A primer probe combination, kit and application for detecting chicken mycoplasma synoviae based on RAA-LFD

Abstract

The application belongs to the technical field of microorganism detection, and particularly relates to a primer probe combination, a kit and application for detecting chicken synovial capsule mycoplasma based on RAA-LFD. The nucleotide sequence of the upstream primer in the primer probe combination is shown in SEQ ID NO. 3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 4, and the nucleotide sequence of the probe is shown in SEQ ID NO. 10. Based on the primer probe combination, the application further develops a kit and a method for detecting chicken synovial capsule mycoplasma. The method has high detection accuracy, good specificity and high sensitivity, can rapidly and accurately realize on-site rapid visual detection of chicken synovial capsule mycoplasma, and is particularly suitable for use in the field.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to a primer-probe combination, kit, and application for detecting Mycoplasma synoviae in chickens based on RAA-LFD. Background Technology

[0002] Mycoplasma synoviae (MS) is a significant pathogenic microorganism affecting poultry farming, infecting chickens, turkeys, and various other avian hosts globally. Clinical manifestations of this pathogen include respiratory inflammation, synovitis, and osteoarthritis. Although MS infection rarely causes acute death in poultry directly, it can lead to infectious synovitis, respiratory disease, growth retardation, decreased egg production, and the production of deformed eggs. Furthermore, infection can weaken the host's immunity, significantly increasing the probability of secondary infections with other pathogens, thereby raising morbidity and mortality rates and causing severe economic losses to the poultry industry. Therefore, accurate and effective detection is a prerequisite for identifying, preventing, and controlling this disease.

[0003] Currently, routine laboratory techniques for MS detection mainly include pathogen isolation, serological analysis, and molecular biology techniques. Although pathogen isolation and culture are the gold standard for clinical diagnosis of MS, this method is time-consuming, typically requiring 3-4 weeks to complete the entire isolation and culture process, making it difficult to meet the needs of rapid diagnosis. Furthermore, the in vitro culture of MS has stringent requirements for culture medium components, further increasing the difficulty of pathogen isolation. Serological detection techniques rely on specific antibodies produced by the host after infection, but these antibodies are usually only effectively detected 2-3 weeks after infection, thus limiting their effectiveness in early infection diagnosis. PCR and qPCR, due to their high sensitivity and specificity, have become the most commonly used molecular diagnostic techniques for MS detection. However, these molecular detection methods require expensive thermal cyclers and have strict requirements for professional laboratory environments. To overcome these shortcomings, researchers have developed isothermal nucleic acid amplification techniques suitable for MS detection, such as polymerase spiral reaction (PSR) and loop-mediated isothermal amplification (LAMP). The amplification products of PSR and LAMP can be directly observed with the naked eye without any instruments. However, both techniques still have shortcomings: the amplification reaction takes 40 to 60 minutes, and loop-mediated isothermal amplification requires the design of 6 complementary primers, which is quite challenging. Therefore, developing and promoting a simple and rapid detection technique for Mycoplasma synovitis is of significant practical importance and urgent need.

[0004] Recombinase-aided amplification (RAA) is a novel isothermal nucleic acid amplification technique. Its basic principle is as follows: the recombinase in the reaction system binds to the primer to form a complex and searches for the target sequence on the template; single-stranded binding proteins assist in the unwinding of the DNA double-strand structure, thereby enabling DNA polymerase to initiate the sequence amplification reaction. This technique can rapidly complete nucleic acid amplification under isothermal conditions of 37–42 °C, with the entire reaction typically taking only 15–30 minutes. If a specific probe is added to the reaction system, the amplified products can be conveniently and visually detected using a lateral flow dipstick (LDF). LFD is a point-of-care testing device based on immunochromatography or nucleic acid hybridization. It relies on capillary action to drive sample migration and achieves rapid target analysis through color development of the detection line (T line) and control line (C line). It consists of components such as a sample pad, a conjugation pad, a nitrocellulose membrane, and an absorbent pad. It is easy to operate, providing results in 5–10 minutes, requiring no complex equipment, and is widely used in clinical pathogen detection, food safety screening, and other scenarios. In recent years, RAA-LFD technology has been successfully applied to the detection of various human and animal pathogens. Therefore, developing a reagent based on the RAA-LFD detection method that can sensitively and specifically detect Mycoplasma synovitis is of great significance for the visual on-site detection of Mycoplasma synovitis. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a primer-probe combination, kit, and application for detecting Mycoplasma synoviae in chickens based on RAA-LFD, which facilitates rapid and efficient detection of Mycoplasma synoviae in chickens in grassroots breeding environments.

