Primer group and kit for detecting bovine mycobacterium paratuberculosis based on recombinase isothermal amplification technology and application

Abstract

The invention provides a primer group and a kit for detecting bovine mycobacterium paratuberculosis based on a recombinase isothermal amplification technology and application, and relates to the technical field of biological detection.The primer group comprises a forward primer MAP-F3, a reverse primer MAP-R3 and a specific fluorescent probe MAP-P3 and has good specificity; the sensitivity reaches 10fg / mu L, which is more sensitive than that of a colloidal gold test strip, the dependence on equipment is broken through, and the detection result is more accurate. The technical problem that in the prior art, it is difficult to distinguish bovine mycobacterium paratuberculosis from other pathogens through a detection method, or the detection process is tedious is solved. According to the prepared reagent or kit, the liquid preparation process is simple, the whole amplification process can be completed only in 30 min, the amplification yield can reach 109-1010 copies, the method is rapid and efficient, the amplification result can be directly judged according to fluorescence data, other auxiliary means are not needed, and the method is suitable for field detection.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a primer set and a kit for detecting bovine Mycobacterium paratuberculosis based on a recombinase isothermal amplification technology and an application thereof. Background Art

[0002] Bovine paratuberculosis (PTB) is a chronic infectious disease caused by Mycobacterium avium subsp. Paratuberculosi (MAP), characterized by granulomatous enteritis, diarrhea, progressive emaciation and death. The disease is one of the most serious infectious diseases affecting the global dairy industry, which can cause reduced milk production, weight loss, culling / death and increased disease control costs. The disease is also a potential zoonosis. Studies have shown that MAP may be related to human Crohn's disease (Mendoza et al., 2009). Therefore, rapid detection of MAP is very important for the prevention and control of paratuberculosis and purification, as well as the control of possible food safety issues.

[0003] One of the main ways of transmission of MAP is from infected cows to calves through the fecal-oral route. It can also be transmitted through pathogenic milk and contaminated feed. The disease is currently widely distributed throughout the world, with a high infection rate, but a low morbidity rate and a long incubation period. Once it occurs in a cattle farm, it is difficult to purify, which will cause significant economic losses to the cattle and sheep breeding industry. People can also develop allergic enteritis by consuming dairy products contaminated with Mycobacterium paratuberculosis, which poses a huge threat to public health. There is no effective treatment for the disease, and the current way to eradicate the disease is to quarantine, isolate or eliminate sick animals. The isolation and culture of Mycobacterium paratuberculosis in cattle is a highly specific detection method and is often used as a reference standard for diagnosing the disease. However, the detection cost of this method is relatively high, the experimental cycle is relatively long, and the best elimination time may be missed, thereby bringing transmission risks and endangering healthy livestock. In addition, the operation steps of this method are complicated and cumbersome, and there are also high requirements for the professional skills and theoretical knowledge of the detection personnel. At present, other methods for detecting MAP, such as microscopy, skin allergy (SDTH), colloidal gold, complement fixation test (CFT), ELISA, agar diffusion test (AGID), etc., are limited in diagnostic value for detecting subclinical infected animals due to the complexity of the test or the low sensitivity of the method; while more sensitive diagnostic methods such as Northern blotting, qPCR, cDNA microarray and mass spectrometry are more complicated, time-consuming and labor-intensive, and have a certain misdiagnosis rate. Therefore, it is necessary to develop a more sensitive, accurate and rapid detection method to meet the challenge of bovine paratuberculosis.

[0004] In recent years, with the development of molecular biology technology, recombinase polymerase amplification (RPA) technology, as an emerging nucleic acid amplification detection technology, has been widely used to detect pathogens of various diseases, including viruses, bacteria and fungi. RPA technology has the advantages of fast detection speed, simple operation, and no need for expensive instruments and equipment. Therefore, it is very suitable for rapid detection of bovine paratuberculosis. However, there is still a lack of RPA systems for detecting bovine paratuberculosis, especially RPA systems suitable for industrial production and wide application.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a primer set for detecting bovine Mycobacterium paratuberculosis, so as to solve the technical problems in the prior art that the detection method is difficult to distinguish bovine Mycobacterium paratuberculosis from other diseases or the detection process is cumbersome.

