Multiplex PCR (Polymerase Chain Reaction) detection kit and method for rapidly detecting common antibiotic-resistant bacteria

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) detection kit and a multiplex PCR detection method for rapidly detecting common antibiotic-resistant bacteria. The kit comprises a primer pair blaCTX-M-F and a primer pair blaCTX-M-R, wherein the primer pair is used for specifically amplifying a beta-lactam drug-resistant gene blaCTX-M of avian pathogenic escherichia coli; the primer pairs oqxA-F and oqxA-R are used for specifically amplifying the fluoroquinolone drug-resistant gene oqxA of the avian pathogenic escherichia coli; the primer pair aphh (3 '')-F and aphh (3'')-R is used for specifically amplifying the avian pathogenic escherichia coli aminoglycoside drug-resistant gene aphh (3 ''); the primer pairs tet (A)-F and tet (A)-R are used for specifically amplifying the tetracycline drug-resistant gene tet (A) of the avian pathogenic escherichia coli; the invention relates to a primer pair mcr-1-F and mcr-1-R for specifically amplifying a colistin drug-resistant gene mcr-1 of avian pathogenic escherichia coli. The multiplex primer provided by the invention has the advantages of difficulty in forming a dimer and a hairpin structure, uniform Tm value, moderate primer length and GC content and the like, and is beneficial to obtaining a high-quality amplification product, so that a detection result is accurate and reliable.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical drug-resistant bacteria detection, and specifically relates to a multiplex PCR detection kit and method for rapid detection of clinically common antibiotic-resistant avian pathogenic Escherichia coli. Background Art

[0002] Avian pathogenic Escherichia coli is an important pathogen that causes Escherichia coli diseases such as respiratory tract infections, pericarditis, and sepsis in poultry. Avian pathogenic Escherichia coli reduces the live weight of broilers by 2%, reduces egg production by 20% in laying hens, and has a mortality rate of 20% and 53.5% for adult chickens and chicks, respectively, causing hundreds of millions of dollars in economic losses to the global breeding industry. Antibiotics are currently the main way to prevent and treat avian pathogenic Escherichia coli infections, but their abuse has led to the continuous emergence of drug-resistant bacteria. In addition, Escherichia coli disease is a zoonosis, and the continuous emergence of drug-resistant bacteria in avian pathogenic Escherichia coli has also brought great challenges to human public health.

[0003] According to domestic and foreign research, drug-resistant avian pathogenic Escherichia coli mainly comes from two aspects. On the one hand, high-concentration antibiotics have a killing effect on sensitive bacteria, but no killing effect on resistant bacteria. Resistant bacteria gradually replace sensitive bacteria as the dominant flora, making avian pathogenic Escherichia coli gradually evolve into drug-resistant avian pathogenic Escherichia coli; on the other hand, in order to adapt to the antibiotic environment, the genes in the body of avian pathogenic Escherichia coli continue to mutate and evolve resistance genes to different antibiotics. These resistance genes can be transmitted horizontally and vertically, so that avian pathogenic Escherichia coli gradually acquire resistance. At present, the detection of drug-resistant avian pathogenic Escherichia coli mainly relies on drug sensitivity tests, such as paper diffusion method and minimum inhibitory concentration, but this method has high professional and technical requirements for instruments and operators, making it difficult to achieve the purpose of rapid detection. With the continuous development of PCR technology and in-depth research on drug-resistant genes of avian pathogenic Escherichia coli, it is possible to map the bacterial resistance by detecting the carrying status of drug-resistant genes of avian pathogenic Escherichia coli.

