CN110305975B - RPA kit for rapidly detecting mycoplasma synoviae and application thereof - Google Patents
RPA kit for rapidly detecting mycoplasma synoviae and application thereof Download PDFInfo
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Abstract
The invention discloses an RPA kit for rapidly detecting chicken bursal mycoplasma and a detection method thereof. The kit comprises: a reaction tube containing RPA freeze-dried particles, a reaction buffer solution, magnesium acetate, a pair of primers and a probe. Experiments prove that the sensitivity and specificity of the kit are equivalent to those of a qPCR method, the whole reaction time is finished within 20min, and the detection result is judged only by changing a fluorescence curve. Therefore, the kit has the advantages of rapidness, high efficiency, sensitivity and the like, not only provides a technical means for effectively detecting and identifying the mycoplasma synoviae, but also has simple and rapid operation, and is suitable for clinical veterinary field diagnosis and scientific research on the mycoplasma synoviae.
Description
Technical Field
The invention belongs to the specific field of veterinary medicine prevention inspection, relates to a kit for detecting mycoplasma synoviae and application thereof, and particularly relates to an RPA detection kit for rapidly detecting the mycoplasma synoviae and application thereof.
Background
Mycoplasma Synoviae (MS) belongs to Mycoplasmatales, Mycoplasmataceae and Mycoplasma, is an important pathogen for infecting chickens, and brings serious economic loss to poultry breeding in China. After MS infection, the chicken group can cause upper respiratory diseases, air sacculitis, joint synovitis and tenosynovitis, and the symptoms of joint swelling, lameness, slow growth and development of chicks and reduction of egg yield of laying hens are mainly clinically manifested; the disease can be propagated in contact with each other and vertically and can spread in chicken flocks; since 2008, MS infection was detected in farms in several provinces of China; when MS is combined with other pathogenic factors, such as Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), low pathogenic avian influenza virus or Escherichia coli infection, the condition of the disease is worsened, and the development of the chicken industry is seriously harmed. Considering the breeding mode of chicken flocks in China, the safety of commercial MS attenuated seedlings and the drug resistance of antibiotics, the prevention and treatment difficulty of MS is increasingly high, so that only by quickly diagnosing the disease and eliminating positive chickens, time can be won for the prevention and control of the disease, and the spread of epidemic diseases and the economic loss are reduced to the maximum extent.
Current methods of detecting MS include: the method comprises the steps of mycoplasma separation culture, serological antibody detection and molecular gene diagnosis, wherein the separation culture is a gold standard for diagnosing mycoplasma infectious diseases, but the mycoplasma has high nutrient requirement on a culture medium, slow growth and high separation difficulty and is only suitable for laboratory detection; serological tests such as a plate agglutination test (SPA) and a hemagglutination inhibition test (HI) relate to the problem of strain source for preparing the antigen, and have the characteristics of low sensitivity, poor specificity, strong subjectivity of result judgment, unsuitability for large-scale detection and the like, so that the wide application of the two methods in clinic is limited. ELISA diagnosis is more directed to adhesion proteins, but some epitopes of these Mycoplasma membrane associated antigens are susceptible to cross-reactivity and false positives with other Mycoplasma. At present, commercial MS indirect ELISA kits mainly come from the United states and Europe, although the specificity is high, the detection sensitivity of antibodies infected by different strains is different, the price is high, the detection cost of one blood sample is up to 11.5 yuan, the use of farmers is limited, and the kit is only limited for laboratory research. In addition, the phenomenon of weak serological reaction is frequently seen in clinic after MS infects the upper respiratory tract, so that the method is a good method which is accurate, convenient and fast by selecting a molecular biological detection means. At present, the traditional Polymerase Chain Reaction (PCR) and real-time fluorescent quantitative PCR (real-time PCR) are the most applied molecular diagnosis methods, but both the two technologies need expensive thermocyclers and skilled operators, are time-consuming and labor-consuming, are difficult to operate on site and popularize to the basic level, for example, patent CN 104263858A discloses a double fluorescent quantitative RT-PCR detection kit and primers for MS and avian reovirus, the nucleic acid detection kit designs the primers and probes by MS hemagglutinin genes, but the method needs a fluorescent PCR instrument with a plurality of detection channels, is long in time consumption, needs professional operators to operate and interpret results, and is only suitable for detection in a laboratory. For example, Duncontext et al (2014) disclose a LAMP rapid visual detection method for MS heat shock ATP dependent protease, which can rapidly and conveniently detect MS. However, the method needs to design three pairs of primers for amplification test, which increases the difficulty of test design; in addition, the method has the advantages of amplification of a plurality of bands, non-specific amplification, high detection temperature (62 ℃) and long detection time (1 h). Yang bin et al (2019) disclose a dual PCR method for detecting an MS heat shock ATP-dependent protease gene and for detecting a Mycoplasma gallisepticum gapA gene, which has high specificity and strong sensitivity, but requires nucleic acid electrophoresis to detect an amplification product, increases the possibility of pollution, and is time-consuming and labor-consuming.