[0006] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides a primer-probe combination for detecting Mycoplasma synoviae in chickens based on RAA-LFD. The primer-probe combination consists of an upstream primer, a downstream primer, and a probe. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the probe is shown in SEQ ID NO.10. The 5' end of the downstream primer is labeled with biotin.

[0007] Furthermore, the 5' end of the probe is labeled with a FAM fluorescent group, and the bases at least 30 bp from the 5' end and at least 15 bp from the 3' end are replaced with tetrahydrofuran (THF), and the 3' end is phosphorylated using Spacer C3.

[0008] Furthermore, the 31st site at the 5' end of the probe is modified with tetrahydrofuran (THF).

[0009] A second aspect of the present invention provides the application of the above-described primer-probe combination in the preparation of a detection kit for Mycoplasma synoviae or in the detection of Mycoplasma synoviae.

[0010] A third aspect of the present invention provides a kit for detecting Mycoplasma synoviae in chickens, the kit comprising the primer-probe combination described above.

[0011] Furthermore, the kit also includes a reaction complex buffer, deoxyribonucleoside triphosphate, an enzyme mixture, and a side-flow chromatography test strip.

[0012] Furthermore, the side-flow chromatography test strip includes: a sample pad, a detection line, and a control line.

[0013] A fourth aspect of this invention provides a method for detecting Mycoplasma synoviae in chickens based on RAA-LFD, the method not intended for disease diagnosis, and includes the following steps: Extract DNA from the sample to be tested; Using the DNA of the sample to be tested as a template, a recombinase-mediated isothermal amplification reaction was carried out using the primer and probe combination described above to obtain the amplification product; The amplification product is diluted with a diluent, and the diluted amplification product is inserted into the sample pad end of the side-flow chromatography test strip. After the reaction is completed, the results are read and determined: when both the detection line and the control line show bands, it indicates that the sample to be tested contains Mycoplasma synoviae.

[0014] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 30℃~41℃ for a time of 10min~30min.

[0015] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 37℃~41℃ for a time of 20min~30min.

[0016] Furthermore, the recombinase-mediated isothermal amplification reaction is carried out at a temperature of 37°C for 20 minutes.

[0017] Furthermore, each 50 μL recombinase-mediated isothermal amplification reaction system comprises: 25 μL A Buffer, 12.9 μL ddH2O, 2 μL 0.5 μmol / L~4 μmol / L upstream primer, 2 μL 0.5 μmol / L~4 μmol / L downstream primer, 0.6 μL 0.5 μmol / L~4 μmol / L probe, 5 μL DNA, 2.5 μL B Buffer, and one tube of RAA reaction general dry powder.

[0018] Furthermore, the concentrations of the upstream and downstream primers are 2.0 μmol / L.

[0019] Furthermore, the concentration of the probe is 2.0 μmol / L.

[0020] Furthermore, each 25 μL of A Buffer contains 10 w / v% polyethylene glycol.

[0021] Furthermore, each 2.5 μL of B Buffer contains 280 mM magnesium acetate.

[0022] Furthermore, the RAA reaction universal dry powder includes dNTPs, recombinase, single-stranded DNA binding protein, and DNA polymerase.

[0023] Furthermore, the system for the recombinase-mediated isothermal amplification reaction is prepared by the following steps: (1) After mixing the A buffer, upstream primer, downstream primer, probe and ddH2O evenly, a premixed solution is obtained; (2) Add the premixed solution to the detection unit tube containing the RAA reaction universal dry powder; (3) Add DNA and B buffer to the detection unit tube from step (2); (3) Cover the tube and mix well. Centrifuge briefly to obtain the system of recombinase-mediated isothermal amplification reaction.