[0004] Extended-spectrum β-lactamase bla CTX-MIt is a class of enzymes that can hydrolyze a variety of β-lactam antibiotics, including penicillins and cephalosporins, such as ampicillin and cefotaxime. It is one of the most common resistance genes in Enterobacteriaceae and spreads rapidly around the world. oqxA is an efflux pump rotase protection repeat peptide encoded by the fluoroquinolone resistance gene. The resistance determinant cluster OqxAB encoded by it not only reduces the sensitivity of bacteria to quinolones (such as ciprofloxacin), but also reduces its sensitivity to other drugs (such as trimethoprim and chloramphenicol). It coexists with other resistance genes on plasmids, allowing resistance genes to spread rapidly between bacteria through horizontal gene transfer. aph(3") is a gene encoding aminoglycoside resistance. This gene encodes an aminoglycoside transferase, which can inactivate aminoglycoside antibiotics such as gentamicin and amikacin by transferring phosphate groups, thereby making bacteria resistant to these drugs. tet(A) is a gene encoding tetracycline resistance. It can make bacteria resistant to tetracycline antibiotics such as tetracycline, trimethoprim and doxycycline. The colistin resistance gene mcr-1 gene encodes a phosphotransferase that can transfer phosphate groups to the lipid A part of the bacteria, thereby changing its structure and reducing the ability of colistin to attach to the bacterial cell membrane, allowing bacteria to resist the bactericidal effect of colistin. Colistin is an important last resort antibiotic widely used to treat multidrug-resistant bacterial infections. The emergence of mcr-1 has attracted widespread attention in global public health.

[0005] In the current poultry farming industry, the problem of drug-resistant avian pathogenic Escherichia coli caused by the abuse of antibiotics is becoming increasingly serious, especially bacteria resistant to β-lactams, tetracyclines, aminoglycosides, fluoroquinolones and polymyxins. The existing methods for identifying drug-resistant bacteria mainly rely on drug sensitivity tests, but this method has high requirements for experimental equipment and personnel and cannot meet the actual needs of production. Therefore, the present invention provides a method for rapid detection of clinical drug-resistant avian pathogenic Escherichia coli, which uses multiple PCR to specifically amplify the β-lactam resistance gene bla of avian pathogenic Escherichia coli. CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3”), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1 to achieve the above objectives and guide the use of drugs in breeding enterprises. Summary of the invention

[0006] The purpose of the present invention is to provide a multiplex PCR detection method for rapidly detecting common antibiotic-resistant bacteria, thereby realizing rapid and efficient identification of clinical avian pathogenic Escherichia coli resistant bacteria, so as to solve the shortcomings of the existing avian pathogenic Escherichia coli resistant bacteria identification, such as high professional requirements for operators and long identification cycle, and to provide a new method for the identification of avian pathogenic Escherichia coli resistant bacteria.

[0007] The technical solution adopted by the present invention is:

[0008] A method for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M A kit for multiplex PCR of fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1, characterized in that it comprises:

[0009] Specific amplification of the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M Primer pair for bla CTX-M -F and bla CTX-M -R; primer pair oqxA-F and oqxA-R for specific amplification of the fluoroquinolone resistance gene oqxA of avian pathogenic Escherichia coli; primer pair aph(3")-F and aph(3")-R for specific amplification of the aminoglycoside resistance gene aph(3") of avian pathogenic Escherichia coli; primer pair tet(A)-F and tet(A)-R for specific amplification of the tetracycline resistance gene tet(A) of avian pathogenic Escherichia coli; primer pair mcr-1-F and mcr-1-R for specific amplification of the colistin resistance gene mcr-1 of avian pathogenic Escherichia coli;

[0010] The nucleotide sequences of each primer are:

[0011] bla CTX-M -F,5'-TTACAAACCGTTGGTGACGA-3';

[0012] bla CTX-M -R,5'-ATGGTTAAAAAATCACTGCG-3';

[0013] oqxA-F, 5'-GCAGCTTAACCTCGACTTCA-3';

[0014] oqxA-R, 5′-TTTTGTAAGCCGTCGACGAT-3′;

[0015] aph(3”)-F, 5’-TCCGTACTCCTGATGATGCATG-3’;

[0016] aph(3”)-R,5’-GATTCCGACTCGTCCAACATC-3’;

[0017] tet(A)-F,5'-TGAAACCCAACAGACCCCTG-3';

[0018] tet(A)-R,5'-CCACGATCCGCCCGATATAG-3';

[0019] mcr-1-F, 5'-CGATGCTACTGATCACCACGC-3';

[0020] mcr-1-R, 5'-CTTGGTCGGTCTGTAGGG-3'.