The RPA technique was established by Babraham institute, Cambridge, UK and invented by TwistDx Biotech corporation (http:// www.twistdx.co.uk /). The technology takes a nucleic acid replication mechanism of T4 bacteriophage as a principle, and the raw materials required by the reaction comprise recombinase proteins uvsX and uvsY coded by the T4 bacteriophage, a single-chain binding protein gp32, DNA polymerase and oligonucleotide; simultaneously, a primer, a probe, a template and ddH are required2O and Mg2+And the like. The technology is based on the recombinase polymerase-mediated amplification principle, simulates DNA replication in organisms, can perform isothermal amplification on target fragments at normal temperature, gets rid of the requirement on a thermal cycler, can rapidly amplify the target fragments in a short time, and has the advantages of simplicity, convenience, rapidness, sensitivity and the like. Currently, the RPA technology is applied to the molecular detection of a series of pathogenic microorganisms, such as mycobacterium tuberculosis, foot and mouth disease virus, canine parvovirus, African swine fever and the like. However, as far as we know, the application of the RPA technology in MS is not seen at home and abroad.
Disclosure of Invention
Aiming at solving the problems in the prior art, the invention establishes and evaluates a rapid diagnosis kit based on RPA aiming at the assumed protein VY93_00965 gene of the mycoplasma synoviae, the kit can rapidly, specifically and simply identify the mycoplasma synoviae, the purpose of rapid diagnosis is realized, and the kit can be used for scientific research of the mycoplasma synoviae.
In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:
the invention designs primers and probes according to the assumed protein VY 93-00965 gene of mycoplasma synoviae; through Blast software comparison analysis in NCBI, the gene is found to have high conservation with different strains of mycoplasma synoviae uploaded in GenBank; the gene is found to exist in different clinical isolates through proteome sequencing in a laboratory, and the homology of the gene is up to more than 98.5 percent as proved by PCR amplification and sequencing results. Therefore, one segment (701bp-920bp) in the whole length of the gene is selected as a target segment design primer and a probe so as to detect as many mycoplasma synoviae as possible. Aiming at the detection characteristics of RPA, the invention respectively designs and synthesizes three pairs of upstream primers and three pairs of downstream primers, and finally selects a primer and probe combination which can generate strong amplification signals to be applied to the invention by evaluating the amplification effect of the primer and probe combination. All primers and probes were synthesized by kingsry (south kyo, china).
The invention provides an RPA kit for rapidly detecting chicken bursal disease mycoplasma, which comprises: reaction tube containing RPA freeze-dried particles, reaction buffer solution, magnesium acetate and ddH2O; the kit also comprises a pair of primers and a probe, wherein the primer pair and the probe are as follows:
an upstream primer: 5'-GTTGCGTTTTTGGCAGCAG-3'
A downstream primer: 5'-ATGGAACTAGCGGGCTTCAAA-3'
And (3) probe: 5 '-GGCAGATTTGATG [ dT-Fam ] C [ dSpacer ] A [ dT-BHQ1] TTAAGAGCTGAAGC [ C3-Spacer ] -3'.