[0024] Further, the diluent is sterile water or phosphate-buffered saline; the volume ratio of the diluent to the amplification product is 1:5.5~6.5.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a primer-probe combination, kit, and application for detecting Mycoplasma synoviae in chickens based on RAA-LFD. The nucleotide sequence of the upstream primer in the primer-probe combination is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, the 5' end of the downstream primer is labeled with biotin, and the nucleotide sequence of the probe is shown in SEQ ID NO.10. Based on this primer-probe combination, this invention also developed a kit and a method for detecting Mycoplasma synoviae in chickens. This method completes the RAA reaction at 37°C for 20 minutes, and the amplified product can be detected by LFD within 5 minutes. The RAA-LFD detection method has low requirements for the detection environment and equipment, is easy to operate, can report results in a short time, and makes the detection results visual, thus having widespread application value.

[0026] This invention compares the sensitivity of three methods: RAA-LFD, PCR, and qPCR. Experimental data show that the limits of detection for the three methods are 2.63 × 10⁻⁶, respectively. 0copies / μL, 2.63×10 3 copies / μL and 2.63×10 0 The RAA-LFD method, with its copies / μL, exhibits sensitivity similar to qPCR and 1000 times higher than PCR. Furthermore, the method provided by this invention does not cross-react with other common pathogens, and repeatability tests consistently demonstrate good amplification, indicating excellent specificity and reproducibility. Using this method to test 100 samples, the results showed a 96% concordance rate with PCR and a 100% concordance rate with qPCR, with a kappa value of 0.92 (K>0.75). This indicates that the method provided by this invention has high detection accuracy, maintaining the high sensitivity and specificity of qPCR while possessing the low equipment dependence and ease of operation of isothermal amplification technology. It also overcomes the drawbacks of traditional methods (long processing times), serological methods (long processing times), and complex LAMP / PSR primer design, providing an integrated solution for MS detection that is "precise, rapid, low-cost, and easy to operate," enabling rapid, real-time, visual detection of nucleic acids on-site. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 Selection of primers and probes for MS amplification. A: Electrophoresis image of primer selection; M: DNA standard molecular weight DL3000; 1-4: Amplification products of primer pairs 1-4; N: Blank control. B: UV image of primer selection; M: DNA molecular weight standard DL3000; 1-4: Positive amplification products of primer pairs 1-4; N: Blank control. C: Primer-probe combination selection; 1: Primer pair 2 + probe 1; 2: Primer pair 2 + probe 1 (negative control, no template added); 3: Primer pair 2 + probe 2; 4: Primer pair 2 + probe 2 (negative control, no template added).

[0029] Figure 2 Results of MS RAA-LFD detection reaction condition optimization: A: Primer concentration screening; B: Probe concentration screening; C: Reaction temperature screening; D: Reaction time screening.

[0030] Figure 3 RAA-LFD test results initiated by body temperature. 1: Negative control; 2: RAA-LFD test sample initiated by body temperature.

[0031] Figure 4 1-4: MS RAA-LFD specificity test results; 5-14: RAA-LFD detection results for Mycoplasma gallisepticum, Mycoplasma bovis, Pasteurella multocida, Mansonia hemolyticus, Staphylococcus aureus, Salmonella pullorum, Escherichia coli, Bacillus subtilis, Enterococcus faecalis, and Enterococcus faecium; 15: Negative control.

[0032] Figure 5 The results show the sensitivity comparison of RAA-LFD, PCR, and qPCR. A: MS RAA-LFD sensitivity test results; B: MS PCR sensitivity test results; C: MS qPCR sensitivity test results. 1-8: MS DNA template concentrations were 2.63 × 10⁻⁸. 7 copies / μL, 2.63×10 6 copies / μL, 2.63×10 5 copies / μL, 2.63×10 4 copies / μL, 2.63×10 3 copies / μL, 2.63×10 2 copies / μL, 2.63×10 1 copies / μL, 2.63×10 0 copies / μL; 9: negative control; M: standard molecular weight DL 3000; N: negative control.

[0033] Figure 6 Results of intragroup repeatability tests for MS RAA-LFD; 1-3: Template concentration was 2.63 × 10⁻⁶ 4 copies / μL; 4-6: template concentration is 2.63×10 3 copies / μL; 7-9: template concentration is 2.63×10 2 copies / μL.