[0021] In a preferred embodiment of the present invention, it is characterized in that:

[0022] Primer pair bla CTX-M -F and bla CTX-M -R The size of the target fragment used for amplification is 876 bp;

[0023] The primer pair oqxA-F and oqxA-R was used to amplify the target fragment with a size of 600 bp;

[0024] The size of the target fragment amplified by primer pair aph(3″)-F and aph(3″)-R was 404 bp;

[0025] The primer pair tet(A)-F and tet(A)-R was used to amplify the target fragment with a size of 315 bp;

[0026] The primer pair mcr-1-F and mcr-1-R was used to amplify a target fragment of 162 bp.

[0027] Another aspect of the present invention also relates to a method for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M A multiplex PCR detection method for detecting fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1, wherein the method is for non-diagnostic purposes and is characterized in that it comprises the following steps:

[0028] Step 1, preparing a PCR template based on the bacteria to be detected;

[0029] Step 2, preparing a specific primer set, using the primers described in any one of claims 1 or 2 to prepare a specific primer set;

[0030] Step 3, performing a PCR amplification reaction, based on the PCR template obtained in step 1, using the specific primer set obtained in step 2 to establish a PCR reaction system including the primer set, and then using the PCR reaction system to perform a PCR amplification reaction under predetermined PCR reaction parameter conditions to obtain a PCR amplification product;

[0031] Step 4, performing electrophoresis detection, performing electrophoresis detection on the amplified product to obtain an electrophoresis result, and determining whether the avian pathogenic Escherichia coli β-lactam resistance gene bla is present according to the electrophoresis result CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3”), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1.

[0032] In a preferred embodiment of the present invention, it is characterized in that:

[0033] The PCR reaction system of step 3 includes:

[0034] 2.5 μL of 10× reaction buffer, 0.4 μL of r Taq polymerase at a concentration of 5,000 U / mL, 2 μL of dNTP at a concentration of 2.5 mM each, 2.5 μL of Mg2+ at a concentration of 25 mM, 1 μL of the PCR template, 11.6 μL of ultrapure water, and the primer set,

[0035] In the primer set, the primer pair bla CTX-M -F and bla CTX-M -R was 0.5 μL each, the volume of primer pair oqxA-F and oqxA-R was 0.5 μL each, the volume of primer pair aph(3”)-F and aph(3”)-R was 0.5 μL each, the volume of primer pair tet(A)-F and tet(A)-R was 0.5 μL each, and the volume of primer pair mcr-1-F and mcr-1-R was 0.5 μL each.

[0036] In a preferred embodiment of the present invention, it is characterized in that:

[0037] When preparing a specific primer set, the concentration of each primer used in the primer set was 10 μM.

[0038] In a preferred embodiment of the present invention, it is characterized in that:

[0039] The predetermined PCR reaction parameter conditions are: pre-denaturation at 94° C. for 4 min; denaturation at 94° C. for 30 s, annealing at 56° C. for 30 s, extension at 72° C. for 1 min, for 30 cycles; and extension at 72° C. for 10 min.

[0040] In a preferred embodiment of the present invention, it is characterized in that:

[0041] The specific process of step 4 is:

[0042] 5 μL of 6× loading buffer was added to the PCR amplification product and mixed to obtain a mixed product. 10 μL of the mixed product was spotted in a 1.0% agarose gel electrophoresis plate well. The electrophoresis was performed at 170 V for 20 min. The electrophoresis result was obtained by taking a picture under a gel imaging system. The detection result was determined based on the electrophoresis result.

[0043] The present invention also relates to the use of the above-mentioned kit in the preparation of a method for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli. CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1 in a multiplex PCR detection kit.

[0044] The beneficial effects of the present invention are:

[0045] (1) Based on the primer set provided by the present invention, the present invention can simultaneously and specifically amplify the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M Gene; specifically amplify the oqxA gene, a fluoroquinolone resistance gene of avian pathogenic Escherichia coli; specifically amplify the aph(3") gene, an aminoglycoside resistance gene of avian pathogenic Escherichia coli; specifically amplify the tet(A) gene, a tetracycline resistance gene of avian pathogenic Escherichia coli; specifically amplify the mcr-1 gene, a colistin resistance gene of avian pathogenic Escherichia coli.