The probe sequence is provided with a fluorescent group FAM and a quenching group BHQ1, the two groups are separated by dSpacer, and the 3' end is modified by C3 Spacer.
The RPA freeze-dried particles (twist Dx exo kit, Cambridge, United kingdom) comprise phage recombinase UvsX, cofactor UvsY, DNA polymerase, single-stranded DNA binding protein, dNTPS.
The reaction Buffer is a Rehydration Buffer.
Preferably, the final concentration of the forward primer, the reverse primer and the probe in the RPA kit is 10 μ M.
Preferably, the 50 μ L reaction system of the kit is as follows:
mu.L of forward primer, 2. mu.L of reverse primer, 0.5. mu.L of probe, 29.5. mu.L of reaction buffer, 4. mu.L of Mycoplasma DNA template, 9.5. mu.L of ddH2O and 2.5. mu.L of magnesium acetate.
The invention also provides the application of the RPA in detecting the mycoplasma synoviae, and the application does not aim at the diagnosis and treatment of diseases.
The invention also provides a detection method of the mycoplasma synoviae, which does not aim at the diagnosis and treatment of diseases and comprises the following steps: using the DNA to be detected as a template, performing recombinase polymerase amplification by using the kit of claim 1, and analyzing the amplification result: if the fluorescence curve is obviously increased, the chicken mycoplasma synoviae genome DNA is contained, otherwise, the chicken mycoplasma synoviae genome DNA is not contained.
Preferably, the recombinase polymerase is used for amplification, the reaction temperature is 37-40 ℃, and the reaction time is 20min-1 h.
Preferably, the recombinase polymerase is used for amplification at a reaction temperature of 37 ℃ for a reaction time of 20 min.
Compared with the prior art, the method has the following advantages:
A. time is saved: the whole process of RPA only needs 20min, which is far lower than 1.5h of qPCR.
B. And (3) reducing the reaction temperature: the RPA completed the experiment at a constant temperature of 37 ℃ which is much lower than 60-95 ℃ for qPCR.
C. The method is simple, and the kit is convenient to carry: the enzyme and other necessary substances required by amplification are freeze-dried and stored, the kit can be placed at normal temperature, only reaction buffer solution, primers, probes and templates need to be added during amplification, and magnesium ions are added to initiate reaction, the professional requirement on operators is low, and the kit is suitable for field diagnosis of primary veterinarians and can also be used for scientific research on mycoplasma synoviae.
D. The sensitivity is high: the RPA primer and the probe of the mycoplasma synoviae designed by the invention can detect a template with at least 100 copies at 37 ℃ under the condition of amplifying for 20 min.
E. The specificity is strong: the kit is added with the probe, so that the detection specificity is improved, and the conformity with the existing qPCR method is 100%.
F. Is not easy to pollute: the kit provided by the invention is added with exonuclease III to cut the amplified product, so that the possibility of product pollution is reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the sensitivity analysis of the real-time fluorescence detection method for RPA.
FIG. 2 shows the specificity of the real-time fluorescence detection method for RPA.
Detailed Description
Modifications and substitutions in detail and form may be made to the present invention without departing from the spirit and scope thereof, but it is intended that all such modifications and substitutions fall within the scope of the present invention. Unless otherwise specified, the raw materials and chemical reagents used in the examples are all conventional commercial products, and the technical means used are conventional means known to those skilled in the art.
Example 1 establishment of RPA kit for rapid diagnosis of Mycoplasma synoviae and detection method
1. Design and preparation of primer and probe sequence
The invention designs primers and probes according to the assumed protein VY 93-00965 gene of mycoplasma synoviae; through Blast software comparison analysis in NCBI, the gene is found to have high conservation with different strains of mycoplasma synoviae uploaded in GenBank; the gene is found to exist in different clinical isolates through proteome sequencing in a laboratory, and the amplification and sequencing results prove that the homology of the gene is up to more than 98.5 percent. Therefore, one segment (701bp-920bp) in the whole length of the gene is selected as a target segment design primer and a probe so as to detect as many mycoplasma synoviae as possible. All primers and probes were synthesized by kingsry (south kyo, china). The invention respectively designs and synthesizes three pairs of upstream primers, three pairs of downstream primers and a probe sequence, which are shown in Table 1.