[0034] Figure 7 Results of the MS RAA-LFD intergroup repeatability test; 1: Template concentration was 2.63 × 10⁻⁶ 4 1: copies / μL; 2: template concentration is 2.63×10 3 3: Template concentration is 2.63 × 10⁻⁶ copies / μL; 2 copies / μL. Detailed Implementation

[0035] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0036] The materials and methods used in this invention are as follows: 1. Strains and clinical samples Mycoplasma synovitis ( Mycoplasma synoviae MS, catalog number BNCC393281), Mycoplasma gallisepticum ( Mycoplasma gallisepticum MG, product number BNCC382031), bovine mycoplasma ( Mycoplasma bovis Mb, ATCC 25523), Pasteurella multocida ( Pasteurella multocida Pm, catalog number BNCC270568), hemolytic Manskov ( Mannheimia haemolytica Mh, product number BNCC376043), Staphylococcus aureus ( Staphylococcus aureus Salmonella pullorum (Sa, product number BNCC391765) Salmonella pullorum Sp, ATCC 9120), Escherichia coli ( Escherichia coli Product No. BNCC336902), Bacillus subtilis ( Bacillus subtilis Product No. BNCC109047), Enterococcus faecalis ( Enterococcusfaecium Product No. BNCC137649), Enterococcus faecalis ( Enterococcus faecalis The samples (item number BNCC102668) were all purchased from Beina Chuanglian Biotechnology Co., Ltd. A total of 100 pharyngeal swab samples from suspected MS-infected chickens were collected from Shihezi City, Xinjiang, and stored at -80℃.

[0037] 2. Experimental reagents and equipment RAA nucleic acid amplification reagent (test strip type) and single-target HybriDetect lateral chromatography test strip (rainbow type) were purchased from Nanjing Wobo Biotechnology Co., Ltd. Gel extraction kit, DNA purification kit, plasmid miniprep kit, and pMD19-T vector were purchased from Vazyme Biotechnology Co., Ltd. V (phenol): V (Chloroform): VIsoamyl alcohol (25:24:1) and Biowest agarose were purchased from Xinjiang Hengchao Biotechnology Co., Ltd. Escherichia coli DH5α competent cells were purchased from Weidi Biotechnology Co., Ltd. Ampicillin was purchased from Shanghai Yuanye Biotechnology Co., Ltd. 2× Universal BlueSYBR Green qPCR Master Mix, DNA molecular weight standard markers, and PBS buffer were all purchased from Wuhan Saiweier Biotechnology Co., Ltd. Gold View nucleic acid dye was purchased from Beijing Bio-Top Technology Co., Ltd. 2× Es Taq PCR Master Mix (Dye) and ddH2O were both purchased from Beijing Kangwei Century Biotechnology Co., Ltd.

[0038] 3. Main equipment The water bath was purchased from Zhejiang Qun'an Scientific Instruments Co., Ltd. The high-speed benchtop centrifuge was purchased from Nanjing Haidixi Equipment Co., Ltd. The PCR instrument was purchased from Thermo Fisher Scientific. The real-time PCR instrument was purchased from Tianlong Technology Co., Ltd. The electrophoresis gel imaging system was purchased from Bio-Rad (USA). The electrophoresis apparatus was purchased from Wuhan Saiweier Biotechnology Co., Ltd.

[0039] 4. Nucleic acid extraction DNA templates for MS, MG, Mb, Pm, Mh, Sa, Salmonella pullorum, Escherichia coli, Bacillus subtilis, Enterococcus faecalis, and Enterococcus faecium were extracted according to the instructions of the DNA extraction kit. All DNA samples were stored at -20°C.

[0040] Example 1: Development of primers and probes According to MS published in GenBank vlhA The complete sequence of the CDS region of the gene (accession number: AF035624.1), combined with the RAA reaction principle, was used to design four pairs of specific amplification MS using Oligo 7.0 software. vlhA Primers for conserved segments in the CDS region of the gene. Primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd., and their sequences are shown in Table 1.

[0041] Table 1 Primer and probe sequences According to the RAA basic nucleic acid amplification kit instructions, a 50 μL RAA reaction system was established, in which the premix consisted of 25 μL A Buffer, 13.5 μL ddH2O, 2 μL upstream primer (10 μmol / L) and 2 μL downstream primer (10 μmol / L).

