Primer combination for detecting staphylococcus aureus and drug-resistant gene and application of primer combination

Abstract

The invention belongs to the technical field of gene engineering, and discloses a primer combination for detecting staphylococcus aureus and a drug-resistant gene and application of the primer combination, the primer combination comprises primer pairs which are respectively designed by taking a species-specific identification gene of the staphylococcus aureus and at least two antibiotic drug-resistant genes as target genes, the primer pair is composed of a common primer and a molecular beacon probe with primer and probe functions. When the primer combination is used as a primer of the RAA kit, the detection efficiency can be remarkably improved, and the amplification specificity and sensitivity are improved. The RAA method based on the primer combination can realize triple fluorescent quantitative detection, and pathogenic microorganisms and drug-resistant genes thereof can be detected at the same time. The method is suitable for normal-temperature amplification and detection of target genes.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and relates to a primer combination for detecting Staphylococcus aureus and drug-resistant genes and an application thereof. Background Art

[0002] Staphylococcus aureus is an important infectious pathogen that causes milk mastitis, accounting for more than 50% of the total number of pathogens. At present, antibiotics are still the main drugs for the treatment of staphylococcal bovine mastitis. However, due to the irrational use of antibiotics, the resistance of Staphylococcus aureus to commonly used antibiotics in clinical practice has become increasingly serious. Staphylococcus aureus gradually develops adaptive resistance to antimicrobial drugs through changes in its own phenotype and metabolic pathways. At the same time, it can also acquire resistance genes through horizontal transfer, thereby showing resistance to various antimicrobial drugs, leading to the emergence of multi-drug resistant strains and rapid spread.

[0003] Among Staphylococcus aureus that has acquired drug-resistant genes, methicillin-resistant Staphylococcus aureus (MRSA) has been infected almost all over the world since its discovery. At present, the detection of MRSA is mainly carried out around the two aspects of drug resistance phenotype and genotype. The Clinical and Laboratory Standards Institute (CLSI) recommends the use of drug sensitivity paper method, oxacillin (OXA) salt agar screening method, β-lactamase detection, E-test and test tube double dilution method to identify MRSA. However, since the expression of methicillin resistance phenotype is usually uneven, it is easily affected by various culture conditions, resulting in missed detection or false detection, and the above detection methods have a long experimental cycle and high requirements on the operation level and equipment of technicians.

[0004] In addition to MRSA, tetracycline antibiotics have a broad antibacterial spectrum. Due to their low price, wide range of action and low toxicity, they are also widely used as antimicrobial growth promoters for livestock and poultry. Therefore, for Staphylococcus aureus that has both oxacillin resistance genes and tetracycline resistance genes, if the species-specific identification genes, oxacillin resistance genes and tetracycline resistance genes of Staphylococcus aureus are tested one by one, there will be problems such as large sample size, complex and time-consuming process, which is not conducive to promotion at the grassroots and on-site. Summary of the invention

[0005] One purpose of the present invention is to provide a primer combination for detecting Staphylococcus aureus and drug-resistant genes and a fluorescent quantitative recombinase-mediated isothermal amplification method based on the primer combination, wherein one primer of the recombinase-mediated isothermal amplification detection (RAA) technology is an improved molecular beacon (IMB) probe having a single-stranded region after forming a stem-loop structure, and a specific identification gene of Staphylococcus aureus and two or more drug-resistant genes are respectively used as target genes, and a corresponding "molecular beacon probe + common primer" is designed to form a primer combination for isothermal amplification, so as to realize the simultaneous detection of two or more drug-resistant genes without having to detect them one by one, thereby achieving the purpose of improving detection efficiency and enhancing amplification specificity and sensitivity;

[0006] Another object of the present invention is to provide the use of the above primer combination for detecting Staphylococcus aureus and drug-resistant genes in a recombinase-mediated isothermal amplification detection kit to achieve the purpose of convenient use and reduce the requirements for detection personnel and equipment.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] A primer combination for detecting Staphylococcus aureus and drug-resistant genes, the primer combination comprising a primer pair Y1 designed with a species-specific identification gene I of Staphylococcus aureus as a target gene, a primer pair Y2 designed with an antibiotic resistance gene II as a target gene, and a primer pair Y3 designed with an antibiotic resistance gene III as a target gene;

[0009] The primer pairs are composed of a molecular beacon probe and a primer;

[0010] The molecular beacon probe comprises, from the 5' end to the 3' end, an N1 region for forming a stem portion of a stem-loop structure, an M region for forming a loop portion of a stem-loop structure, an N2 region for forming a stem portion of a stem-loop structure, and a single-stranded region;

[0011] The bases of the N1 region and the N2 region are complementary to each other; the 5' end of the N1 region has a fluorescent group binding site, and the 3' end of the N2 region has a quenching group binding site that matches the fluorescent group. The excitation light wavelengths of the fluorescent groups of the molecular beacon probes in different primer pairs are different;

[0012] The M region used to form the loop of the stem-loop structure, the N2 region used to form the stem of the stem-loop structure, and the single-stranded region are sequentially complementary to the bases at one end of the target gene. That is, the M region, the N2 region, and the single-stranded region assume the function of pairing the common primer with the target gene during the amplification reaction.