[0007] The second object of the present invention is to provide the use of the above primer set in preparing a product for detecting or assisting in detecting bovine Mycobacterium paratuberculosis.

[0008] The third object of the present invention is to provide a reagent for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, comprising the above-mentioned primer set.

[0009] A fourth object of the present invention is to provide a kit for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, comprising the above-mentioned primer set or the above-mentioned reagent.

[0010] A fifth object of the present invention is to provide a method for detecting or assisting in detecting bovine Mycobacterium paratuberculosis for non-therapeutic and non-diagnostic purposes.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a primer set for detecting bovine Mycobacterium paratuberculosis, comprising a forward primer MAP-F3, a reverse primer MAP-R3 and a specific fluorescent probe MAP-P3;

[0013] The nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.9, the nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.10, the structure of the MAP-P3 is SEQ ID NO.11-tetrahydrofuran residue-SEQ ID NO.12, the 30th nucleotide T of SEQ ID NO.11 is labeled with a fluorescent reporter group, the 1st nucleotide T of SEQ ID NO.12 is labeled with a fluorescent quencher group, and the 3' end is blocked and modified.

[0014] Furthermore, the molar ratio of MAP F, MAP R and MAP P is 2-5:2-5:1, preferably 3:3:1.

[0015] Further, the fluorescent reporter group is selected from at least one of FAM, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705;

[0016] The fluorescence quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA;

[0017] The 3' end of SEQ ID NO. 4 is connected to C3-spacer.

[0018] In a second aspect, the present invention provides use of the above primer set in preparing a product for detecting or assisting in detecting bovine Mycobacterium paratuberculosis.

[0019] In a third aspect, the present invention provides a reagent for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, comprising the above-mentioned primer set.

[0020] In a fourth aspect, the present invention provides a kit for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, comprising the above primer set or the above reagent.

[0021] Furthermore, it also includes PCR reaction reagents, MgAc, positive controls and negative controls required for isothermal amplification of the recombinase;

[0022] Preferably, the PCR reaction reagents include at least one of dNTP, recombinase, nuclease, polymerase, creatine kinase, single-stranded DNA binding protein or excipient;

[0023] Preferably, the positive control is a positive plasmid sequence containing a partial sequence of a gene in the conserved region of Mycobacterium bovis paratuberculosis;

[0024] Preferably, the negative control is DEPC-treated water;

[0025] Preferably, the recombinase is selected from bacteriophage UvsX protein and / or Escherichia coli recA protein;

[0026] Preferably, the nuclease is selected from exonucleases and / or endonucleases;

[0027] Preferably, the polymerase is selected from at least one of bsu polymerase, klenow polymerase or phi polymerase;

[0028] Preferably, the single-stranded DNA binding protein is selected from E. coli SSB and / or GP32 protein;

[0029] Preferably, the excipient is selected from at least one of 20% polyethylene glycol, 100 mmol / L Tricine, 5 mmol / L dextran or 5 mmol / L dithiothreitol.

[0030] Furthermore, the primer set described in any one of claims 1 to 3 or the reagent, PCR reaction reagent and MgAc described in claim 5 are all in dry powder form.

[0031] In a fifth aspect, the present invention provides a method for detecting or assisting in detecting bovine paratuberculosis Mycobacterium for non-therapeutic and non-diagnostic purposes, comprising performing recombinase isothermal amplification on a sample using the above-mentioned primer set, the above-mentioned reagent or the above-mentioned kit, and determining whether bovine paratuberculosis Mycobacterium is present in the sample based on a fluorescent signal.

