CN111270015A - Primer composition, kit and method for detecting CSFV and PRRSV - Google Patents
Primer composition, kit and method for detecting CSFV and PRRSV Download PDFInfo
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Abstract
The invention discloses a primer composition, a kit and a method for detecting CSFV and PRRSV. The primer composition designed and screened by the invention comprises 2 groups of specific primers and probes, can simultaneously identify and detect the swine fever and the porcine reproductive and respiratory syndrome in the same reaction tube under the preferable reaction system and reaction conditions, and has the characteristics of good specificity, high sensitivity, convenience, rapidness and the like.
Description
Technical Field
The application relates to the technical field of virus detection, in particular to a primer composition, a kit and a method for detecting CSFV and PRRSV.
Background
Classical Swine Fever (CSF) is an acute and highly contagious disease of pigs caused by Classical Swine Fever Virus (CSFV), characterized by high fever, subcutaneous hemorrhage, and splenic infarction, with extremely high mortality. The epidemic disease has been epidemic for decades in China, and China has adopted a plurality of strict epidemic prevention measures including the application of swine fever vaccines and the like to the disease all the time, so that the wide epidemic of swine fever is greatly controlled, but the disease still exists in China and many pig-raising countries in the world at present.
Porcine Reproductive and Respiratory Syndrome (PRRS) is a porcine acute infectious disease caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), commonly called porcine reproductive and respiratory syndrome, and the sick pigs have the symptoms of inappetence, high body temperature, obvious redness of ear roots and abdomen, extremely high morbidity and mortality, extremely high harm to the pigs and extremely high economic loss.
Classical Swine Fever (CSF) and Porcine Reproductive and Respiratory Syndrome (PRRS) are two common infectious diseases with great harm to pigs, the clinical symptoms of pigs caused by infection of CSFV and PRRSV are sometimes similar, and clinically, cases of mixed infection of CSFV and PRRSV often occur, the common symptoms are body temperature rise, acute death, pregnant abortion, stillbirth and the like, and the judgment of sows is difficult to make by clinical diagnosis, and the diagnosis of sows can be confirmed by means of laboratories. At present, pathogen isolation and identification, a PCR (polymerase chain reaction) method, a fluorescent quantitative PCR (polymerase chain reaction) method, an ELISA (enzyme-linked immunosorbent assay) method, a LAMP (loop-mediated isothermal amplification) method, a GeXP method and the like are common in the laboratory diagnosis methods of the two diseases, but the methods have advantages and disadvantages. Pathogen separation and identification are long and tedious, the PCR method needs electrophoresis to obtain a result, the ELISA method is not sensitive enough to detect pathogens, the fluorescent quantitative PCR method and the GeXP method need expensive instruments and equipment, and only one pathogen can be detected by the single LAMP method.
Disclosure of Invention
The invention provides a primer composition for detecting CSFV and PRRSV, which comprises 2 groups of specific primers and probes, wherein the first group of specific primers and probes comprises CSFV-F3, CSFV-B3, CSFV-FIP, CSFV-BIP and CSFV-Probe, the second group of specific primers and probes comprises PRRSV-F3, PRRSV-B3, PRRSV-FIP, PRRSV-BIP and PRRSV-Probe, the first group and the second group of specific primers and probes are nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in a sequence table, or nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the sequence table are substituted and/or deleted and/or added by one or more nucleotides and have the same functions as the nucleotide sequences shown in the sequence table; the 5 'end of the CSFV-probe is marked with FAM fluorescent group, the 3' end is marked with BHQ1 quenching group, and the FAM fluorescent group is green under the wavelength of 520 nm; the 5 'end of the PRRSV-probe is marked with a CY5.5 fluorescent group, the 3' end is marked with a BHQ2 quenching group, and the CY5.5 fluorescent group is red at the wavelength of 690 nm.
The invention also provides a kit for detecting CSFV and PRRSV, which comprises the primer composition.
Furthermore, the concentration ratio of the inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP in the kit, the outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3 and the probes CSFV-Probe and PRRSV-Probe is 80: 10: 1.
further, each 25ul of the kit of the present inventionThe reaction system comprises 2 mu L of template RNA, 12.5 mu L of 2 Xbuffer solution, 320U of Bst3.0DNA polymerase, 40pmol of each of inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP, 5pmol of each of outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3, 0.5pmol of each of probes CSFV-Probe and PRRSV-Probe, and double distilled water is added to complement to 25 mu L; the 2 Xbuffer solution has a composition of 100mM KCl, 200mM Tris-HCl, pH8.8, 100mM (NH)4)2SO4，80mMMgSO4，8Mbetaine，1％Tween-20，14mMdNTPs。
The invention also provides a method for detecting the CSFV and PRRSV, which adopts the primer composition or the kit to carry out the loop-mediated isothermal amplification reaction.