[0013] As a limitation of the present invention, the N1 region and the single-stranded region are both composed of 5 to 8 base sequences complementary to one end of the target gene.

[0014] As another limitation of the present invention, the N1 region and the N2 region contain 1-2 locked nucleic acid modification sites respectively.

[0015] As a limitation of the present invention, the species-specific identification gene I is a gene that distinguishes Staphylococcus aureus from other species of bacteria, including the gyrB gene or the nuc gene;

[0016] The antibiotic resistance gene II and the antibiotic resistance gene III are respectively one of aminoglycoside antibiotic resistance genes, tetracycline antibiotic resistance genes, β-lactam antibiotic resistance genes and macrolide antibiotic resistance genes.

[0017] Furthermore, the antibiotic resistance gene II is the mecA gene; and the antibiotic resistance gene III is the tetM gene.

[0018] As a further limitation of the present invention, the fluorescent group is selected from one of JOE, TET, CY3, CY5, ROX, Texas Red and LCRED460, and the quenching group is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 and TaMRa.

[0019] Furthermore, the fluorescent group is FAM, VIC or JUN.

[0020] As a further limitation of the present invention, the primer pair Y1 is a molecular beacon probe nucF having a sequence of SEQ ID NO: 1 and a primer nucR having a sequence of SEQ ID NO: 2;

[0021] Primer pair Y2 is a primer mecAF having a sequence of SEQ ID NO: 3 and a molecular beacon probe mecAR having a sequence of SEQ ID NO: 4;

[0022] Primer pair Y3 is a primer tetMF having a sequence of SEQ ID NO:5 and a molecular beacon probe tetMR having a sequence of SEQ ID NO:6.

[0023] The present invention also provides a kit for detecting the above-mentioned primer combination of Staphylococcus aureus and drug-resistant genes, the kit comprising the above-mentioned primer combination, a reagent system suitable for recombinase-mediated isothermal amplification reaction, and a single-stranded DNA binding protein, a recombinase and a polymerase.

[0024] The present invention also provides the use of the primer combination in preparing a kit for detecting Staphylococcus aureus by recombinase-mediated isothermal amplification.

[0025] The present invention also provides a kit based on molecular beacon probes, which comprises a primer combination, a reagent system suitable for recombinase-mediated isothermal amplification reaction, and a single-stranded DNA binding protein, a recombinase and a polymerase.

[0026] The present invention also provides a fluorescence quantitative recombinase-mediated isothermal amplification method, using the primer combination for detecting Staphylococcus aureus and drug-resistant genes to perform fluorescence quantitative recombinase-mediated isothermal amplification reaction, in which the total volume fraction of the primer pair is 10-15% and the concentration is 40 μM. The volumes of the upstream primer (or molecular beacon probe) and the downstream primer (or molecular beacon probe) are the same.

[0027] As a further limitation of the present invention, the reaction system of the RAA reaction is 25 μL, and the reaction system comprises the above-mentioned primer combination, buFFer, A buFFer, B buFFer, recombinase, recombinase auxiliary protein, single-stranded binding protein, polymerase, dNTPs, template DNA and distilled water; the reaction conditions are a constant temperature of 38-45° C. for 20-40 min.

[0028] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0029] The present invention designs a primer combination for detecting Staphylococcus aureus and drug-resistant genes. The primer pair is composed of "molecular beacon probe + matching common primer", and the primer pair is labeled with different fluorescent groups. Through multiple fluorescent quantitative RAA reactions, multiple target genes can be detected simultaneously, providing a basis for the synchronous detection of multiple antibiotic resistance genes, reducing the sample amount, and realizing rapid and efficient detection of samples. It is of great significance for the synchronous detection of multiple antibiotic residues in livestock products such as milk.

[0030] Each primer pair in the primer combination contains a molecular beacon probe, which is a modified molecular beacon (IMB) probe with a "single-stranded tail" after one of the primers of the RAA reaction is structurally modified to form a stem-loop structure. The modified IMB probe has both probe and primer functions, reducing the number of primers required for the RAA reaction to 2, and only using the IMB probe and another unmodified common primer can perform fluorescence quantitative amplification. The molecular beacon probe includes an N1 region for forming the stem of the stem-loop structure, an M region for forming the loop of the stem-loop structure, an N2 region for forming the stem of the stem-loop structure, and a single-stranded region, wherein the 5' end of the N1 region has a fluorescent group binding site, and the 3' end of the N2 region has a quenching group binding site matching the fluorescent group. The single-stranded region is a 5-8 base sequence at the 3' end. The remaining sequence to the 5' end can form a stem-loop structure after folding, and the binding force of the IMB probe stem is enhanced by locked nucleic acid modification. The single-stranded region does not participate in the stem-loop structure and exists in the form of a "single-stranded tail".

[0031] Compared with the national standard GB4789.10-2016 method and the drug-sensitive paper agar diffusion method, the fluorescent quantitative recombinase-mediated isothermal amplification method of the present invention can significantly shorten the experimental cycle. It only takes 20 minutes to perform the RAA reaction using the primer combination of the present invention. Amplification and detection are completed simultaneously in one tube and one step. There is no need to open the lid of the amplified product for detection, which significantly improves the detection efficiency and achieves a more efficient analysis of the matching rate of drug-resistant genes and drug-resistant phenotypes. The detection limit of positive plasmid DNA within 20 minutes is 1.2×10 2 The 10-fold serial dilution DNA amplification curve has a good linear relationship, and the linear coefficient is within R 2 >0.99, the amplification efficiency was 100% <E <110%; the detection limit of positive plasmids in artificially contaminated skim milk was 1.3×10 2 The coefficient of variation of the Ct value of 10-fold serial dilution DNA amplification was less than 2%, with good repeatability.