[0032] Furthermore, the reaction temperature of the recombinase isothermal amplification is 33-42°C, preferably 37°C;

[0033] Preferably, the sample is a DNA sample.

[0034] The primer set for detecting bovine paratuberculosis provided by the present invention detects the MAP F57 conservative sequence, can distinguish bovine paratuberculosis from bovine mycoplasma, Klebsiella pneumoniae, Mannheimia (Mh), bovine rotavirus and bovine viral diarrhea virus, and has good specificity; the sensitivity reaches 10fg / μL, which is more sensitive than the colloidal gold test strip, breaks through the dependence on equipment and has more accurate detection results. The technical problem that the detection method in the prior art is difficult to distinguish bovine paratuberculosis from other pathogens or the detection process is cumbersome is solved.

[0035] On the other hand, the reagents or kits provided have a simple preparation process, and the entire amplification process can be completed in just 30 minutes. The amplification yield can reach 10 9 ~10 10 It is fast and efficient, and can directly judge the amplification results based on the fluorescence data without other auxiliary means, making it suitable for on-site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 The MAP F57 gene primer set design map of the MAP RPA method provided in Example 1 of the present invention;

[0038] Figure 2 Amplification curves of different RPA detection primer sets provided in Example 1 of the present invention;

[0039] Figure 3 This is an amplification curve diagram of the sensitivity detection of the MAP RPA method provided in Example 5 of the present invention;

[0040] Figure 4 This is an amplification curve diagram of the stability detection of the MAP RPA method provided in Example 6 of the present invention;

[0041] Figure 5 This is an amplification curve diagram of specific detection of the MAP RPA method provided in Example 7 of the present invention;

[0042] Figure 6 This is an amplification curve diagram of clinical samples detected by the MAP RPA method provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0043] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0044] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0045] In one aspect, the present invention provides a primer set for detecting bovine Mycobacterium paratuberculosis, comprising a forward primer MAP-F3, a reverse primer MAP-R3 and a specific fluorescent probe MAP-P3;

[0046] The nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.9, the nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.10, the structure of the MAP-P3 is SEQ ID NO.11-tetrahydrofuran residue-SEQ ID NO.12, the 30th nucleotide T of SEQ ID NO.11 is labeled with a fluorescent reporter group, the 1st nucleotide T of SEQ ID NO.12 is labeled with a fluorescent quencher group, and the 3' end is blocked and modified.

[0047] The primer set is based on the sequence information of Mycobacterium paratuberculosis in Gen Bank, determines its conservative region, and uses biological software to design specific RPA primers and probes; the length of the probe is 35-55 nucleotide bases, and the T bases on both sides of THF are labeled with fluorescent groups and quenching groups, and the probe is blocked at the 3' end by a blocking agent. The inventors have proved through experiments that the primer set detects the MAP F57 conservative sequence, and can distinguish bovine Mycobacterium paratuberculosis from bovine Mycoplasma, Klebsiella pneumoniae, Mannheimia (Mh), bovine rotavirus and bovine viral diarrhea virus, with good specificity; the sensitivity reaches 10fg / μL, which is more sensitive than colloidal gold test strips, breaking through the dependence on equipment and the test results are more accurate. It solves the technical problems in the prior art that the detection method is difficult to distinguish bovine Mycobacterium paratuberculosis from other pathogens, or the detection process is cumbersome.

[0048] In some specific embodiments, the molar ratio of MAP F, MAP R and MAP P is 2-5:2-5:1, preferably 3:3:1.

[0049] In some specific embodiments, the fluorescent reporter group is selected from at least one of FAM, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 or LCRED705; the fluorescent quencher group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA; the 3' end of SEQ ID NO.4 is connected to C3-spacer.

[0050] According to another aspect of the present invention, there is also provided use of the above primer set in preparing a product for detecting or assisting in detecting bovine Mycobacterium paratuberculosis.