[0042] After thoroughly mixing the premixed solution in a clean PCR reaction tube, add it to a detection unit tube containing the reaction powder. Then, add 5 μL of the DNA sample to be tested to the detection unit tube, and add 2.5 μL of B Buffer to the inside of the cap. Tightly cap the tube, gently invert it, and tap the tube wall 6 times to mix thoroughly. Centrifuge rapidly for 10 s, and incubate at 39°C for 30 min. After the reaction is complete, add 50 μL of [unspecified reagent] to the detection unit tube. V (Phenol): V (Chloroform): V A mixture of isoamyl alcohol (25:24:1) was thoroughly mixed and centrifuged at 12,000 rpm for 5 min. The supernatant was then subjected to 2 w / v agarose gel electrophoresis to screen for the optimal primer pair.

[0043] The results are as follows Figure 1 As shown, the amplification products of primer pair 2 have high band brightness, good clarity, and the fewest non-specific bands.

[0044] Subsequently, biotin was labeled at the 5' end of the downstream primer MS-R2 and named MS R2b; at the same time, two detection probes were designed according to the instructions of the RAA-LFD detection kit (see Table 1). The 5' end of each probe was labeled with a FAM fluorescent group, a tetrahydrofuran (THF) site was embedded in the middle of the probe, and the 3' end was blocked by C3 phosphorylation.

[0045] Specifically: the 5' end of probe 1 is labeled with a FAM fluorescent group, and the 3' end is blocked by C3 phosphorylation. Probe 1 is counted starting from the first T at the 5' end, and tetrahydrofuran replaces the base at position 35; the 5' end of probe 2 is labeled with a FAM fluorescent group, and the 3' end is blocked by C3 phosphorylation. Probe 2 is counted starting from the first A at the 5' end, and tetrahydrofuran replaces the base at position 31.

[0046] To detect the possibility of the primer-probe combination generating a potential false positive signal on the test strip, each probe was validated by the RAA-LFD assay without the addition of MSDNA template.

[0047] The probe screening results showed that both probes could produce positive signal bands on the test strip, but probe 1 exhibited false positives. Ultimately, the primer-probe combination of primer pair 2 and probe 2 was selected for subsequent experiments.

[0048] Example 2: Establishment of the RAA-LFD reaction system Based on the optimal primer pairs and probes, a 50 μL RAA reaction system was established according to the instructions of the RAA test strip nucleic acid amplification kit. The premixed solution included 25 μL A Buffer, 12.9 μL ddH2O, 2 μL upstream primer (2 μmol / L), 2 μL downstream primer (2 μmol / L), and 0.6 μL probe (2 μmol / L).

[0049] After thoroughly mixing the premixed solution in a clean PCR reaction tube, add it to a detection unit tube containing RAA reaction universal dry powder (RAA reaction universal dry powder includes dNTPs, recombinase, single-stranded DNA binding protein, and DNA polymerase). Then, add 5 μL of the DNA sample to be tested to the detection unit tube, and add 2.5 μL of B Buffer to the inside of the tube cap. Each 25 μL of A Buffer contains 10 w / v% polyethylene glycol, and each 2.5 μL of B Buffer contains 280 mM magnesium acetate. Tightly cap the tube, gently invert it, and tap the tube wall 6 times to mix thoroughly. Centrifuge rapidly for 10 s, and incubate in a 39°C water bath for 30 min. After the reaction, dilute 50 μL of the reaction mixture with diluent at a 1:6 (reaction mixture: diluent) volume ratio, and insert it into a single-target HybriDetect lateral chromatography strip (rainbow type). The diluent is sterile water.

[0050] Example 3: Optimization of RAA-LFD Reaction Conditions The best primer pairs were diluted, and with other components of the reaction system unchanged, different concentrations of MS-F2 and MS-R2 were added to the reaction system at final primer concentrations of 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 3 μmol / L and 4 μmol / L to screen for the optimal primer concentration.