[0032] The fluorescent quantitative recombinase-mediated isothermal amplification method of the present invention was used to amplify the nuc gene and identified 17 strains of S. aureus, with a detection rate of 18.89%, which was 100% consistent with the GB4789.10-2016 method; the detection rates of mecA and tetM genes in the 17 isolated S. aureus strains were 11.76% and 5.88%, respectively, and the consistency rates with the oxacillin-resistant and tetracycline-resistant phenotypes were both 100%.

[0033] In summary, the primer combination of the present invention is suitable for RAA technology, the method of the present invention is suitable for industrial production, and can be used for the preparation of RAA detection kits to simultaneously detect pathogenic microorganisms and their drug-resistant genes.

[0034] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the formation of a stem-loop structure of the IMB probe in Example 1

[0036] Figure 2 This is a schematic diagram of the working principle of the IMB probe in Example 1;

[0037] Figure 3 This is a diagram of the specificity verification results in Example 1;

[0038] Figure 4 This is a fluorescence value detection result diagram of the serial dilution of G58691-2 plasmid DNA in Example 1;

[0039] Figure 5 This is the standard curve result diagram in Example 1. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0041] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Some of the reagents are as follows:

[0042] ①LB liquid medium: 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, 500 mL distilled water, adjust the pH to 7.4 with 1 mol / L HCl.

[0043] ②LB solid medium: Add 2% agar to LB liquid medium.

[0044] 0.85% normal saline: 8.5 g NaCl, 1000 mL distilled water, divided into test tubes, 9 mL per tube.

[0045] All prepared culture media and reagents were sterilized by autoclave at 121°C for 15 min.

[0046] ③RAA nucleic acid amplification reagent (basic type): Hangzhou Zhongce Biotechnology Co., Ltd.; dNTP Mixture (10mM): Bao Bioengineering (Dalian) Co., Ltd.

[0047] ④ Gene recombination positive plasmid pUC57 nuc gene: 600bp, mecA gene: 1400bp and tetM gene: 1400bp seamlessly cloned pUC57): 6110bp; plasmid transformation glycerol bacteria G58691-2 (pUC57 transformed into TOP10 competent cells): Jiangsu Saisuofei Biotechnology Co., Ltd. 16SrDNA universal primer sequence 341F: 5'CCTACGGGNGGCWGCAG3', 805R: 5'GACTACHVGGGTATCTAATCC3': Shanghai Jierui Bioengineering Co., Ltd.

[0048] ⑤ Oxacillin and tetracycline drug sensitivity papers, Hangzhou Microbiological Reagent Co., Ltd. Tiangen Plasmid Mini Extraction Kit, Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0049] Some of the instruments and equipment include: PQuantStudio3 real-time fluorescence PCR instrument (Applied Biosystems (ABI) Company, USA); BSC-1304ⅡA2 biological safety cabinet (Suzhou Antai Air Technology Co., Ltd.); Milli-Q Synthesis ultrapure water machine (Merck Millipore, Germany); micropipette gun (Eppendorf AG, Germany).

[0050] The primers and molecular beacon probes synthesized and prepared in the embodiments of the present invention were all in the form of freeze-dried powder, centrifuged at 4°C, 6000r / min for 5min, diluted with TE to a 100μM stock solution, and diluted with TE to a 40μM working solution when used.

[0051] The molecular biology experimental operations in the examples, including plasmid construction, culture medium preparation, etc., were mainly performed with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002).

[0052] Example 1 A primer combination for detecting Staphylococcus aureus and drug resistance genes

[0053] This embodiment is a primer combination Z1 for detecting Staphylococcus aureus and drug-resistant genes, which is composed of primer pair Y1, primer pair Y2 and primer pair Y3. The design method is as follows:

[0054] In GenBank, the sequence of S. aureus heat-resistant nucleic acid protein nuc gene (ACC No: EF529596.1: 648bp) was used as the species-specific identification gene I of Staphylococcus aureus, the sequence of penicillin-binding protein mecA gene (ACCNo: FJ810877.1: 1459bp) was used as the antibiotic resistance gene II, and the sequence of tetracycline resistance protein TetM gene (ACCNo: AY057894.1: 1862bp) was used as the antibiotic resistance gene III. The above three gene sequences were used as target genes, and primers and molecular beacon probes were designed using Primer Premier v5.00 software, following the general principles of primer design, and primer probe structure analysis was performed using DNAMAN V 6.0. The design results and analysis are as follows:

[0055] Table 1 Primer pair sequences

[0056]

[0057] In the above sequence, the underline indicates that the site is fluorescently labeled, and the emphasis indicates that the site is modified with locked nucleic acid (LNA). The schematic diagram of the stem-loop structure formed by three molecular beacon probes is shown in Figure 1 .