[0046] (2) The multiple primers provided by the present invention have the advantages of being difficult to form dimers and hairpin structures, having uniform Tm values, and having moderate primer lengths and GC contents, which are conducive to obtaining high-quality amplification products, making the detection results accurate and reliable.

[0047] (3) The drug resistance prediction range of the method provided by the present invention includes commonly used clinical antibiotics such as ampicillin, cefotaxime, ciprofloxacin, amikacin, gentamicin, tetracycline, doxycycline, and polymyxin.

[0048] (4) The method provided by the present invention can guide the medication procedures of farms, prevent the spread of drug-resistant bacteria caused by misuse and abuse of antibiotics, and reduce the economic losses of breeding enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a single-plex PCR amplification result diagram, where M. DNA standard DL 2,000; 1.bla CTX-M ; 2.oqxA; 3.aph(3”); 4.tet(A); 5.mcr-1; 6.negative control;

[0050] Figure 2This is a graph showing the optimization results of dNTP dosage in multiplex PCR, where M. DNA standard DL 2,000; 1-7. 0.5μL, 0.75μL, 1μL, 1.25μL, 1.5μL, 1.75μL, 2μL; 8. Negative control;

[0051] Figure 3 This is the result diagram of multiplex PCR 25mM MgCl2 dosage optimization, where M. DNA standard DL 2,000; 1-7. 1.5μL, 1.75μL, 2μL, 2.25μL, 2.5μL, 2.75μL, 3μL; 8. Negative control;

[0052] Figure 4 This is the result of optimizing the dosage of rTaq enzyme in multiple PCR, where M. DNA standard DL 2,000; 1-5. 0.1μL, 0.2μL, 0.3μL, 0.4μL, 0.5μL; 6. Negative control;

[0053] Figure 5 This is the result of multiplex PCR primer dosage optimization, where M. DNA standard DL 2,000; 1-4. 0.5μL, 1μL, 1.5μL, 2μL; 5. Negative control;

[0054] Figure 6 This is a graph showing the optimization results of multiplex PCR cycle conditions, where M. DNA standard DL 2,000; 1. 20 cycles; 2. 25 cycles; 3. 30 cycles; 4. 35 cycles; 5. Negative control;

[0055] Figure 7 It is a graph of the optimization results of multiplex PCR annealing temperature, where M. DNA standard DL 2,000; 1-6. 53°C, 54°C, 55°C, 56°C, 57°C, 58°C; 7. Negative control;

[0056] Figure 8 It is a graph of the results of a multiplex PCR specificity test, wherein, M. DNA standard DL 2,000; 1-15. APEC clinical isolates; 16. negative control;

[0057] Fig. 9 This is the result of multiplex PCR sensitivity test, where M. DNA standard DL 2,000; 1-10. bacterial genome quantity 10 1 ng, 10 -1 ng, 10 -2 ng, 10 -3 ng, 10 -4 ng, 10 -5 ng, 10 -6 ng, 10 -7ng, 10 -8 ng, 10 -9 ng; 11. Negative control. DETAILED DESCRIPTION

[0058] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention. In the following examples, if no specific techniques or conditions are specified, they are all carried out according to the techniques or conditions described in the literature in the art or according to the product instructions; if the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0059] Example 1

[0060] This example provides a primer set for detecting drug-resistant genes of avian pathogenic Escherichia coli. For the primer set, how to make multiple primers not easy to form dimers and hairpin structures, have uniform Tm values, and have moderate primer lengths and GC contents, so as to be conducive to obtaining high-quality amplification products and prevent the detection results of amplification primers of different genes from interfering with each other, so as to make the detection results accurate and reliable is one of the problems to be solved urgently by technicians in this field. The primer set acquisition method of the present invention comprises the following steps:

[0061] (1) Download the corresponding gene sequence according to the gene ID of each drug resistance gene from NCBI.