[0051] According to another aspect of the present invention, a reagent for detecting or assisting in detecting bovine Mycobacterium paratuberculosis is provided, comprising the above-mentioned primer set.

[0052] According to another aspect of the present invention, a kit for detecting or assisting in detecting bovine Mycobacterium paratuberculosis is provided, comprising the above primer set or the above reagent.

[0053] The test kit can be used for on-site instant testing and combined with miniaturized equipment to realize POCT. It can also be used for on-site testing of suspected diseased ruminants and laboratory confirmation testing; and for early diagnosis of animals before or in the early stages of disease (without obvious symptoms).

[0054] In some specific embodiments, the PCR reaction reagents, MgAc, positive controls and negative controls required for isothermal amplification of the recombinase are also included;

[0055] In some specific embodiments, the PCR reaction reagents include at least one of dNTP, recombinase, nuclease, polymerase, creatine kinase, single-stranded DNA binding protein or excipient.

[0056] Wherein, the recombinase is selected from bacteriophage UvsX protein and / or Escherichia coli recA protein; the nuclease is selected from exonuclease and / or endonuclease; the polymerase is selected from at least one of bsu polymerase, klenow polymerase or phi polymerase; the single-stranded DNA binding protein is selected from Escherichia coli SSB and / or GP32 protein; the excipient is selected from at least one of 20% polyethylene glycol, 100mmol / L Tricine, 5mmol / L dextran or 5mmol / L dithiothreitol.

[0057] In some specific embodiments, the positive control is a positive plasmid sequence containing a partial sequence of a gene in the conserved region of Mycobacterium bovis paratuberculosis.

[0058] In some specific embodiments, the negative control is DEPC-treated water.

[0059] In some specific embodiments, the primer set or the above reagents, PCR reaction reagents and MgAc are all in dry powder form. It is more convenient to store and use, and the liquid preparation process is simple. Just add the DNA template to be tested into the RPA freeze-dried reagent A tube, cover it with the RPA freeze-dried reagent B tube cap, mix well and put it on the machine; the whole amplification process only takes 30 minutes to complete, and the amplification yield can reach 10 9 -10 10 Suitable for industrial production and large-scale applications.

[0060] According to another aspect of the present invention, a method for detecting or assisting in detecting bovine paratuberculosis for non-therapeutic and non-diagnostic purposes is also provided, comprising performing recombinase isothermal amplification on a sample using the above primer set, the above reagent or the above kit, and determining whether bovine paratuberculosis is present in the sample based on a fluorescent signal. The amplification result can be directly determined based on the fluorescent data without the need for other auxiliary means, and is suitable for on-site detection.

[0061] In some specific embodiments, the reaction temperature of the recombinase isothermal amplification is 33-42°C, preferably 37°C. Specifically, the fluorescence RPA reaction procedure can be: place the fluorescence isothermal amplification instrument at 37°C, preheat for 5 minutes, take out the reaction tube, mix it with a vortex instrument, and then centrifuge it instantly; 37°C, 25 minutes, a total of 25 cycles. The FAM channel fluorescence signal is collected once every 1 minute, and a total of 25 times.

[0062] In some specific embodiments, the sample is a DNA sample. Using the DNA of the sample to be tested as a template, a real-time fluorescence RPA reaction is performed in the presence of a forward primer, a reverse primer, a specific fluorescent probe, an RPA dry powder reagent A, an RPA dry powder reagent B and DEPC water for Mycobacterium paratuberculosis bovis, and the sample to be tested is analyzed according to the real-time fluorescence RPA amplification curve.

[0063] In some specific embodiments, determining whether bovine paratuberculosis Mycobacterium is present in the sample according to the fluorescence signal includes using real-time fluorescence RPA instrument analysis software to analyze the sample according to the amplification curve of real-time fluorescence RPA. Further, the analysis of the sample includes judging that when the fluorescence curve of the FAM channel of the sample to be tested presents a typical "S" curve and the Tt value is ≤20, it is judged as a positive result for bovine paratuberculosis; when the curve of the sample to be tested does not present a typical "S" curve or the Tt value is >20, it is judged as a negative result for bovine paratuberculosis.