[0058] The sequence of the molecular beacon probe nucF is SEQ ID NO: 1, which sequentially comprises the N1 region for forming the stem of the stem-loop structure, and the sequence is The sequence of the M region used to form the loop of the stem-loop structure is AGGTTTTTCTTTTTCG; the sequence of the N2 region used to form the stem of the stem-loop structure is And a single-stranded region composed of 7 base sequences complementary to one end of the target gene, the sequence is GTTGCTT; wherein, the bases of the N1 region and the N2 region are complementary and paired; the "T" at the 5' end of the N1 region is marked with a fluorescent group FAM, and contains 2 locked nucleic acid modification sites, specifically locked nucleic acid modification is performed at "G" and "A"; the "A" at the 3' end of the N2 region is marked with a quenching group DABCYL matching the fluorescent group, and contains 2 locked nucleic acid modification sites, specifically locked nucleic acid modification is performed at "A" and "T".

[0059] The sequence of the molecular beacon probe mecAR is SEQ ID NO: 4, which sequentially comprises the N1 region for forming the stem of the stem-loop structure, and the sequence is The sequence of the M region used to form the loop of the stem-loop structure is ATACTCTTTTG; the sequence of the N2 region used to form the stem of the stem-loop structure is And a single-stranded region composed of 6 base sequences complementary to one end of the target gene, the sequence is AGCATC; wherein, the bases of the N1 region and the N2 region are complementary to each other; the "A" at the 5' end of the N1 region is marked with a fluorescent group VIC, and contains 2 locked nucleic acid modification sites, specifically locked nucleic acid modification is performed at "A" and "T"; the "T" at the 3' end of the N2 region is marked with a quenching group DABCYL matching the fluorescent group, and contains 2 locked nucleic acid modification sites, specifically locked nucleic acid modification is performed at "T" and "C".

[0060] The molecular beacon probe tetMR has a sequence of SEQ ID NO: 6, which sequentially comprises the N1 region for forming the stem of the stem-loop structure, and the sequence is The sequence of the M region used to form the loop of the stem-loop structure is CCATCTAAAACT; the sequence of the N2 region used to form the stem of the stem-loop structure is And a single-stranded region composed of 5 base sequences complementary to one end of the target gene, the sequence is GAACG; wherein, the bases of the N1 region and the N2 region are complementary and paired; the "A" at the 5' end of the N1 region is marked with a fluorescent group JUN, containing 1 locked nucleic acid modification site, specifically, locked nucleic acid modification is performed at "A"; the "T" at the 3' end of the N2 region is marked with a quenching group BHQ2 matching the fluorescent group, containing 2 locked nucleic acid modification sites, specifically, locked nucleic acid modification is performed at "A" and "T".

[0061] The working principle of the molecular beacon probe of the present invention is as follows Figure 2 As shown, during the reaction, an unmodified primer for amplifying the target gene and a modified molecular beacon probe IMB probe rely on multiple enzymes (recombinase, recombinase auxiliary protein, single-strand binding protein, DNA polymerase) to bind and recognize, bind to and extend the DNA double strand, and start the replication process. At the beginning of the RAA reaction, common primers and IMB probes bind to the recombinase to form a complex, which is specifically recognized with the help of the recombinase auxiliary protein. After recognition, one single-stranded DNA binds to the complex, while the other single-stranded DNA binds to the single-stranded binding protein (SSB) to prevent the DNA from reforming into a double-stranded structure. The recombinases on the 3' ends of the primer and the IMB probe fall off, and the DNA polymerase replaces the recombinase to bind to the primer at the 3' end of the primer, thereby extending the primer forward. The quenching group and fluorescent group marked at the 5' and 3' ends of the molecular beacon stem of the IMB probe complex are pulled apart to show fluorescence, and the replication process begins immediately. This procedure is repeated, and the target gene fragment is exponentially amplified, which is proportional to the fluorescence intensity, that is, the amplification result can be displayed by a fluorescence curve.

[0062] In other embodiments, the sequence of the molecular beacon probe in the primer combination is substantially the same as that in Example 1, except that the N1 region and the single-stranded region are respectively composed of 6 or 8 base sequences complementary to one end of the target gene.

[0063] In other embodiments, the fluorescent group can be selected from one of FAM, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LCRED460 as needed, and the quenching group can be selected from one of the matching BHQ1, BHQ2, BHQ3, Dabcy1 or TaMRa.

[0064] 1) Verification of the specificity of primer combination Z1

[0065] 1) Cultivation of the bacteria to be tested.

[0066] Four strains of S. aureus and 13 strains of non-S. aureus glycerol-preserved bacteria were inoculated into LB liquid culture medium and cultured at 37°C for 18 h. G58691-2 was inoculated into LB liquid culture medium containing ampicillin and cultured at 37°C for 18 h. The bacteria were then transferred and subcultured once for later use.

[0067] Table 2 Information of strains to be tested

[0068]

[0069]

[0070] Note: ATCC is the American Type Culture Collection; CICC is the China Industrial Microbiology Culture Collection Center; CGMCC is the China General Microbiology Culture Collection Center; “+” means resistant, and “-” means sensitive.

[0071] The other strains are preserved in the Food Science and Engineering Laboratory of Shijiazhuang University, and all of the above strains are available to the public.

[0072] 2) Preparation of DNA samples of bacteria to be tested.