[0062] (2) Import the reference sequences into the primer design software Primer5 and set the parameters. Select the output mode of the positive chain and complementary chain; the sequence amplification length is 150-1000bp; Haripin: None; Dimer: None; False Priming: None; Cross Dimer: None. Run the program to obtain a pair of specific primer sequences for the positive chain and antisense chain of each drug-resistant gene. After the primer design is completed, perform Primer-BLAST on each primer on the NCBI website to ensure the specificity of the primer. The primer sequences are shown in Table 1.

[0063] Table 1 Primer information for detection of drug resistance genes of avian pathogenic Escherichia coli

[0064]

[0065]

[0066] Example 2

[0067] This example provides a protocol for extracting complete genomic DNA of avian pathogenic Escherichia coli. The specific implementation steps are as follows:

[0068] (1) Avian pathogenic Escherichia coli was inoculated into 5 ml of autoclaved LB liquid culture medium, cultured overnight at 37°C and 180 rpm in a shaking incubator, and the bacteria were collected.

[0069] (2) Use the bacterial genomic DNA extraction kit (product number DP302) of Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract bacterial genomic DNA according to its operating procedures.

[0070] (3) The extracted bacterial genomic DNA was stored at -20°C for future use.

[0071] Example 3

[0072] Based on the primer set provided in Example 1, this example provides a reagent for detecting drug-resistant genes of avian pathogenic Escherichia coli, including:

[0073] (1) Primer sets shown in Table 1.

[0074] (2)dNTP(10mM), 10×PCR Buffer(Mg 2+ free), 25 mM MgCl2, rTaq enzyme.

[0075] (3) Negative control: nuclease-free water.

[0076] (4) Avian pathogenic Escherichia coli containing the above-mentioned drug-resistant genes is used as the positive quality control product.

[0077] Example 4

[0078] Based on the primer set in Example 1, this example provides a single-plex PCR amplification reaction system for detecting drug-resistant genes of avian pathogenic Escherichia coli to verify the feasibility of the primers. The specific reaction system is shown in Table 2.

[0079] (1) Vortex the prepared reaction system, centrifuge it briefly, place it in a PCR instrument and run the following program: initial denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 54°C for 30 s, extension at 72°C for 60 s, and final extension at 72°C for 10 min, for 30 cycles.

[0080] (2) After PCR amplification, 5 μL of PCR product was electrophoresed on a 1% agarose gel at 170 V for 20 min and the results were observed in a gel imaging system. Figure 1 As shown, the primers provided by the present invention can specifically amplify the drug-resistant genes of avian pathogenic Escherichia coli.

[0081] Table 2 Single-plex PCR amplification reaction system

[0082]

[0083] Example 5

[0084] Based on the primer set provided in Example 1 and the reaction system provided in Example 4, the reaction system was optimized according to the following steps:

[0085] (1) Optimize the amount of dNTP (10mM): The amount of other reaction systems remains unchanged, and the amount of dNTP (10mM) is set to 0.5μL, 0.75μL, 1μL, 1.25μL, 1.5μL, 1.75μL, and 2μL. Vortex the prepared reaction system, centrifuge it momentarily, place it in a PCR instrument and run the program: pre-denaturation, 94℃ for 10min; denaturation at 94℃ for 30s, annealing and extension at 54℃ for 30s, extension at 72℃ for 60s, final extension at 72℃ for 10min, 30 cycles. After PCR amplification, take 5μL of PCR product and perform electrophoresis on 1% agarose gel at 170V for 20min. Place it in a gel imaging system to observe the results. The results are as follows Figure 2 As shown, the optimal amount of dNTP (10 mM) is 2 μL.

[0086] (2) Optimize the dosage of 25mM MgCl2: The dosage of other reaction systems remains unchanged, and the dosage of 25mM MgCl2 is set to 1.5μL, 1.75μL, 2μL, 2.25μL, 2.5μL, 2.75μL, and 3μL. Vortex the prepared reaction system, centrifuge it momentarily, place it in a PCR instrument and run the program: pre-denaturation, 94℃ for 10min; denaturation at 94℃ for 30s, annealing and extension at 54℃ for 30s, extension at 72℃ for 60s, final extension at 72℃ for 10min, 30 cycles. After PCR amplification, take 5μL of PCR product and perform electrophoresis on 1% agarose gel at 170V for 20min. Place it in a gel imaging system to observe the results. The results are shown in the figure. Figure 3 As shown, the optimal amount of 25 mM MgCl2 is 2.5 μL.