[0064] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Mycoplasma bovis, Klebsiella pneumoniae, Mannheimia, bovine rotavirus and bovine viral diarrhea virus in the following examples are all preserved by the research and development laboratory of Beijing Biaochi Zehui Biotechnology Co., Ltd.

[0066] Example 1: Design and synthesis of primer sets

[0067] According to the gene sequence of Mycobacterium paratuberculosis F57 published by NCBI, MAP F57 was used as the target gene, and the sequence of the highly conserved region was selected to design specific primers and probes. A total of 3 primer sets were screened. The specific primers are shown in Table 1, and the primer set design map is shown in Figure 1 The third primer set was verified to have good specificity and sensitivity. The primer sequences involved in this study were synthesized by Shanghai Shenggong Biotechnology Co., Ltd.

[0068] Table 1 MAP F57 genome nucleotide sequence

[0069]

[0070] Take quantitative reference materials to conduct amplification comparison test on the above primer probes, and verify them from the aspects of primer amplification efficiency, probe signal-to-noise ratio and amplification curve morphology. Figure 2 As shown, a is the amplification curve diagram of the first group of RPA detection primer groups provided in this embodiment; b is the amplification curve diagram of the second group of RPA detection primer groups provided in this embodiment; c is the amplification curve diagram of the third group of RPA detection primer groups provided in this embodiment.

[0071] Depend on Figure 2 It can be seen that both the second group of RPA detection primers and the third group of RPA detection primers showed good curve morphology and sensitivity. Compared with the third group of RPA detection primers, the sensitivity of the second group of RPA detection primers was weaker; and the test results of the first group of primer probes showed that the amplification efficiency of the primer group and the signal-to-noise ratio of the probe were worse than the second group, and the sensitivity was low. If applied to the test, it may affect the detection of clinical samples. The third group of RPA detection primers are the best in curve morphology, sensitivity and probe sequence matching. Therefore, the primer probes of the third group were finally screened for the following embodiments.

[0072] Example 2: Kit for detecting or assisting in detecting bovine Mycobacterium paratuberculosis

[0073] The kit provided in this embodiment includes RPA dry powder reagent A, RPA dry powder reagent B, bovine Mycobacterium paratuberculosis positive control and negative control. Among them, RPA dry powder reagent A includes the RPA detection primer set and PCR reaction reagent screened in Example 1, the RPA detection primer set includes 450nM forward primer, 450nM reverse primer and 150nM specific fluorescent probe, and the PCR reaction reagent includes 10mM dNTP, 100ng / μL recombinase, 30ng / mL nuclease, 30ng / μL polymerase, 100ng / μL creatine kinase, 100ng / μL single-stranded DNA binding protein and excipient; RPA dry powder reagent B includes 14mM MgAc; the positive control is a positive plasmid sequence containing a partial sequence of the gene in the conservative region of bovine Mycobacterium paratuberculosis, and its nucleotide sequence is shown in SEQ IDNO.13; the negative control is DEPC treated water.

[0074] Example 3: Isothermal amplification method mediated by recombinase of Mycobacterium bovis

[0075] This example uses the kit provided in Example 2 to perform bovine Mycobacterium paratuberculosis recombinase-mediated isothermal amplification. The specific method is performed according to the following steps:

[0076] S1. Sample collection: Collect samples such as nasal swabs, anal swabs, and vaginal swabs from infected cattle; collect environmental samples such as animal urine, feces, milk, and cotton swab sticky materials from contaminated areas.

[0077] S2. Sample pretreatment: Place the sample in a 2 mL centrifuge tube, add 1.2 mL of DEPC water and mix thoroughly, centrifuge at 8000 g for 5 minutes; take the above sample and extract it according to the steps of the nucleic acid extraction kit, and take the eluted sample for later use.