[0073] The subcultured bacterial liquid was inoculated into LB liquid medium and cultured at the corresponding temperature for 18 h. 1 mL of the bacterial liquid was taken for 10-fold serial dilutions. 1 mL of the dilutions of three continuous concentration gradient bacteria were taken and mixed with LB solid medium and cultured at the corresponding culture temperature for 24 h. The plate counts were performed and the concentration of 1 mL of the bacterial liquid of each strain was recorded.

[0074] At the same time, 1 mL of bacterial solution was used to extract genomic DNA according to the Tiangen Bacterial Genomic DNA Extraction Kit (DP302), and 4 mL of bacterial solution was used to extract G58691-2 plasmid DNA according to the Tiangen Plasmid Mini Extraction Kit (DP103). The extracted genomic and plasmid G58691-2 DNA were measured using Nanodrop 2000 ultra-micro spectrophotometer, and the DNA concentration was 40-100 ng / uL, and was frozen at -20°C for later use.

[0075] Calculate the copy number, dilute the plasmid with deionized water to a final concentration of 108-101 copies / μL, and store at -20°C for later use. Calculation formula: plasmid copy number (copies·mL-1) = 6.02×1023×plasmid concentration (g·mL-1) / plasmid length×660.

[0076] 3) Specificity verification method.

[0077] 1 μL of genomic DNA from 4 strains of S. aureus from different sources and 13 strains other than S. aureus and recombinant plasmid G58691-2 plasmid DNA were taken respectively, and 1 μL of sterile ddH2O was used as blank control to perform RAA detection with primers of primer pair Z1 to verify the specificity of the primers.

[0078] Among them, the RAA detection system and reaction conditions are as follows:

[0079] The 25 μL reaction system was prepared as follows: 10 μL of system buFFer, 6.25 μL of A buFFer, 1.8 μL of premix of four proteins (the volume ratio of recombinase, recombinase auxiliary protein, single-stranded binding protein and polymerase was 0.28:0.09:0.82:0.21); the working concentration of primers and molecular beacon probes was 40 μM, and the total volume fraction was 14.4%, of which nucF and nucF were 0.5 μL, mecAF and mecAR were 0.5 μL, tetMF and tetMR were 0.8 μL, respectively; dNTPs working concentration was 10 mM, 0.9 μL, template DNA was 1 μL, and sterile distilled water was 1.3 μL, and 1.25 μL of B buFFer was added to the inner wall of each reaction tube cap;

[0080] After instantaneous centrifugation of the reaction system, it was immediately put on the machine and the reaction conditions were set to 39°C constant temperature reaction time for 30 minutes for RAA amplification reaction.

[0081] 4) Specificity verification results and comparison.

[0082] like Figure 3As shown, the results showed that only the DNAs of 4 different sources of S. aureus and G58691-2 showed an S-shaped amplification curve for nuc gene amplification, and the DNA amplification of 13 non-S. aureus strains did not show an S-shaped amplification curve, indicating that the primer combination of the present invention has good specificity.

[0083] The drug sensitivity paper agar diffusion method was used to determine that, except for the positive plasmid G58691-2, the S. aureus CICC25138 strain was resistant to oxacillin, and the mecA gene amplification showed an S-type amplification curve. The DNA amplification of the other 16 strains of S. aureus and non-S. aureus bacteria did not show an S-type amplification curve, indicating that the primer combination of the present invention is consistent with its phenotype in detecting the oxacillin-resistant mecA gene, but is not species specific.

[0084] The drug sensitivity paper agar diffusion method was used to determine that the DNA amplification of S. aureus CICC21600 and G58691-2 strains with tetracycline-resistant tetM gene showed an S-shaped amplification curve, while the DNA amplification of the other 16 strains of S. aureus and non-S. aureus bacteria did not show an S-shaped amplification curve, indicating that the primer combination of the present invention is consistent with its phenotype in detecting the tetracycline-resistant tetM gene, but is not species-specific.

[0085] II) Sensitivity of primer combination Z1 for detecting pure cultured S. aureus

[0086] The recombinant plasmid G58691-2 DNA was diluted 10-fold from 1.2 × 10 8 copies.μL －1 to 1.2×10 1 copies·μL －1 As a template, three replicates were made for each concentration to perform RAA amplification. The standard curve was drawn with the logarithm of plasmid concentration as the horizontal axis and the cycle threshold (Ct) as the vertical axis, and its linear equation (y = ax + b, where a is the slope of the linear equation and b is the intercept) and amplification efficiency (E = 10 -1 / a -1, a is the slope of the linear equation), to indicate the sensitivity of the primer combination in detecting pure culture of S. aureus.

[0087] The results are as follows Figure 4 As shown, the G58691-2 plasmid DNA concentration was 1.2×10 7 copies·μL -1 ~1.2×10 1 copies·μL -1The highest fluorescence intensity of the amplified S-shaped curves was different. The fluorescence intensity of mecA gene amplification was the highest, and the fluorescence values ​​of nuc gene and tetM gene were not much different. Within 20 min, the triple NFQ-RAA could amplify the G58691-2 plasmid with a 10-fold serial dilution DNA concentration of 1.2×10 2 copies·μL -1 .