TABLE 1 Mycoplasma synoviae RPA primers and probes
2. Strains, viral genomes and clinical samples
The genomes of Mycoplasma synoviae (WVU1385), Mycoplasma gallisepticum (MG Rlow) strains, and Salmonella gallinarum (CVCC541), Escherichia coli (CVCC1560), Staphylococcus aureus (ATCC25923), infectious bronchitis virus (H120) used in the present invention were maintained by the laboratory. The inventor of the application collects 70 clinical cotton swab samples from Gansu province, Henan province and Shandong province and 10 cotton swabs of healthy chickens for testing.
3. Mycoplasma genome extraction
The mycoplasma DNA was extracted using a high-purity mycoplasma nucleic acid extraction Kit (QIAamp cador Pathologen Mini Kit) according to the instructions and finally eluted with 50. mu.L of RNase-free water. The extracted DNA was stored at-20 ℃ for further use.
4. Standard plasmid preparation
Extracting the mycoplasma synoviae DNA. A segment (367bp-1178bp) in the full length of the amplified VY 93-00965 is designed to be used as a target fragment (SEQ No.1), and primers are as follows:
an upstream F: 5'-ACTAACTGCTTGTGGCAGGTT-3' the flow of the air in the air conditioner,
downstream R: 5'-CTTCGCCAGTTACAAACGCT-3'
Pre-denaturing for 5min at 94 ℃ under the amplification condition by taking the extracted mycoplasma synoviae DNA as a template; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 50s, and extension at 72 ℃ for 80s for 35 cycles; further extension at 72 ℃ for 10min and at 4 ℃ for 5 min. And (3) carrying out electrophoresis identification on the PCR product in 10g/L agarose gel to confirm that the target gene fragment with the size of 812bp is obtained by amplification. PCR products were recovered using a gel recovery kit, ligated with pMD-19T cloning vector, transformed into Escherichia coli competent cell JM109, plated on LB medium plate containing 100mg/L ampicillin, and cultured at 37 ℃ for 12 hours. Screening positive single bacteria, dropping into liquid culture medium containing ampicillin, culturing at 37 deg.C, collecting 2mL of bacteria liquid, extracting plasmid, and determining sequence positive as pMG-19T-812.
SEQ No.1:
ACTAACTGCTTGTGGCAGGTTAAATATGTCTTTATTACCTTGAGCTACTGAATACAATGTATTTTTGACAAATTCTTTGAAGTTGTCAAAGCTTGTAAAGTTTGTATATTGTTGAATTGAAATTTTATCTTTAGAAACTACTAAATATGCATTAGGCATATCTTTAATTGTATAAATAAATGAAACTAAATTATTTCCACCAACATTTTTAACTAGCTGATTTGTATATTGTCTAACTAAAAGAGATGAAAATTCGGCATTAGACATATTGGTTAAATATGATTCGATAAATTGATTTAACTCTTTGGTTTTATTAAATGCATCTTGATCTGAGGTTGCGTTTTTGGCAGCAGTTAAAAGAGTTGAAAATCTTGAATAAGCGCTAGGATCATTAGTTCTTATATTTTCAAAGTCAAAGACTTTACTTAAATCAACTTGTGGCAGATTTGATGCATTAAGAGCTGAAGCTAAAACTTTGCTTCAAATAGATGATAGCCCTTGCTCGGTTCCATTTTGAAATACGCTTTCAATGCTTTGAAGCCCGCTAGTTCCATAACTATCACCTTTAGTTAAAGTTAGTGTATTTAAAAAGGTTTGATAGTTTCCTTGATTAACTGTAATTTGAGATTGTGAAAGAGCTTCTGTTGGAGTTGGGAAATAAGAAGTAGCTGGCTTTAGTTGTTTTAAAATTTCGCTTCCGCTTAAAATTTGTCTTTGTGAATTAGTATTTTTAACAGATCAATATTTAGCAAGGTTGATAAATTTAGCTTTAGCGTCAACATCTAAAGTAATAGCGTTTGTAACTGGCGAAG
Optimization of Real-time RPA test amplification conditions
The amplification test is carried out by taking the extracted and purified mycoplasma synoviae DNA or standard plasmid as a template, and the experimental system is as follows: mu.L of reaction buffer (rehydration buffer, twist Dx exo kit, Cambridge, United Kingdom), 2. mu.L of upstream and downstream primers (10. mu.M), 0.5. mu.L of probe (10. mu.M), 9.5. mu.L of deionized water, 4. mu.L of template, and 2.5. mu.L of 280mmol/L magnesium acetate solution were added to the reaction tube containing the RPA lyophilized particles.