[0078] S3. Sample preparation: Take out the components in the packaging box and place them at room temperature. Take out 50μL of the sample processed in the above steps and add it to the pre-packed RPA dry powder reagent A reaction tube. After the bottom of the corresponding reaction tube is completely dissolved, use a pipette to repeatedly blow and mix it for later use; then take the pre-packed RPA dry powder reagent B reaction tube and cover it. At the same time, prepare negative and positive controls according to the same steps. Invert all samples to mix well and transfer them to the amplification area by instant centrifugation.

[0079] S4, amplification on the machine: put the PCR reaction tube into the sample slot of the thermal amplification instrument, set the negative control, positive control and the name of the sample to be tested in the corresponding order, and perform RPA constant temperature amplification;

[0080] S5. Result analysis and judgment: After the reaction procedure is completed, record the sample Tt value and amplification curve. The negative control has no Tt value and no S-type amplification curve; the positive control Tt value should be ≤20, and a typical S-type amplification curve should appear. If the negative and positive controls do not meet the above conditions, the experiment will be considered invalid. If the Tt value of the FAM channel in the test results of the tested sample is ≤20, and a typical S-type amplification curve appears, it will be reported as positive for bovine Mycobacterium paratuberculosis nucleic acid; if the FAM channel in the test results of the tested sample has no Tt value and no characteristic amplification curve, it will be reported as negative for bovine Mycobacterium paratuberculosis nucleic acid; if the Tt value of the FAM channel in the test results of the tested sample is >20 and a typical S-type amplification curve appears, it is recommended to retest the sample. If the above results still appear after retesting, it will be reported as positive, otherwise it will be invalid. If a Tt value appears but there is no characteristic amplification curve, it will be reported as negative.

[0081] Example 4: Optimization of bovine Mycobacterium paratuberculosis recombinase-mediated isothermal amplification conditions

[0082] 1. Sample screening

[0083] MAP genome; 10 samples each from bovine nasal swab, anal swab and vaginal swab.

[0084] 2. Extraction of genome of samples to be tested

[0085] The samples were all subjected to DNA extraction using the Bacterial Genomic DNA Extraction Kit (50preps) of Kangwei Century Biotechnology Co., Ltd. and stored at -20°C for future use.

[0086] 3. Optimization and establishment of RPA methods

[0087] Different experimental conditions were set to investigate the primer concentration, reaction temperature, and mg 2+ The concentration and reaction time were optimized, and the specific optimization range is shown in Table 2.

[0088] Table 2RPA reaction temperature, primer concentration, mg 2+ Optimization of concentration and reaction time

[0089] Serial number Experimental conditions scope 1 Reaction temperature (℃) 28、33、37、42、45 2 Forward / reverse primer (nM) 300、350、400、450、500 3 Specific fluorescent probe (nM) 100、150、200、250、300 4 <![CDATA[mg 2+ Concentration (mM)]]> 8、10、12、14、16 5 Reaction time (min) 15、20、25、30、40

[0090] After a series of experimental condition optimization, the components of the 50 μL system for MAP RPA constant temperature PCR amplification were determined as follows: 450 nM forward primer, 450 nM reverse primer, 150 nM specific fluorescent probe, 10 mM dNTP, 14 mM mg 2+ Concentration, reaction time 25min, template cDNA 50μL, made up with DEPC water.

[0091] The experimental parameters are as follows: set the reaction system to 50 μL; place the fluorescence isothermal amplification instrument at 37°C, preheat for 5 minutes, take out the reaction tube, mix it with a vortex instrument, and then centrifuge it instantly; 37°C, 25 minutes, a total of 25 cycles. Collect the FAM channel fluorescence signal every 1 minute, for a total of 25 times.