Further, the concentration ratio of inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP in the loop-mediated isothermal amplification reaction system, outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3 and probes CSFV-Probe and PRRSV-Probe is 80: 10: 1.
furthermore, every 25ul of the reaction system of the loop-mediated isothermal amplification reaction comprises 2 ul of template RNA, 12.5 ul of 2 Xbuffer solution, 320U of Bst3.0DNA polymerase, 40pmol each of inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP, 5pmol each of outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3, 0.5pmol each of probes CSFV-Probe and PRRSV-Probe, and double distilled water to make up to 25 ul; the 2 Xbuffer solution has the composition of 100 MKCl, 200 MpH8.8 Tris-HCl, 100mM (NH)4)2SO4，80mMMgSO4，8Mbetaine，1％Tween-20，14mMdNTPs。
Further, the reaction process of the loop-mediated isothermal amplification reaction includes reaction at 62 ℃ for 90 minutes and reaction at 85 ℃ for 5 minutes.
The invention also provides an application of the primer composition for detecting CSFV and PRRSV, a kit for detecting CSFV and PRRSV and a method for detecting CSFV and PRRSV in the simultaneous detection of classical swine fever and porcine reproductive and respiratory syndrome.
The beneficial effects of the invention include: the primer composition and the kit for detecting CSFV and PRRSV provided by the invention have the advantages of good detection specificity, higher sensitivity, convenience and rapidness, accurate and visual result directly through the color judgment result of the reaction product under fluorescence, can be used for detecting clinical samples, and provide a plurality of selection methods for clinically and rapidly detecting and distinguishing CSFV and PRRSV.
The double LAMP of the invention only needs 2 outer primers, 2 inner primers and 1 ring primer with a fluorescent group, while the single conventional LAMP needs six primers comprising 2 outer primers, 2 inner primers and 2 ring primers. The ring primer of the invention is provided with different luminescent fluorescent groups and quenching groups, the fluorescent groups are closely adjacent to the quenching genes before reaction, the fluorescent groups in the probe do not emit light, in the double LAMP reaction, probe molecules are hybridized to a target sequence, the primer is extended, the probe is broken, the fluorescent groups are separated from the quenching groups, and the fluorescent groups emit light. The result can detect and identify 2 pathogens at one time through different colors, and is quick.
The invention adopts Bst3.0DNA polymerase to carry out double fluorescence LAMP amplification, and has better elongation capability activity, stronger reverse transcription activity and strong strand displacement activity compared with the double fluorescence LAMP adopting Bst2.0DNA polymerase and Bst DNA polymerase large fragments. The primer group and the detection kit constructed by the enzyme can complete LAMP amplification by adding the template once, do not need to invert the RNA template into cDNA independently in advance, and are particularly convenient for detecting the mixed template of DNA and RNA (or RNA containing a secondary complex structure). For the RNA template with the secondary complex structure, double fluorescence LAMP using Bst2.0DNA polymerase or BstDNA polymerase large fragment cannot amplify.
Drawings
FIG. 1 is a diagram showing the detection result of the specificity of duplex fluorescence LAMP, wherein A is a 520nm fluorescence channel diagram, B is a 690nm fluorescence channel diagram, and C is a 520nm and 690nm fluorescence channel diagram; FIGS. 1A-1C show 1 CSFV Shimen strain, 2 PRRSV CH1R,3 control water, 4 CSFV C and PRRSV CH1R, 5 CSFV N and PRRSV CH1R,6 CSFV B and PRRSV CH1R,7 CSFV L and PRRSV CH1R,8 CSFV GXH-2 and PRRSV CH1R,9 FMDV type, 10 BVDVOrevgonC24V,11 PPV,12 JEV,13 PCV2BH5,14 PRV,15 BS2, 16 SS 2;
FIG. 2 is a diagram showing the result of detection of duplex fluorescence LAMP sensitivity, wherein A is a 520nm fluorescence channelFIG. B is a 690nm fluorescence channel map, and C is a 520nm and 690nm fluorescence channel map; 1-8 in FIGS. 2A-2C are 10 in sequence7～100Copy/mul CSFV and PRRSV RNA mixed template standard substance;
FIG. 3 is a graph showing the results of dual fluorescence LAM interference detection, in which A is a graph of 520nm fluorescence channel, B is a graph of 690nm fluorescence channel, and C is a graph of 520nm and 690nm fluorescence channels; 1-7 in FIGS. 3A-3C are samples 1-7 in that order.