[0087] (3) Optimize the dosage of rTaq enzyme (5U / μL): The dosage of other reaction systems remains unchanged, and the dosage of rTaq enzyme (5U / μL) is set to 0.1μL, 0.2μL, 0.3μL, 0.4μL, and 0.5μL. Vortex the prepared reaction system, centrifuge it instantaneously, place it in the PCR instrument and run the program: pre-denaturation, 94℃ for 10min; denaturation at 94℃ for 30s, annealing and extension at 54℃ for 30s, extension at 72℃ for 60s, final extension at 72℃ for 10min, 30 cycles. After PCR amplification, take 5μL of PCR product and perform electrophoresis on 1% agarose gel at 170V for 20min. Place it in the gel imaging system to observe the results. The results are as follows Figure 4 As shown, the optimal amount of rTaq enzyme (5U / μL) is 0.4 μL.

[0088] (4) Optimize the amount of primer set (10 μmol / L): The amount of other reaction systems remains unchanged, and the amount of primer set (10 μmol / L) is set to 0.5 μL, 1 μL, 1.5 μL, and 2 μL. Vortex the prepared reaction system, centrifuge it, place it in the PCR instrument and run the program: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 54°C for 30 s, extension at 72°C for 60 s, final extension at 72°C for 10 min, 30 cycles. After PCR amplification, take 5 μL of PCR product and perform electrophoresis on 1% agarose gel at 170 V for 20 min. Place it in the gel imaging system to observe the results. The results are as follows Figure 5 As shown, the optimal dosage of the primer set (10 μmol / L) is 0.5 μL.

[0089] In summary, the present invention provides a multiplex PCR amplification reaction system for detecting drug-resistant genes of avian pathogenic Escherichia coli. The specific reaction system is described in Table 3.

[0090] Table 3 Multiplex PCR amplification reaction system

[0091]

[0092] Example 6

[0093] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the reaction procedure was optimized according to the following steps:

[0094] (1) Optimize the reaction cycle conditions: According to the reaction system provided in Example 5, the prepared reaction system was vortexed and centrifuged, then placed in a PCR instrument and run the program: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 54°C for 30 s, final extension at 72°C for 10 min, and the cycle conditions were set to 20, 25, 30, and 35 cycles, respectively. After the PCR amplification, 5 μL of the PCR product was electrophoresed on a 1% agarose gel at 170 V for 20 min, and the results were observed in a gel imaging system. The results are shown in Figure 2. Figure 6 As shown, the optimal reaction cycle condition of the present invention is 30 cycles.

[0095] (2) Optimize the reaction annealing temperature: According to the reaction system provided in Example 5, the prepared reaction system was vortexed and centrifuged, then placed in a PCR instrument and run the program: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension were set at 53°C for 30 s, 54°C for 30 s, 55°C for 30 s, 56°C for 30 s, 57°C for 30 s, and 58°C for 30 s, extension at 72°C for 60 s, and final extension at 72°C for 10 min, 30 cycles, and after PCR amplification, 5 μL of the PCR product was electrophoresed on a 1% agarose gel at 170 V for 20 min, and the result was observed in a gel imaging system. The results are shown in Figure 2. Figure 7 As shown, the optimal annealing condition of the present invention is 56° C. for 30 s.

[0096] In summary, the present invention provides a multiplex PCR amplification reaction program for detecting drug-resistant genes of avian pathogenic Escherichia coli. The specific reaction program is: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 56°C for 30 s, extension at 72°C for 60 s, and final extension at 72°C for 10 min, for 30 cycles.