[0088] According to the concentration of G58691-2 plasmid DNA at 1.2×10 7 copies·μL -1 ~1.2×10 1 copies·μL -1 The logarithmic values ​​of the series concentrations were plotted as the horizontal axis, and the Ct value (1 Cycle = 1 min) was used as the vertical axis to draw the standard curve. The linear equations of the standard curves of the single nuc gene, mecA resistance gene and tetM resistance gene were as follows: Figure 5 As shown, they are nuc-y=-3.2603x+25.618, mecA-y=-3.2961x+25.932, tetM-y=-3.2424x+26.202, where x is the reaction time (min), y is the logarithm of the initial concentration of plasmid DNA (log10), nuc-R 2 =0.9994, mecA-R 2 =0.9961,tetM-R 2 =0.9957, R of 3 genes 2 >0.99, all with good linear correlation, and the amplification efficiencies were E(nuc)=102.64%, E(mecA)=101.09%, E(tetM)=103.43%, and 100%<E<110%.

[0089] The above results show that the primer combination of the present invention is 1.2×10 7 copies / μL-1.2×10 1 The detection range of copies / μL is high in sensitivity and good in repeatability.

[0090] III) Detection limit of primer combination Z1 for artificially contaminated G58691-2 plasmid DNA in skim milk powder

[0091] Take 25mL skim milk powder and homogenize it in 225mL sterile saline under sterile conditions to make 1:10 skim milk. Before adding, test the skim milk powder to ensure that S. aureus is negative. Take G58691-2 bacterial suspension and concentrate it 10 times, take 1mL and add it to 9mL 1:10 skim milk. Take 5mL recombinant plasmid G58691-2 bacterial suspension and add 1mL anhydrous ethanol, 1mL ammonia water and 1mL petroleum ether to mix. Centrifuge the mixture at 12000rpm for 10min, collect the bacteria and discard the supernatant. The cells were suspended in 1 mL of sterile deionized water, shaken with an oscillator, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded; the cells were treated with 500 μL of lysozyme (50 mg / mL) for 5 min, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded; the cells were suspended in 1 mL of sterile deionized water, shaken with an oscillator, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded; the precipitate was dissolved in 1 mL of sterile deionized water, and 1.3 × 10 of the plasmid DNA extracted at the same time and at different times were taken. 7 copies·μL -1 ~1.3×10 2 copies·μL -1 1 μL of each was added to the same reaction tube, and the primer combination was used for RAA reaction to detect nuc gene, mecA gene and tetM gene at the same time, and the detection limit of artificially added S. aureus was determined by amplification. Each sample was repeated 3 times, and the intra-group and inter-group repeatability tests were performed. The coefficient of variation (CV%) of the intra-group and inter-group repeatability tests was calculated to evaluate the repeatability of the method.

[0092] The results showed that the detection limit of plasmid DNA in artificially contaminated G58691-2 bacteria in skim milk powder was 1.3×10 2 copies·μL -1 The coefficient of variation (CV) of the intra-group and inter-group repeatability tests for each sample was <2.00%, as shown in Table 3.

[0093] Table 3 Results of repeatability test of nuc, mecA and tetM gene plasmid DNA

[0094]

[0095]

[0096] The above results show that the detection limit of artificial contamination plasmid DNA in skim milk is 1.3×10 2 The above-mentioned specificity, detection limit and sensitivity were tested on the primer combinations of other embodiments of the present invention, and similar results were obtained.

[0097] Example 2 A kit for detecting Staphylococcus aureus and drug-resistant genes

[0098] The kit comprises the primer combination for detecting Staphylococcus aureus and drug-resistant genes of Example 1; reagents suitable for recombinase-mediated isothermal amplification reaction: system buFFer, AbuFFer, B buFFer; and single-stranded DNA binding protein, recombinase, recombinase auxiliary protein and polymerase.

[0099] Example 3 Fluorescence quantitative recombinase-mediated isothermal amplification method based on primer combination

[0100] This example is a fluorescent quantitative recombinase-mediated isothermal amplification method based on primer combination, using the primer combination Z1 of Example 1, and simultaneously detecting Staphylococcus aureus and drug resistance genes (oxacillin resistance gene mecA and tetracycline resistance gene tetM) in 90 milk samples of cows with mastitis collected from three dairy farms in Hebei Province. The details are as follows:

[0101] S1. Extract DNA from the sample to be tested as a template:

[0102] 1 mL of milk samples of 90 cases of mastitis collected from three dairy farms in Hebei Province was mixed with 1 mL of anhydrous ethanol, 1 mL of ammonia water and 1 mL of petroleum ether. The mixture was centrifuged at 12,000 rpm for 10 min, and the bacteria were collected and the supernatant was discarded. The bacteria were suspended with 1 mL of sterile deionized water, shaken with an oscillator, centrifuged at 12,000 rpm for 1 min, and the supernatant was discarded; the bacteria were treated with 500 μL of lysozyme (50 mg / mL) for 5 min, centrifuged at 12,000 rpm for 1 min, and the supernatant was discarded; the bacteria were suspended with 1 mL of sterile deionized water, shaken with an oscillator, centrifuged at 12,000 rpm for 1 min, and the supernatant was discarded; the precipitate was dissolved with 1 mL of sterile deionized water, the precipitate was fully suspended, 50 μL of the suspension was boiled in a boiling water bath for 10 min, and centrifuged at 1,2000 rpm for 1 min. The supernatant was the template DNA.