[0051] The results are as follows Figure 2 As shown in Figure A, clear target bands were detected in primer concentrations ranging from 0.5 μmol / L to 4 μmol / L, with the bands being brighter at concentrations of 2 μmol / L to 4 μmol / L. Ultimately, 2 μmol / L was determined to be the optimal primer concentration.

[0052] Based on the optimal primer concentration, the concentration of probe 2 was diluted to 0.5 μmol / L, 1 μmol / L, 1.5 μmol / L, 2 μmol / L, 3 μmol / L, and 4 μmol / L to screen for the optimal probe concentration.

[0053] The results are as follows Figure 2As shown in B, bands were detectable in the probe concentration range of 0.5 μmol / L to 4 μmol / L, with better band brightness at concentrations of 1 μmol / L to 4 μmol / L. Therefore, the lowest effective concentration of 1 μmol / L was selected as the probe concentration for subsequent experiments.

[0054] Based on the optimal primer and probe concentrations, the reaction was carried out at 25℃, 30℃, 35℃, 37℃, 39℃, and 41℃ for 30 min to screen for the optimal reaction temperature.

[0055] The results are as follows Figure 2 As shown in C, the bands can be detected in the reaction temperature range of 30℃ to 41℃. The bands are brighter at temperatures of 37℃ to 41℃, so 37℃ was determined to be the reaction temperature for subsequent experiments.

[0056] Finally, the reaction times were set to 10 min, 15 min, 20 min, 25 min, and 30 min to optimize the best reaction time.

[0057] The results are as follows Figure 2 The D-display in the image showed that a clearly discernible target band appeared within a reaction time range of 10 to 30 minutes, with the band brightness reaching its optimal level between 20 and 30 minutes. Therefore, the shorter optimal reaction time of 20 minutes was chosen as the reaction time for subsequent experiments.

[0058] Additionally, in the absence of heating equipment, simply hold the centrifuged test unit tube in your palm for 15 minutes. Figure 3 This allows for efficient amplification and accurate detection.

[0059] Example 4: Specificity Test Using the DNA of MS, MG, Mb, Pm, Mh, Sa, Salmonella pullorum, Escherichia coli, Bacillus subtilis, Enterococcus faecalis, and Enterococcus faecium as templates, and ddH2O as a negative control, RAA-LFD amplification was performed under the optimal reaction conditions obtained in Example 3 to detect the specificity of the method.

[0060] The results showed that the DNA of all MS strains could amplify the target band, while the DNA of other pathogens did not amplify the band. Figure 4 This indicates that the MS RAA-LFD detection method established in this study has good specificity and no cross-reactivity with other common pathogens.

[0061] Example 5: Sensitivity Test 1. Recombinant plasmid pMD19- vlhA Preparation MS was amplified by PCR. vlhA Genes. MS vlhAThe sequences of the primers for full-length gene PCR amplification are shown in SEQ ID NO.11 and SEQ ID NO.12.

[0062] Upstream primer: 5'-TTGCAATGTCAGAAGAAGCT-3', SEQ ID NO.11; Downstream primer: 5'-GCTTGGAATAATGAAGATTC-3', SEQ ID NO.12.

[0063] Each 20 μL PCR amplification reaction system contains: 10 μL PCR Mix, 6 μL ddH2O, 1 μL of 10 μmol / L upstream primer, 1 μL of 10 μmol / L downstream primer, and 2 μL MS DNA template.

[0064] The PCR amplification reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 32 cycles; 72℃ final extension for 7 min.

[0065] After the reaction was completed, the amplification product was subjected to agarose gel electrophoresis, the target band was cut off and purified.

[0066] The amplification products were ligated into the pMD19-T vector. Each 10 μL ligation system contained: gel-recovered... vlhA 4 μL of gene, 1 μL of pMD19-T vector, and 5 μL of Solution I ligase were added. The ligation system was incubated at 4°C for 12 h to obtain the ligation product.

[0067] Thaw E. coli DH5α competent cells stored at -80℃ on ice. In a clean bench, transfer 10 μL of ligation product into the competent cells, gently shake to mix, incubate on ice for 30 min, perform heat shock at 42℃ for 90 s, and then quickly incubate on ice for 3 min. Add 1 mL of LB liquid medium without Amp resistance and incubate at 37℃ and 200 rpm for 1 h. Take the culture, centrifuge at low speed for 1 min, remove the supernatant, and take 200 μL of the bacterial culture to spread evenly on LB solid medium (Amp resistant). After the bacterial culture is completely absorbed, incubate upside down for 12 h. Pick a single colony from the medium and transfer it to 5 mL of LB liquid medium containing Amp resistance. Incubate at 37℃ and 220 rpm for 12 h.