[0097] Example 7

[0098] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the multiplex PCR provided by the present invention was specifically verified using the clinically isolated strains of avian pathogenic Escherichia coli in Table 4 as positive templates (Note: the strains selected in the table have been subjected to whole genome sequencing analysis to determine the drug resistance gene carrying status), and the specific verification steps are as follows:

[0099] Table 4 Drug resistance gene carriers of clinical isolates of avian pathogenic Escherichia coli

[0100]

[0101] (1) The genome of each avian pathogenic Escherichia coli strain was extracted according to the complete genomic DNA extraction protocol in Example 2.

[0102] (2) Using the genomic DNA in the above step as a template, the prepared reaction system was vortexed and centrifuged briefly, then placed in a PCR instrument and run the program: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension at 54°C for 30 s, and final extension at 72°C for 10 min, for 30 cycles. After PCR amplification, 5 μL of the PCR product was electrophoresed on a 1% agarose gel at 170 V for 20 min, and the results were observed in a gel imaging system. The results were as follows: Figure 8 shown.

[0103] (3) The results show that the multiplex PCR provided by the present invention can specifically detect the β-lactam resistance gene bla CTX-MThe specific detection of fluoroquinolone resistance gene oqxA gene has a detection accuracy of 100%; the specific detection of aminoglycoside resistance gene aph(3") gene has a detection accuracy of 100%; the specific detection of tetracycline resistance gene tet(A) gene has a detection accuracy of 100%; the specific detection of colistin resistance gene mcr-1 gene has a detection accuracy of 100%.

[0104] Therefore, the multiplex PCR detection kit for detecting drug-resistant genes of avian pathogenic Escherichia coli provided by the present invention has excellent detection specificity.

[0105] Example 8

[0106] Based on the primer set provided in Example 1 and the reaction system provided in Example 5, the sensitivity verification of the multiplex PCR provided by the present invention is specifically performed in the following steps:

[0107] (1) The genome of avian pathogenic Escherichia coli was extracted according to the complete genomic DNA extraction protocol in Example 2.

[0108] (2) The extracted genome concentration was measured using a NanoDrop spectrophotometer, and its concentration was adjusted to 100 ng / μl, and then diluted step by step. In this way, the genomic DNA added to the corresponding reaction system was 10 ng, 1 ng, 0.1 ng, 0.01 ng, 1 picogram, and 0.1 picogram, respectively. The prepared reaction system was vortexed and centrifuged briefly, then placed in a PCR instrument and run the program: pre-denaturation, 94°C for 10 min; denaturation at 94°C for 30 s, annealing and extension were set to 54°C for 30 s, and final extension was set to 72°C for 10 min, for 30 cycles. After PCR amplification, 5 μL of PCR product was electrophoresed on a 1% agarose gel at 170V for 20 min, and the results were observed in a gel imaging system. The results are shown in the figure. Figure 8 Shown

[0109] (3) The results showed that the detection sensitivity for genomic DNA was 0.1 nanogram.

[0110] Therefore, the multiplex PCR detection kit for detecting drug-resistant genes of avian pathogenic Escherichia coli provided by the present invention has high detection sensitivity.

Claims

1. A method for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M , a multiplex PCR kit for fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1, characterized in that: include: Specific amplification of the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M Primer pair for bla CTX-M -F and bla CTX-M -R; primer pair oqxA-F and oqxA-R for specific amplification of the fluoroquinolone resistance gene oqxA of avian pathogenic Escherichia coli; primer pair aph(3")-F and aph(3")-R for specific amplification of the aminoglycoside resistance gene aph(3") of avian pathogenic Escherichia coli; primer pair tet(A)-F and tet(A)-R for specific amplification of the tetracycline resistance gene tet(A) of avian pathogenic Escherichia coli; primer pair mcr-1-F and mcr-1-R for specific amplification of the colistin resistance gene mcr-1 of avian pathogenic Escherichia coli; The nucleotide sequences of each primer are: bla CTX-M -F,5’-TTACAAACCGTTGGTGACGA-3’; bla CTX-M -R,5'-ATGGTTAAAAAATCACTGCG-3'; oqxA-F, 5'-GCAGCTTAACCTCGACTTCA-3'; oqxA-R, 5′-TTTTGTAAGCCGTCGACGAT-3′; aph(3”)-F, 5’-TCCGTACTCCTGATGATGCATG-3’; aph(3”)-R,5’-GATTCCGACTCGTCCAACATC-3’; tet(A)-F,5'-TGAAACCCAACAGACCCCTG-3'; tet(A)-R,5'-CCACGATCCGCCCGATATAG-3'; mcr-1-F, 5'-CGATGCTACTGATCACCACGC-3'; mcr-1-R, 5'-CTTGGTCGGTCTGTAGGG-3'.

