then the target fragment was connected to the pMD18-T vector, and then transformed into DH5α chemical competent cells. After culturing at 37° overnight, PCR identification was performed. Positive clones were picked and inoculated into LB liquid medium containing Amp, and an appropriate amount of bacterial liquid of the positive recombinant plasmid was sent to Qingke Biotechnology Co., Ltd. for sequencing. Except for the lnuA gene, which was synthesized by Qingke Biotechnology Co., Ltd., the other 16 types were constructed.

[0050] 4. Synthesis and modification of liquid chip primers and probes

[0051] According to the information provided in the Luminex cookbook, magnetic microspheres suitable for the Luminex 200TM liquid chip detector were selected. The specific primers and probes of 17 drug resistance genes were modified as follows: biotin was added to the 5' end of the downstream primer, and NH2-C 12 The modified primers and probes were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0052] 5. Liquid chip detection method

[0053] 5.1 PCR amplification

[0054] (1) PCR system: The total volume of the reaction system was 50 μL, which contained: 2× Taq Master Mix 25 μL, 1 μL of each of the 16 primer pairs (10 μmol / L) except for mecA (0.2 μL of the primer for the mecA resistance gene), 5 μL of the positive plasmid template, and sterile deionized water to 50 μL.

[0055] (2) PCR amplification procedure: pre-denaturation at 95°C for 10 min; 35 cycles of amplification (denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 30 s); and final extension at 72°C for 10 min.

[0056] 5.2 Probe and microsphere coupling

[0057] (1) After thoroughly vortexing the microsphere stock solution with a vortex mixer and sonicating for 10 to 20 seconds, take 50 μL into a low-absorption centrifuge tube, place the centrifuge tube on a magnetic rack for 2 minutes, and discard the supernatant.

[0058] (2) Add 50 μL of 0.1 M MES (PH 4.5) to resuspend the microspheres, vortex to mix, and discard the supernatant.

[0059] (3) Add 7 μL of 0.1 M MES (pH 4.5) and vortex to mix.

[0060] (4) Add 3 μL of 5′-terminal amino-modified oligonucleotide probe (probe concentration: 20 μM / μL) and vortex to mix.

[0061] (5) Add 2.5 μL of freshly prepared EDC solution (10 mg / mL) and shake immediately after addition.

[0062] (6) Incubate at room temperature in the dark for 30 min.

[0063] (7) Add 2.5 μL of freshly prepared EDC solution (10 mg / mL) again, shake immediately after addition, and incubate at room temperature in the dark for 30 min (or repeat steps 5 and 6).

[0064] (8) Add 1 mL of 0.02% Tween-20 solution, vortex to mix, place the centrifuge tube on a magnetic rack for 2 min, and discard the supernatant.

[0065] (9) Add 1 mL of 0.1% SDS solution, vortex to mix, place on a magnetic stand for 2 min, and discard the supernatant.

[0066] (10) Add 100 μL TE (pH 8.0) solution and vortex to mix.

[0067] (11) Store at 4°C away from light until needed.

[0068] 5.3. Hybridization incubation

[0069] Adjust the microsphere concentration to 75 microspheres / μL with 1.5×TMAC hybridization buffer, take the mixed microsphere working solution (33 μL) and mix it with the multiple PCR product (5 μL), and add TE (pH=8) buffer to the total volume (50 μL). After oscillation and mixing, put it into the PCR instrument, denature at 95℃ for 5 minutes, and hybridize and incubate at 50℃ for 15 minutes.

[0070] SAPE was diluted to a working concentration of 6 μg / mL using 1× TMAC hybridization buffer, and 25 μL of SAPE reporter solution was added after the first incubation, followed by hybridization incubation at 50°C for 5 min.

[0071] 5.4. On-the-job analysis

[0072] Turn on the liquid chip detection equipment, preheat the copper plate to 50°C in advance, set the program, and test on the machine. Judge the results according to the MFI value. The liquid chip qualitative ratio result (LQRR) is equal to the ratio of the average value of the median fluorescence intensity (MFI) of the sample to the average value of the median fluorescence intensity (MFI) of the blank control. When the MFI ratio of the positive control to the blank control is ≥6, the experiment is established.