Detailed Description
The present invention will be further illustrated and described with reference to the following examples, but the examples described are only a part of the examples of the present invention, and not all of the examples. All other inventions and embodiments based on the present invention and obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Wherein the Loopamp degree instrument is a product of Japan Rongyan company, and the model is Loopamp LA-320C; the nucleic acid analyzer is a product of ThermoFisher Scientific, USA, model NanoDrop 2000; the multicolor fluorescence imaging analysis system is a product of BIO-RAD company in the United states; RT-LAMP kit was purchased from Japan Rongyan Co; the RNA/DNA co-extraction kit and the plasmid small-amount extraction kit are purchased from Beijing all-purpose gold biotechnology limited; whole blood RNA extraction kit was purchased from hundred generations of Bio Inc. (Cat. No. 51025, 50 reactions); the premixTaq PCR kit and the PMD-20T vector were purchased from Dalibao Bio, Bst3.0DNA polymerase from NEB.
Hog cholera strain: shimen strain (F114) was purchased from the institute of veterinary drugs in China and kept in the laboratory. The hog cholera lapinized virulent strain (strain C) was provided by Guangxi Liansheng Probiotics GmbH. Local strains N strain/1986, B strain/1987, L strain/1988 and GXH-2/2000, which were isolated, identified and stored by the Guangxi veterinary institute. Porcine reproductive and respiratory syndrome strain CH-1R (American type) was purchased from Harbin Vitaceae Biotechnology development Inc. Control strains: porcine foot and mouth disease type O inactivated vaccine (FMDV type O) was purchased from the lanzhou veterinary institute, bovine viral diarrhea mucosal virus Orevgon C24V strain (BVDVOrevgon C24V) was provided by the chinese veterinary institute, Porcine Parvovirus (PPV), porcine japanese encephalitis b virus (JEV), porcine circovirus-2 BH5 strain (PCV-2BH5), porcine pseudorabies virus (PRV), porcine brucella 2 (BS2), and porcine streptococcus 2 (SS2) was provided by the Guangxi Zhuang nationality veterinary institute.
The molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.
Example 1 design of primer composition
According to the conserved regions of the sequences of the classical swine fever strain E2 gene (accession number: MK124646.1) and the porcine reproductive and respiratory syndrome virus NSP2 gene (accession number: MF772778.1) published on Genebank, 2 sets of primers and probe composition for LAMP specific amplification are respectively designed by MEGA 5.0 online comparison analysis and then Primer premier5.0 and Primer expore V4 software. Each set of primer composition comprises 4 primers and 1 probe: outer primers F3 and B3, inner primers FIP (FIP ═ F1c + F2) and BIP (BIP ═ B1c + B2), Probe. The designed Probe Probe sequence is between F1c and B1c, and the two ends of the Probe Probe are respectively marked with a fluorescent group and a quenching group. The 5 'end of the Probe CSFV-Probe is marked with a fluorescent group FAM, the 3' end is marked with a quenching group BHQ1, and the free FAM fluorescent group can excite green fluorescence under a 520nm wavelength channel. The 5 'end of the Probe PRRSV-Probe is marked with a fluorescent group CY5.5, the 3' end is marked with a quenching group BHQ2, and free Cy5.5 fluorescent group can excite red under a wavelength channel of 690nm up and down. The primers were synthesized by Dalibao Biopsis, purified by HPLC, and the sequences of the primers in the primer composition are shown in Table 1.
TABLE 1 primer sequences
Example 2 verification test of Duplex fluorescent LAMP detection method for CSFV and PRRSV viruses
1. Extraction of pathogenic nucleic acids
Extracting pathogenic nucleic acid according to the steps required by the specification of the RNA/DNA co-extraction kit, firstly sucking 250 mu L of virus liquid or the supernatant of a treated clinical sample, putting the virus liquid or the supernatant into a 1.5mL centrifuge tube, adding 200 mu L of virus lysate, mixing, then extracting the pathogenic RNA/DNA according to the steps, finally adding 25 mu L of TE elution buffer solution, centrifugally eluting the RNA/DNA, subpackaging and storing at-80 ℃ for later use.