2. The kit according to claim 1, characterized in that: Primer pair bla CTX-M -F and bla CTX-M -R The size of the target fragment used for amplification is 876 bp; The primer pair oqxA-F and oqxA-R was used to amplify the target fragment with a size of 600 bp; The size of the target fragment amplified by primer pair aph(3″)-F and aph(3″)-R was 404 bp; The primer pair tet(A)-F and tet(A)-R was used to amplify the target fragment with a size of 315 bp; The primer pair mcr-1-F and mcr-1-R was used to amplify a target fragment of 162 bp.

3. A method for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1, wherein the method is for non-diagnostic purposes and is characterized in that: The following steps are involved: Step 1, preparing a PCR template based on the bacteria to be detected; Step 2, preparing a specific primer set, using the primers described in any one of claims 1 or 2 to prepare a specific primer set; Step 3, performing a PCR amplification reaction, based on the PCR template obtained in step 1, using the specific primer set obtained in step 2 to establish a PCR reaction system including the primer set, and then using the PCR reaction system to perform a PCR amplification reaction under predetermined PCR reaction parameter conditions to obtain a PCR amplification product; Step 4, performing electrophoresis detection, performing electrophoresis detection on the amplified product to obtain an electrophoresis result, and determining whether the avian pathogenic Escherichia coli β-lactam resistance gene bla is present according to the electrophoresis result CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3”), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1.

4. The multiplex PCR detection method according to claim 3, characterized in that: The PCR reaction system of step 3 includes: 2.5 μL of 10× reaction buffer, 0.4 μL of r Taq polymerase at a concentration of 5,000 U / mL, 2 μL of dNTP at a concentration of 2.5 mM each, 2.5 μL of Mg2+ at a concentration of 25 mM, 1 μL of the PCR template, 11.6 μL of ultrapure water, and the primer set, In the primer set, the primer pair bla CTX-M -F and bla CTX-M -R was 0.5 μL each, the volume of primer pair oqxA-F and oqxA-R was 0.5 μL each, the volume of primer pair aph(3”)-F and aph(3”)-R was 0.5 μL each, the volume of primer pair tet(A)-F and tet(A)-R was 0.5 μL each, and the volume of primer pair mcr-1-F and mcr-1-R was 0.5 μL each.

5. The multiplex PCR detection method according to claim 3 or 4, characterized in that: When preparing a specific primer set, the concentration of each primer used in the primer set was 10 μM.

6. The multiplex PCR detection method according to claim 3 or 4, characterized in that: The predetermined PCR reaction parameter conditions are: pre-denaturation at 94° C. for 4 min; denaturation at 94° C. for 30 s, annealing at 56° C. for 30 s, extension at 72° C. for 1 min, for 30 cycles; and extension at 72° C. for 10 min.

7. The multiplex PCR detection method according to any one of claims 1 or 4 to 8, characterized in that: The specific process of step 4 is: 5 μL of 6× loading buffer was added to the PCR amplification product and mixed to obtain a mixed product. 10 μL of the mixed product was spotted in a 1.0% agarose gel electrophoresis plate well. The electrophoresis was performed at 170 V for 20 min. The electrophoresis result was obtained by taking a photo under a gel imaging system. The detection result was determined based on the electrophoresis result.

8. The kit according to any one of claims 1 or 2 is used in the preparation of a kit for detecting the β-lactam resistance gene bla of avian pathogenic Escherichia coli CTX-M , fluoroquinolone resistance gene oqxA, aminoglycoside resistance gene aph(3"), tetracycline resistance gene tet(A) and colistin resistance gene mcr-1 in a multiplex PCR detection kit.

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