[0103] S2. Reaction system configuration and RAA amplification reaction based on the primer combination of the present invention:

[0104] The 25 μL reaction system was prepared as follows: 10 μL of system buFFer, 6.25 μL of AbuFFer, 1.8 μL of premix of four proteins (the volume ratio of recombinase, recombinase auxiliary protein, single-stranded binding protein and polymerase was 0.28:0.09:0.82:0.21); the working concentration of primers and molecular beacon probes was 40 μM, and the total volume fraction was 14.4%, of which nucF and nucF were 0.5 μL, mecAF and mecAR were 0.5 μL, tetMF and tetMR were 0.8 μL, respectively; the working concentration of dNTPs was 10 mM, 0.9 μL, template DNA was 1 μL, and sterile distilled water was 1.3 μL, and 1.25 μL of B buFFer was added to the inner wall of each reaction tube cap;

[0105] After instant centrifugation of the reaction system, it was immediately put on the machine and the reaction conditions were set to 39°C constant temperature reaction time for 20 minutes for RAA amplification reaction.

[0106] S3. Real-time monitoring of fluorescence intensity and analysis of the results:

[0107] Of the 90 samples of mastitis milk from dairy cows, 17 samples were found to contain S. aureus, with a detection rate of 18.89%. Two strains were resistant to oxacillin and one was resistant to tetracycline.

[0108] In other embodiments, the total volume fraction of primers and molecular beacon probes in step S2 is 10%, 13%, 12.5%, 14% or 15%, the reaction conditions are 38°C, 39°C, 42°C, 43°C or 45°C, and the constant temperature reaction time is 25min, 28min, 33min, 37min or 40min. The RAA amplification reaction based on the primer combination of the present invention is successfully carried out and similar effects are achieved.

[0109] Comparative Example 1 The above 90 milk samples were tested by the method of detecting 16SrRNA. According to the operation method and judgment criteria of the antimicrobial susceptibility test of the Clinical & Laboratory Standards Institute (CLSI), the strains isolated from the S. aureus samples were subjected to drug susceptibility tests, and the sensitivity of the strains to aminoglycosides of oxacillin and tetracycline resistance genes was determined by the drug susceptibility paper agar diffusion method. Each drug susceptibility paper was repeated 3 times to analyze the matching rate of resistance genes and resistance phenotypes.

[0110] Comparative Example 2 used the GB4789.10-2016 method to test the above 90 milk samples, and the results of the drug-sensitive paper agar diffusion method in Comparative Example 1 were used as the resistance gene detection results.

[0111] Comparing the results of Example 6, Comparative Example 1 and Comparative Example 2, the coincidence rates of each method in detecting S. aureus were compared, wherein coincidence rate = (number of true positives + number of true negatives) / total number of samples tested.

[0112] The results showed that for the detection of S. aureus: Comparative Example 1 detected 17 samples containing S. aureus, with a detection rate of 18.89%. Comparative Example 2 also identified 17 samples as S. aureus using the GB4789.10-2016 method. The conformity rate of the method in Example 6 with Comparative Examples 1 and 2 was 100%.

[0113] Regarding the detection of drug resistance genes: the drug sensitivity paper agar diffusion method detected 2 strains of oxacillin-resistant and 1 strain of tetracycline-resistant genes, respectively. The method of Example 6 was 100% consistent with the results of the drug sensitivity paper agar diffusion method, and the phenotypic consistency was also 100%.

[0114] The above results show that the present invention can complete the simultaneous detection of pathogenic microorganisms and drug-resistant genes within a reaction time of 20 minutes, and is convenient, efficient, and has high accuracy compared to traditional methods such as GB4789.10-2016, which are time-consuming, complicated, and require higher equipment and operations. It provides support for the early diagnosis of bovine mastitis and the adjustment of medication regimens.

[0115] In some embodiments, the target gene is the gyrB gene of S. aureus as the species-specific identification gene, and a molecular beacon probe is designed to include, from the 5' end to the 3' end, an N1 region for forming a stem portion of a stem-loop structure, an M region for forming a loop portion of the stem-loop structure, an N2 region for forming a stem portion of the stem-loop structure, and a single-stranded region, wherein the bases of the N1 region and the N2 region are complementary to each other; the 5' end of the N1 region has a fluorescent group binding site, and the 3' end of the N2 region has a quenching group binding site matching the fluorescent group.

[0116] In some embodiments, the target gene is a resistance gene to antibiotics such as aminoglycosides, tetracyclines, β-lactams, macrolides, etc., and a molecular beacon probe is designed to include, from the 5' end to the 3' end, an N1 region for forming a stem portion of a stem-loop structure, an M region for forming a loop portion of a stem-loop structure, an N2 region for forming a stem portion of a stem-loop structure, and a single-stranded region, wherein the bases of the N1 region and the N2 region are complementary to each other; the 5' end of the N1 region has a fluorescent group binding site, and the 3' end of the N2 region has a quenching group binding site that matches the fluorescent group.

[0117] Among them, the resistance genes of aminoglycoside antibiotics include streptomycin resistance gene, gentamicin resistance gene, tobramycin resistance gene, kanamycin resistance gene, amikacin resistance gene, neomycin resistance gene, netilmicin resistance gene or isepamicin resistance gene.