[0068] Positive strains were screened by bacterial culture PCR verification. The sequences of the universal primers M13F and M13R for pMD19-T are shown in SEQ ID NO.13 and SEQ ID NO.14, respectively. M13F: 5'-CGCCAGGGTTTTCCCAGTCACGAC-3', SEQ ID NO.13; M13R: 5'-AGCGGATAACAATTTCACACAGGA-3', SEQ ID NO. 14.

[0069] Each 20 μL PCR amplification reaction system contains: 10 μL PCR Mix, 6 μL ddH2O, 1 μL 10 μmol / L M13F, 1 μL 10 μmol / L M13R, and 2 μL bacterial culture.

[0070] The PCR amplification conditions were: 95℃ for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min, 30 cycles; extension at 72℃ for 7 min. The reaction products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing, and the sequencing results were compared with NCBI. Plasmid DNA was extracted using a plasmid miniprep kit (to obtain pMD19-). vlhA The plasmid was stored at -20°C. The correct recombinant plasmid pMD19- was obtained after PCR and sequencing verification. vlhA The concentration was determined using an ultra-micro spectrophotometer, and the copy number was calculated.

[0071] 2. Sensitivity determination of the detection method The constructed positive plasmid standard pMD19- vlhA The concentrations obtained by serially diluting the product 10-fold were 2.63 × 10⁻⁶. 7 copies / μL, 2.63×10 6 copies / μL, 2.63×10 5 copies / μL, 2.63×10 4 copies / μL, 2.63×10 3 copies / μL, 2.63×10 2 copies / μL, 2.63×10 1 copies / μL, 2.63×10 0 The copies / μL were used as a template to determine the sensitivity of three methods: RAA-LFD, PCR, and qPCR.

[0072] RAA-LFD amplification was performed under the optimal reaction conditions obtained in Example 3.

[0073] The primers used for PCR detection were the same as those used for RAA-LFD. The reaction mixture consisted of 10 μL PCR Mix, 6 μL ddH2O, 1 μL 10 μmol / L MS-F2, 1 μL 10 μmol / L MS-R2, and 2 μL DNA template. The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 32 cycles; and a final extension at 72℃ for 7 min.

[0074] qPCR detection was performed using SYBR Green qPCR Mix, with the same primers as RAA. The qPCR reaction system (10 μL) consisted of: 5 μL qPCR Mix, 0.2 μL 10 μmol / L MS-F2, 0.2 μL 10 μmol / L MS-R2, 3.6 μL sterile water, and 1 μL template DNA. The reaction program was as follows: the reaction solution was pre-denatured at 95℃ for 30 s; then 40 cycles were performed, each cycle consisting of denaturation at 95℃ for 15 s, annealing at 60℃ for 10 s, and extension at 72℃ for 30 s.

[0075] Experimental data showed that the limits of detection for RAA-LFD, PCR, and qPCR were 2.63 × 10⁻⁶. 0 copies / μL, 2.63×10 3 copies / μL and 2.63×10 0 copies / μL ( Figure 5 ).

[0076] Example 6: Repeatability Test The MS RAA-LFD method established according to this invention achieves a yield of 2.63 × 10⁻⁶. 4 copies / μL, 2.63×10 3 copies / μL, 2.63×10 2 The three concentrations of positive plasmid standard pMD19- were measured in copies / μL. vlhA Using the template, three intra-group and inter-group repeatability tests were performed to analyze the amplification results.

[0077] Results of intragroup repeatability tests are as follows Figure 6 As shown, the target band was successfully amplified at all concentrations, and the brightness of the amplified bands in each repeated detection at the same concentration was basically the same. The results of the inter-group repeatability test are as follows: Figure 7 As shown, all concentrations successfully amplified the target band in three replicate experiments, and the brightness of the amplified bands was basically the same across different replicate groups.