Wherein, the sequences of the pair of primers and the probe are as follows:
an upstream primer: 5'-GTTGCGTTTTTGGCAGCAG-3'
A downstream primer: 5'-ATGGAACTAGCGGGCTTCAAA-3'
And (3) probe: 5 '-GGCAGATTTGATG [ dT-Fam ] C [ dSpacer ] A [ dT-BHQ1] TTAAGAGCTGAAGC [ C3-Spacer ] -3'.
When the reaction temperature of the RPA primer was determined, the reactants were added as described above. We tested four different reaction temperatures of 37 deg.C, 38 deg.C, 39 deg.C and 40 deg.C, respectively, with a reaction time of 20min, and analyzed the results by real-time fluorescence quantitative PCR instrument software of Bio-Rad after the completion. The result analysis shows that the amplification result at 37 ℃ is optimal. Therefore, the amplification temperature of the RPA of the mycoplasma synoviae designed by the invention is set to be 37 ℃. When PRA reaction time is determined, reactants are added according to the system, 3 different times are set at 37 ℃, namely 20min, 30min and 1h respectively, and the result shows that the negative and positive results of amplification for 20min and amplification for 1h are consistent, so that the amplification time of the RPA of the mycoplasma synoviae is set to be 20 min.
Meanwhile, the combination of three pairs of primers and probes is evaluated by using the amplification system, and the result shows that the combination of one pair of primers (VY 93-00965 RPAF1/R1 in the table 1) and the probes can generate strong amplification signals, so the primer and probe combination is selected by the invention.
Judging the RPA test result: the fluorescence curve of the reaction tube containing Mycoplasma synoviae genomic DNA increased, and the negative control was a smooth straight line.
RPA kits and methods of use
The RPA kit comprises the following components:
a reaction tube containing RPA freeze-dried particles, a reaction buffer (rehydrationbuffer, TwistDx exo kit, Cambridge, United kingdom), an upstream primer, a downstream primer, a probe, deionized water, standard plasmids and a 280mmol/L magnesium acetate solution.
The 50 μ L reaction was as follows:
to a reaction tube containing RPA lyophilized particles, 29.5. mu.L of reaction buffer (dehydration buffer, twist Dx exo kit, Cambridge, United Kingdom), 2. mu.L each of upstream and downstream primers (10. mu.M), 0.5. mu.L of probe (10. mu.M), 9.5. mu.L of deionized water, 4. mu.L of template, and 2.5. mu.L of 280mmol/L magnesium acetate solution were sequentially added.
The primer is VY93_00965RPAF1/R1 in Table 1, and the probe is the probe in Table 1.
And (2) placing the reaction system in a real-time fluorescent quantitative PCR instrument for amplification, wherein the RPA amplification conditions are as follows: amplifying at 37 ℃ for 20min, collecting a primary fluorescence signal for 1min, and analyzing the result by a real-time fluorescence quantitative PCR instrument after the amplification is finished.
And (4) judging the standard: the fluorescence curve of the reaction tube containing the Mycoplasma synoviae genomic DNA is increased, and the reaction tube containing no Mycoplasma synoviae genomic DNA is a smooth straight line.