[0092] Example 5: Sensitivity test of bovine Mycobacterium paratuberculosis recombinase-mediated isothermal amplification

[0093] This example uses the RPA method optimized in Example 4 to detect the sensitivity of the third group of RPA detection primers screened in Example 1. The recombinant plasmid was prepared by connecting the MAP F57 conservative sequence nucleotide fragment shown in SEQ ID NO.13 to the pMD-19T vector through homologous recombination and named pMD-19T-MAP-1, wherein 1.0×10 4 pMD-19T-MAP-1 at 10 copies / μL can be used as a positive control, while DEPC water can be used as a negative control.

[0094] The specific operations are as follows:

[0095] The concentration of the positive plasmid was determined by NanoDrop nucleic acid analyzer, and diluted to concentrations of 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL and 1fg / μL, respectively. Six gradient quantitative references, numbered 1 to 6, were tested according to the operation of Example 2. The amplification curves are shown in FIG. Figure 3As shown in the figure, from left to right are samples 1 to 6, and positive and negative controls are set. The specific Tt value results are shown in Table 3. It can be seen that its sensitivity reaches 10fg / μL.

[0096] Table 3 Sensitivity test results of MAP RPA detection method

[0097] Sample No. concentration Tt value 1 100pg / μL 11:00 2 10pg / μL 11:20 3 1pg / μL 11:30 4 100fg / μL 12:00 5 10fg / μL 12:30 6 1fg / μL NoT 7 Positive Control 11:30 8 Negative control NoT

[0098] Example 6: Stability test of bovine Mycobacterium paratuberculosis recombinase-mediated isothermal amplification

[0099] In this example, the concentration of the solution prepared in Example 5 is 1.0×10 4 The recombinant plasmid of 1000 copies / μL was subjected to three repetitive operations according to the RPA method optimized in Example 4, and positive and negative controls were set. The amplification results were as follows: Figure 4 The specific Tt values ​​are shown in Table 4. It can be seen that it has good stability.

[0100] Table 4 Stability test results of MAP RPA detection method

[0101] serial number Sample name Tt value 1 Sample Replicate 1 11:25 2 Sample replicate 2 11:20 3 Sample replicate 3 11:25 4 Positive Control 11:30 5 Negative control NoT

[0102] Example 7: Specificity test of bovine Mycobacterium paratuberculosis recombinase-mediated isothermal amplification

[0103] In this example, the RPA method optimized in Example 4 was used for specificity testing, and bovine paratuberculosis, mycoplasma bovis, Klebsiella pneumoniae, bovine rotavirus and bovine viral diarrhea virus were detected respectively. Three replicates were set, and the amplification curves were Figure 5 As shown, 1 to 3 are the amplification curves of Mycobacterium paratuberculosis of bovine, and the Tt values ​​are shown in Table 5. It can be seen that it has good specificity.

[0104] Table 5 Specificity test results of MAP RPA detection method

[0105] Sample No. Sample name Tt value 1～3 Mycobacterium bovis paratuberculosis 9:30 / 9:30 / 9:35 4 Mycoplasma bovis NoT 5 Klebsiella pneumoniae NoT 6 Bovine rotavirus NoT 7 Bovine viral diarrhea virus NoT 8 Negative control NoT

[0106] Example 8: Clinical sample testing

[0107] In this embodiment, the clinical sample test was performed according to the RPA method of embodiment 4.

[0108] (1) Collect 6 cotton swab samples from bovine paratuberculosis confirmed in vivo as sample A; at the same time, collect 6 cotton swab samples or environmental samples from healthy cattle in vivo as sample B;

[0109] (2) extracting the sample collected in step (1) for testing;

[0110] (3) to (5), the same method as in Example 3;

[0111] (6) Amplification curve Figure 6 As shown, a is sample A, b is sample B, and the Tt values ​​are shown in Table 6.