[0078] Example 7: Performance Measurement in Practical Applications The test samples in the method for detecting Mycoplasma synoviae in chickens can be samples collected from any environment. In this embodiment, chicken pharyngeal swab samples were used as the test samples: 100 pharyngeal swab samples were collected from chickens suspected of being infected with MS in three broiler farms in Shihezi City, Xinjiang Uygur Autonomous Region. The RAA-LFD detection method, PCR method, and qPCR method in Example 5 were used to detect all samples, and the concordance among the three methods was compared.

[0079] Table 2. Detection results of clinical samples using different methods (n=100) Table 2 shows that the positive rate of RAA-LFD detection was 48% (48 / 100); the positive rate of PCR detection was 44% (44 / 100); and the positive rate of qPCR detection was 48% (48 / 100). The concordance rate between RAA-LFD and PCR was 96%, and the concordance rate between RAA-LFD and qPCR was 100%.

[0080] Table 3 Results of MS detection in samples using RAA-LFD and PCR methods (n=100) RAA-LFD: Recombinase-mediated isothermal amplification combined with lateral flow chromatography strip.

[0081] Table 4 Performance evaluation of the RAA-LFD detection method using PCR as the standard (n=100) Furthermore, as shown in Tables 3 and 4, all 44 samples that tested positive by PCR were also positive in the RAA-LFD test, indicating that the sensitivity of RAA-LFD relative to PCR is 100%. Of the 56 samples that tested negative by PCR, 52 were also negative in the RAA-LFD test, indicating that the specificity of RAA-LFD relative to PCR is 92.9%. Compared to the PCR method, the kappa value of the RAA-LFD detection method established in this invention is 0.92 (K>0.75).

[0082] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0083] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A primer probe combination for detecting Mycoplasma synoviae in chickens based on RAA-LFD, characterized by, The primer-probe combination consists of an upstream primer, a downstream primer, and a probe; the nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4, and the nucleotide sequence of the probe is shown in SEQ ID NO.10; the 5' end of the downstream primer is labeled with biotin.

2. The application of the primer-probe combination according to claim 1 in the preparation of a detection kit for Mycoplasma synoviae or in the detection of Mycoplasma synoviae.

3. A kit for detecting Mycoplasma synoviae in chickens, characterized in that, The kit comprises the primer-probe combination as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also includes reaction buffer, deoxyribonucleoside triphosphate, enzyme mixture, and side-flow chromatography test strips.

5. A method for detecting Mycoplasma synoviae in chickens based on RAA-LFD, characterized in that, Includes the following steps: Extract DNA from the sample to be tested; Using the DNA of the sample to be tested as a template, a recombinase-mediated isothermal amplification reaction was carried out using the primer and probe combination described in claim 1 to obtain the amplification product; The amplification product is diluted with a diluent, and the diluted amplification product is inserted into the sample pad end of the side-flow chromatography test strip. After the reaction is completed, the results are read and determined: when both the detection line and the control line show bands, it indicates that the sample to be tested contains Mycoplasma synoviae.

6. The method according to claim 5, characterized in that, The recombinase-mediated isothermal amplification reaction is carried out at a temperature of 30℃~41℃ for a time of 10min~30min.

7. The method according to claim 5, characterized in that, Each 50 μL recombinase-mediated isothermal amplification reaction system contains: 25 μL A Buffer, 12.9 μL ddH2O, 2 μL 0.5 μmol / L~4 μmol / L upstream primer, 2 μL 0.5 μmol / L~4 μmol / L downstream primer, 0.6 μL 0.5 μmol / L~4 μmol / L probe, 5 μL DNA, 2.5 μL B Buffer, and one tube of RAA reaction general dry powder.

8. The method according to claim 7, characterized in that, Each 25 μL A Buffer contains 10 w / v% polyethylene glycol; each 2.5 μL B Buffer contains 280 mM magnesium acetate; the RAA reaction universal dry powder contains dNTPs, recombinase, single-stranded DNA binding protein and DNA polymerase.

9. The method according to claim 5, characterized in that, The diluent is sterile water or phosphate-buffered saline.

10. The method according to claim 9, characterized in that, The volume ratio of the amplified product to the diluent is 1:5.5~6.5.

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