RPA assay sensitivity and specificity detection:
and (3) sensitivity detection: the concentration of the positive plasmid pMD-19T-812 is determined to be 58 ng/mu L by a nucleic acid determinator, the base number of the pMD-19T vector is 2692bp, the size of an amplification product is 812bp, the average molecular weight of each base is 660 daltons/bp, and the copy number of the detected gene in each mu L sample is calculated according to the formula: sample copy number (copies/. mu.L) ═ avogalois constant (6.02X 10)23) X plasmid concentration ng/. mu.L.times.10-9(660X recombinant plasmid base number), wherein the recombinant plasmid base number ═ vector sequence base number + insertion sequence base number, then the calculated copy number is 1.51X 1010copies/. mu.L. The positive plasmid was diluted 10-fold to a concentration of 107～101Between copies/. mu.L and used as template for RPA reaction. The amplification reaction was carried out in a qPCR apparatus at 37 ℃ for 20min, the fluorescence signal was collected once at 1min, and the amplification curve was collected, and the results are shown in FIG. 1, where the amplification curve time and the fluorescence signal value gradually decreased with the decrease in the template copy number and the copy number was 102Still has an amplification curve when the copy number is 101No obvious amplification curve is generated; is shown at 107～102Samples in the copies/. mu.L range were detected.
FIG. 1 shows the sensitivity analysis of the real-time fluorescence detection method for RPA. Wherein the content of the first and second substances,a: template concentration of 1.51X 107Amplification curves for copies/. mu.L; b: template concentration of 1.51X 106Amplification curves for copies/. mu.L; c: template concentration of 1.51X 105Amplification curves for copies/. mu.L; d: template concentration of 1.51X 104Amplification curves for copies/. mu.L; e: template concentration of 1.51X 103Amplification curves for copies/. mu.L; f: template concentration of 1.51X 102Amplification curves for copies/. mu.L; g: template concentration of 1.51X 101Amplification curves of copies/. mu.L.
And (3) specific detection: and adding reactants according to the system to perform RPA detection, wherein the used templates are mycoplasma synoviae (positive control), mycoplasma gallisepticum (MG Rlow), salmonella gallinarum (CVCC541), colibacillus gallinarum (CVCC1560), staphylococcus aureus (ATCC25923) and infectious bronchitis virus (H120). Performing amplification reaction in a real-time fluorescent quantitative PCR instrument at 37 ℃, wherein the reaction time is 20min, collecting a primary fluorescent signal after 1min, and collecting an amplification curve; as shown in FIG. 2, only Mycoplasma synoviae can be well amplified, and no amplification curve is generated in other cases, which indicates that the method has strong specificity for detecting Mycoplasma synoviae.
FIG. 2 shows the specificity analysis of the real-time fluorescence detection method for RPA: a: a positive control; b: mycoplasma gallisepticum (MG Rlow); c: salmonella gallinarum (CVCC 541); d: chicken escherichia coli (CVCC 1560); e: staphylococcus aureus (ATCC 25923); f: infectious bronchitis virus of chicken (H120); g: negative Control (NC).
Detection of clinical samples by RPA assay
The applicant detected 70 clinical cotton swab samples collected from three provinces of Gansu, Henan and Shandong and 10 cotton swab samples of healthy chickens by an established RPA method, and compared the results with the indirect results of qPCR. The results are shown in table 2: in 70 clinical samples, 38 positive samples are detected by the RPA method and are completely consistent with the qPCR detection result; the RPA detection result and the qPCR detection result of 10 serum samples of healthy chickens are consistent and negative, and the coincidence rate is 100%. Among them, the qPCR method is carried out by the method described in the literature (Raviv Z, Kleven SH. the later of diagnostic real-time TaqMan PCRs for four pathological antigenic mycoplasma. Avian Dis.2009,53(1): 103-.
Table 2 compares the results of clinical sample detection by RPA and qPCR methods
9. Repeatability test
The pMD-19T-812 standard plasmid (1.51X 10) was used in the above reaction system7copies/μL～1.51×101copies/. mu.L) as template for stability detection. 3 replicate wells were made for each dilution of standard plasmid in the same experiment to analyze intra-group differences; each of the 3 independent experiments was repeated under the same conditions as described above, and the difference between groups was analyzed to calculate the Coefficient of Variation (CV) as Standard Deviation (SD)/average (X). SPSS19.0 software is used for statistical analysis, variance analysis is used for comparing differences among different samples, and P is less than 0.05, so that statistical significance is achieved. The results show that: the CV values between the holes in the group are between 0.34% and 0.83%, and the CV values between the repeated determinations of the groups are between 0.41% and 0.93%, and are all less than 1% (Table 3).