[0112] Table 6 Clinical sample test results of MAP RPA detection method

[0113] serial number Sample name Tt value 1 A1 13:30 2 A2 13:00 3 A3 17:30 4 A4 12:00 5 A5 13:00 6 A6 8:30 7 Positive Control 11:30 8 Negative control NoT 1* B1 NoT 2* B2 NoT 3* B3 NoT 4* B4 NoT 5* B5 NoT 6* B6 NoT 7* Positive Control 11:30 8* Negative control NoT

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for detecting bovine Mycobacterium paratuberculosis, characterized in that: It includes forward primer MAP-F3, reverse primer MAP-R3 and specific fluorescent probe MAP-P3; The nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.9, the nucleotide sequence of the MAP-F3 is shown in SEQ ID NO.10, the structure of the MAP-P3 is SEQ ID NO.11-tetrahydrofuran residue-SEQ ID NO.12, the 30th nucleotide T of SEQ ID NO.11 is labeled with a fluorescent reporter group, the 1st nucleotide T of SEQ ID NO.12 is labeled with a fluorescent quencher group, and the 3' end is blocked and modified.

2. The primer set according to claim 1, characterized in that The molar ratio of MAP F, MAP R and MAP P is 2-5:2-5:1, preferably 3:3:

1.

3. The primer set according to claim 1, characterized in that The fluorescent reporter group is selected from at least one of FAM, VIC, HEX, JOE, FITC, TRT, CY3, CY5, ROX, TET, TexasRed, SYTO-13, SYTO-82, LCRED460 and LCRED705; The fluorescence quenching group is selected from at least one of BHQ1, BHQ2, BHQ3, Dabcy1 or TAMRA; The 3' end of SEQ ID NO. 4 is connected to C3-spacer.

4. Use of the primer set according to any one of claims 1 to 3 in preparing a product for detecting or assisting in detecting bovine Mycobacterium paratuberculosis.

5. A reagent for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, characterized in that: The invention comprises the primer set according to any one of claims 1 to 3.

6. A kit for detecting or assisting in detecting bovine Mycobacterium paratuberculosis, characterized in that: The method comprises the primer set according to any one of claims 1 to 3 or the reagent according to claim 5.

7. The kit according to claim 6, characterized in that It also includes PCR reaction reagents, MgAc, positive controls and negative controls required for isothermal amplification of the recombinase; Preferably, the PCR reaction reagents include at least one of dNTP, recombinase, nuclease, polymerase, creatine kinase, single-stranded DNA binding protein or excipient; Preferably, the positive control is a positive plasmid sequence containing a partial sequence of a gene in the conserved region of Mycobacterium bovis paratuberculosis; Preferably, the negative control is DEPC-treated water; Preferably, the recombinase is selected from bacteriophage UvsX protein and / or Escherichia coli recA protein; Preferably, the nuclease is selected from exonucleases and / or endonucleases; Preferably, the polymerase is selected from at least one of bsu polymerase, klenow polymerase or phi polymerase; Preferably, the single-stranded DNA binding protein is selected from E. coli SSB and / or GP32 protein; Preferably, the excipient is selected from at least one of 20% polyethylene glycol, 100 mmol / L Tricine, 5 mmol / L dextran or 5 mmol / L dithiothreitol.

8. The kit according to claim 7, characterized in that The primer set described in any one of claims 1 to 3 or the reagent, PCR reaction reagent and MgAc described in claim 5 are all in dry powder form.

9. A method for detecting or assisting in detecting Mycobacterium paratuberculosis of bovine for non-therapeutic and non-diagnostic purposes, characterized in that: The method comprises performing recombinase isothermal amplification on a sample using the primer set described in any one of claims 1 to 3, the reagent described in claim 5 or the kit described in any one of claims 6 to 8, and determining whether bovine paratuberculosis Mycobacterium is present in the sample according to a fluorescent signal.

10. The method according to claim 9, characterized in that The reaction temperature of the recombinase isothermal amplification is 33-42°C, preferably 37°C; Preferably, the sample is a DNA sample.

Images