[0073] Experimental Example 2

[0074] 1. Specificity test

[0075] The other detection method steps are the same as those in Experimental Example 1, except that the upstream and downstream primers (10 μmol) of the 17 drug-resistant genes are mixed together, and the PCR system is: 12.5 μL of PCRMix, 1 μL of mixed upstream primers (10 μmol), 1 μL of mixed biotin-modified downstream primers (10 μmol), 1 μL of template DNA, and water is added to make up to 25 μL. The other PCR reaction conditions are set unchanged, and PCR amplification is performed. After the gel electrophoresis imaging results are correct, hybridization incubation with the mixed microspheres of the corresponding coupled probes is performed, and the detection results are as follows: Figure 1 As shown. It can be seen that the detection results of the target gene by this method are all positive, indicating that the established method has good specificity.

[0076] 2. Sensitivity test - minimum detection limit

[0077] After the concentration of each drug resistance gene positive plasmid was measured by a nucleic acid concentration meter, it was diluted 10 times to 10 0 (Including 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 102 , 10 1 , 10 0 ), PCR amplification was performed on the 17 drug resistance gene positive plasmids at different concentrations (same as in Experimental Example 1), and after the gel electrophoresis imaging results were correct, microsphere hybridization incubation with the corresponding coupled probes was performed, and the results were obtained as follows: Figure 2 The method has the highest sensitivity for the detection of blaZ, tetB, ant(4')-Ia, tetX, gyrA and ermA, with a minimum detection limit of 10 copies / reaction; the minimum detection limit for tetM, floR, and lnuA is 10 2 copies / reaction; the minimum detection limit for Sul1, mcr-1, mecA, qnrB and aadA was 10 3 copies / reaction; the minimum detection limit for ermB, blaNDM and blaSHV was 10 4 copies / reaction.

[0078] Experimental Example 3

[0079] Fluorescence PCR and ordinary PCR cannot detect 17 drug-resistant gene-positive plasmids in one reaction well at the same time. Currently, the fluorescence PCR instrument can detect up to five different targets in one reaction well. Compared with ordinary PCR, gel electrophoresis is qualitative, while liquid phase is quantitative, and the detection limit is smaller than PCR.

[0080] The common PCR reaction system is PCRMix 12.5μL, upstream primer (10μmol) 0.5μL, biotin-modified downstream primer (10μmol) 0.5μL, template DNA 1μL, and water to 25μL. The amplification program is: 95℃ pre-denaturation for 10min; 95℃ denaturation for 30s; 56℃ annealing for 30s; 72℃ extension for 30s; 35 cycles; and finally 72℃ extension for 10min.

[0081] Gel electrophoresis Figure 3 As shown in the figure (above), the detection limit is 10 4 Copy number / reaction, the figure (below) shows the detection limit of 10 5 Copy number / reaction, 1 to 17 are qnrB, ermB, aadA, tetB, mecA, tetM, ermA, gyrA, tetX, floR, lnuA, blaZ, blaSHV, blaNDM, ant(4')-Ia, Sul1, and mcr-1.

[0082] The specific results are: 10 5 The copy number of lanes 6, 9, 10, 14, and 17 was not detected. 4The copy number lanes 2, 5, 6, 9, 10, 14, and 17 were not detected. At this time, the ordinary PCR results showed that not all targets could be positively detected, while the liquid phase chip method had positive detection under these corresponding template concentration conditions, which shows that the liquid phase chip technology has higher sensitivity.

[0083] Experimental Example 4

[0084] Liquid chip method-optimization of annealing temperature during hybridization incubation

[0085] The experimental method was the same as that of Experimental Example 1, and the effects of different annealing temperatures (95°C, 5 min; 35, 40, 50, 55, 60°C, 15 min) in the hybridization procedure in step (3) on the detection results were investigated.