TABLE 3 Real-time RPA detection of Mycoplasma synoviae in-and between-groups repeatability
Note: in the table
As the mean, S is the standard deviation and CV is the coefficient of variation.
In conclusion, the mycoplasma synoviae detection kit based on recombinase polymerase amplification technology (RPA) can be used for quickly diagnosing the mycoplasma synoviae, and is simple and quick in detection method and real and reliable in detection result.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
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Claims (7)
1. A RPA kit for rapidly detecting mycoplasma synoviae is characterized in that: the kit comprises: reaction tube containing RPA freeze-dried particles, reaction buffer solution, magnesium acetate and ddH2O; the kit also comprises a pair of primers and a probe, wherein the primer pair and the probe are as follows:
an upstream primer: 5'-GTTGCGTTTTTGGCAGCAG-3'
A downstream primer: 5'-ATGGAACTAGCGGGCTTCAAA-3'
And (3) probe: 5 '-GGCAGATTTGATG [ dT-Fam ] C [ dSpacer ] A [ dT-BHQ1] TTAAGAGCTGAAGC [ C3-Spacer ] -3'.
2. The RPA kit according to claim 1, characterized in that: the final concentration of the upstream primer, the downstream primer and the probe in the RPA kit is 10. mu.M.
3. The RPA kit according to claim 1, characterized in that: the 50-mu-L reaction system of the kit is as follows: 29.5. mu.L of reaction buffer, 2. mu.L of each of 10. mu.M of upstream and downstream primers, 0.5. mu.L of 10. mu.M of probe, ddH2O9.5. mu.L, Mycoplasma DNA template or standard plasmid 4. mu.L and 2.5. mu.L of 280mmol/L magnesium acetate solution.
4. Use of the RPA kit according to any one of claims 1-3 for the detection of mycoplasma synoviae, said use not being aimed at the diagnosis and treatment of diseases.
5. A method for detecting mycoplasma synoviae, said method not being aimed at the diagnosis and treatment of diseases, characterized in that: the method comprises the following steps: using the DNA to be detected as a template, performing recombinase polymerase amplification by using the kit of claim 1, and analyzing the amplification result: if the fluorescence curve is obviously increased, the chicken mycoplasma synoviae genome DNA is contained, otherwise, the chicken mycoplasma synoviae genome DNA is not contained.
6. The detection method according to claim 5, characterized in that: when the recombinase polymerase is used for amplification, the reaction temperature is 37-40 ℃, and the reaction time is 20min-1 h.
7. The detection method according to claim 6, characterized in that: and during recombinase polymerase amplification, the reaction temperature is 37 ℃, and the reaction time is 20 min.
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| CN114467855A (en) * | 2022-01-14 | 2022-05-13 | 广东海大集团股份有限公司 | Construction method and application of chick model with multiple-cause ballooning disease |
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| CN103131790A (en) * | 2013-03-15 | 2013-06-05 | 广西壮族自治区兽医研究所 | LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS) |
| CN106399545A (en) * | 2016-11-03 | 2017-02-15 | 武汉科前生物股份有限公司 | Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof |
| CN108359737A (en) * | 2018-02-11 | 2018-08-03 | 苏州先达基因科技有限公司 | Mycoplasma contamination detection method and application |
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| CN103131790A (en) * | 2013-03-15 | 2013-06-05 | 广西壮族自治区兽医研究所 | LAMP (loop-mediated isothermal amplification) visual detection kit for chicken mycoplasma synoviae (MS) |
| CN106399545A (en) * | 2016-11-03 | 2017-02-15 | 武汉科前生物股份有限公司 | Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof |
| CN108359737A (en) * | 2018-02-11 | 2018-08-03 | 苏州先达基因科技有限公司 | Mycoplasma contamination detection method and application |
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