[0118] Resistance genes for tetracycline antibiotics include chlortetracycline resistance gene, oxytetracycline resistance gene, tetracycline resistance gene, minocycline resistance gene, doxycycline resistance gene or doxycycline resistance gene.

[0119] The resistance genes of β-lactam antibiotics include penicillin resistance genes, cephalosporin resistance genes, carbapenem resistance genes, cephamycin resistance genes, thiomycin resistance genes, oxacephem resistance genes,

[0120] The resistance genes of macrolide antibiotics include erythromycin resistance gene, roxithromycin resistance gene, azithromycin resistance gene, clarithromycin resistance gene, dirithromycin resistance gene, fluazifop resistance gene or telithromycin resistance gene.

[0121] The molecular beacon probes can all be used as a primer in the recombinase-mediated isothermal amplification detection technology and can be used to prepare RAA kits to achieve simultaneous detection of Staphylococcus aureus and drug-resistant genes.

[0122] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the above embodiments, those skilled in the art can still modify the technical solutions described in the above embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the scope of protection of the claims of the present invention.

Claims

1. A primer combination for detecting Staphylococcus aureus and drug-resistant genes, characterized in that: The primer combination includes a primer pair Y1 designed with a species-specific identification gene I of Staphylococcus aureus as a target gene, a primer pair Y2 designed with an antibiotic resistance gene II as a target gene, and a primer pair Y3 designed with an antibiotic resistance gene III as a target gene; The primer pairs are composed of a molecular beacon probe and a primer; The molecular beacon probe comprises, from the 5' end to the 3' end, an N1 region for forming a stem portion of a stem-loop structure, an M region for forming a loop portion of a stem-loop structure, an N2 region for forming a stem portion of a stem-loop structure, and a single-stranded region; The bases of the N1 region and the N2 region are complementary to each other; the 5' end of the N1 region has a fluorescent group binding site, and the 3' end of the N2 region has a quenching group binding site that matches the fluorescent group. The excitation light wavelengths of the fluorescent groups of the molecular beacon probes in different primer pairs are different; The M region used to form the loop portion of the stem-loop structure, the N2 region used to form the stem portion of the stem-loop structure, and the single-stranded region are sequentially complementary to the bases at one end of the target gene.

2. The primer combination for detecting Staphylococcus aureus and drug-resistant genes according to claim 1, characterized in that: The N1 region and the single-stranded region are both composed of 5 to 8 base sequences complementary to one end of the target gene.

3. The primer combination for detecting Staphylococcus aureus and drug-resistant genes according to claim 1 or 2, characterized in that: The N1 region and the N2 region contain 1-2 locked nucleic acid modification sites respectively.

4. The primer combination for detecting Staphylococcus aureus and drug-resistant genes according to claim 3, characterized in that: The species-specific identification gene I is a gene that distinguishes Staphylococcus aureus from other species of bacteria, including ikB Gene or nuc Gene; The antibiotic resistance gene II and the antibiotic resistance gene III are respectively one of aminoglycoside antibiotic resistance genes, tetracycline antibiotic resistance genes, β-lactam antibiotic resistance genes and macrolide antibiotic resistance genes.

5. The primer combination for detecting Staphylococcus aureus and drug-resistant genes according to claim 4, characterized in that: Primer pair Y1 is a molecular beacon probe with a sequence of SEQ ID NO: 1 nuc F and a primer having a sequence of SEQ ID NO: 2 nuc R; Primer pair Y2 is a primer having a sequence of SEQ ID NO: 3 mec AF and a molecular beacon probe having a sequence of SEQ ID NO: 4 mec AR; Primer pair Y3 is a primer having a sequence of SEQ ID NO: 5 tet MF and a molecular beacon probe having a sequence of SEQ ID NO: 6 tet MR.

6. The primer combination for detecting Staphylococcus aureus and drug-resistant genes according to claim 5, characterized in that: The fluorescent group is selected from one of JOE, TET, CY3, CY5, ROX, Texas Red and LCRED460, and the quenching group is selected from one of BHQ1, BHQ2, BHQ3, Dabcy1 and TaMRa.

7. Use of the primer combination for detecting Staphylococcus aureus and drug-resistant genes according to any one of claims 1 to 6 in the preparation of a recombinase-mediated isothermal amplification detection kit.

8. A kit for detecting Staphylococcus aureus and the primer combination for drug resistance gene according to any one of claims 1 to 6, characterized in that: The kit comprises the primer combination, a reagent system suitable for recombinase-mediated isothermal amplification reaction, and a single-stranded DNA binding protein, a recombinase and a polymerase.

9. A fluorescence quantitative recombinase-mediated isothermal amplification method, characterized in that: The primer combination for detecting Staphylococcus aureus and drug-resistant genes is used to carry out a fluorescent quantitative recombinase-mediated isothermal amplification reaction. In the reaction system, the total volume fraction of the primer pair is 10-15%, and the concentration is 40 μM.

10. A fluorescent quantitative recombinase-mediated isothermal amplification method according to claim 9, characterized in that: The reaction system comprises a primer combination, buFFer, A buFFer, B buFFer, recombinase, recombinase auxiliary protein, single-stranded binding protein, polymerase, dNTPs, template DNA and distilled water; The reaction conditions are constant temperature at 38-45°C for 20-40 min.

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