[0086] After the construction of 17 drug-resistant gene plasmids was completed, the annealing temperature was the same at 56°C in the previous study, but during the hybridization incubation stage of liquid chip technology, e.g. Figure 4 As shown, most drug-resistant genes have an optimal MFI value at 50°C. There are also drug-resistant genes such as blaNDM whose optimal temperature is not 50°C, but the MFI values ​​are not much different. Considering that they are in the same system, 50°C is selected as the annealing temperature in the hybridization incubation stage of this method.

[0087] Experimental Example 5

[0088] Reaction system optimization

[0089] The MFI value of mecA resistance gene was relatively low in this method, so the reaction system was optimized and two systems were made in total.

[0090] ① PCR system: PCR Mix 12.5 μL, mixed upstream primer (10 μmol) 0.1 μL, mixed biotin-modified downstream primer (10 μmol) 0.1 μL, template DNA 1 μL, add water to make up to 25 μL. PCR reaction conditions remain unchanged.

[0091] ②PCR system: PCR Mix 12.5 μL, mixed upstream primer (30 μmol) 0.5 μL, mixed biotin-modified downstream primer (30 μmol) 0.5 μL, template DNA 1 μL, add water to make up to 25 μL. Other steps are the same as those in Experimental Example 1.

[0092] The results are as follows Figure 5 As shown, it is appropriate to use the first type of gene initial amplification reaction system optimization scheme.

[0093] It can be seen from the above embodiments and test examples that the detection method of the present invention, which designs specific primers and probes for 17 common drug-resistant genes and uses liquid phase chip technology to identify pathogenic strains, can simultaneously detect 17 common drug-resistant genes, quickly lock the infected genes, require a small amount of sample, and has high sensitivity and strong specificity.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid composition for rapid simultaneous detection of 17 drug resistance genes in raw milk, characterized in that: The following primer and probe sequences were included: Table 1 2. The nucleic acid composition according to claim 1, characterized in that The 5' end of the downstream primer of the nucleic acid composition is connected to biotin, and the 5' end of the probe is connected to NH2-C 12 .

3. A kit for rapid simultaneous detection of 17 drug-resistant genes in raw milk, characterized in that: The method comprises the nucleic acid composition and microspheres as described in claim 1 or 2.

4. The kit according to claim 3, characterized in that It also includes multiplex PCR Mix, MES solution, EDC solution, Tween 20 solution, SDS solution, TE solution and SAPE reporter solution.

5. A method for simultaneously and rapidly detecting 17 drug-resistant genes in raw milk, characterized in that: The steps include: (1) using the sample DNA sequence as a template, performing multiplex PCR amplification using the upstream primer and the downstream primer described in claim 1 or 2 to obtain multiplex PCR products; (2) coupling the probe described in claim 1 or 2 with the microspheres at a volume ratio of (1-5):50 to obtain a liquid chip working solution; (3) The multiplex PCR product is hybridized with the liquid chip working solution in a volume ratio of (4-6):33, and then incubated with SAPE to detect the MFI value; the detection result is judged according to the MFI value; if the qualitative ratio result is ≥6, the result is positive.

6. The detection method according to claim 5, characterized in that: The total system of the multiplex PCR amplification is 50 μL, including 25 μL of multiplex PCRMix, 0.5 μL of each upstream primer of each drug resistance gene, 0.5 μL of each downstream primer of each drug resistance gene, 5 μL of template DNA, and then ddH2O is added to 50 μL.

7. The detection method according to claim 5, characterized in that: The multiplex PCR amplification program was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s; annealing at 56°C for 30 s; extension at 72°C for 30 s; 35 cycles; and finally extension at 72°C for 10 min.

8. The detection method according to claim 5, characterized in that: The qualitative ratio result = MFI value of the test sample / MFI value of the blank control.

9. The detection method according to claim 6, characterized in that: Except for the upstream primer of mecA, which was added at an amount of 0.1 to 0.4 μL, the concentration of the upstream primer of each of the other drug resistance genes was 8 to 12 μmol / L; Except for the downstream primer of mecA, which was added in an amount of 0.1 to 0.4 μL, the concentration of the downstream primer of each of the other drug-resistant genes was 8 to 12 μmol / L.

10. The detection method according to claim 5, characterized in that: The hybridization procedure in step (3) is 95°C, 5 min; 50°C, 15 min.

Images