2. Preparation of reaction Standard template
The extracted hog cholera lapinized virus strain (CSFVC) and PRRSV CH-1R nucleic acid are reversely transcribed into cDNA, then CSF outer primer (CSFV-B3, CSFV-F3) and PRRSV outer primer (PRRSV-F3, PRRSV-B3) are respectively used for amplification, the amplified fragment size is respectively about 224bp and 228bp, PCR product is cut and recovered by agarose gel electrophoresis, and is connected with PMD-20T carrier to transform competent cell DH5 α, recombinant bacteria are identified by PCR and double digestion, plasmid in cultured positive recombinant bacteria is extracted by plasmid small amount kit, the concentration of extracted plasmid is determined by NanoDrop 2000, then the copy number is calculated according to a formula, namely copy number (copies/mu L)-1)＝6.02×1023/660×3000×10-9X plasmid concentration (g/. mu.L)-1). The two templates with the same quantity are proportioned according to different concentrations or diluted according to gradient, and the prepared standard template is stored at-70 ℃ for standby or direct application.
3. Optimization of double-fluorescence LAMP reaction system and reaction conditions
And establishing a double fluorescence LAMP reaction system by using the extracted RNA templates of the CSFV and the PRRSV CH-1R together, and optimizing the primer concentration, the probe concentration and the annealing extension temperature range in the reaction system. The optimized primer concentration, probe concentration and temperature ranges are as follows: 5-60 pmoL/. mu.L of inner primer, 1-20 pmoL/. mu.L of outer primer, 0.1-1 pmoL/. mu.L of probe, annealing and extension temperature range 60-68 ℃.
The optimized optimal reaction system is as follows: 2 times LAMP buffer 12.5. mu.L (product of SIGMA Tokyo, Japan, containing 100mM KCl, 200. mu.L)Tris-HCl, 100mM (NH), pH8.8 in mM4)2SO4，80mMMgSO48Mbetaine, 1% (v/v) Tween-20, 14mM dNTPs), Bst3.0DNA polymerase 320U, 1. mu.L each of 40 pmoL/. mu.L inner primers (CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP) (final working concentration is 1.6 pmoL/. mu.L, respectively), 1. mu.L each of 5 pmoL/. mu.L outer primers (CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3) (final working concentration is 0.2 pmoL/. mu.L, respectively), 1. mu.L each of 0.5 pmoL/. mu.L Probe (CSFV-Probe and PRRSV-Probe) (final working concentration is 0.02 pmoL/. mu.L, respectively), and 2. mu.L of template RNA is complemented with sterile ultra pure water. The reagents were mixed gently and then placed in a Loopamp turbidimeter or a thermostated water bath.
The optimal annealing extension temperature after optimization is 62 ℃, the reaction is carried out for 90 minutes, and the reaction is finished at 85 ℃ for 5 minutes.
4. Determination of double fluorescence LAMP reaction result
And (4) taking out the reaction product obtained according to the optimized reaction system and reaction conditions in the step (3), and placing the reaction product in a multicolor fluorescence imaging analysis system.
The channel of 520nm and 690nm is selected for color development, the CSFV sample containing the positive control under the channel of 520nm is green, the PRRSV sample and the negative control sample are colorless, the PRRSV sample containing the positive control under the channel of 690nm is red, the CSFV and the negative control sample are colorless, the mixed sample of the CSFV and the PRRSV containing the positive control under the common channel of 520nm and 690nm is yellow, and the negative control sample is colorless. Under the condition that the standard positive and negative samples are established, the result of the detection sample is judged according to the color, and the result is visual and accurate and is easy to judge.
5. Dual LAMP specificity test
Extracting RNA of CSFV phylum, CSFV C, CSFV N/1986, CSFV B/1987, CSFV L/1988, CSFV GXH-2/2000 and PRRSV CH-1R, simultaneously extracting RNA or DNA of control strains FMDV O type, BVDVOrevgon C24V, PCV-2BH5, PPV, JEV, PRV, BS2 and SS2, determining the concentration, carrying out double fluorescence LAMP amplification detection on the extracted RNA or DNA by using the optimized reaction condition and method established in the step 3, and verifying the specificity of the method.
As shown in figure 1, only CSFV shimen, CSFVC, CSFV N/1986, CSFV B/1987, CSFV L/1988, CSFV GXH-2/2000 and PRRSV CH-1R can be amplified by the double fluorescence LAMP detection method of the invention, the reference strains FMDV O type, BVDVOrevgon C24V, PPV, JEV, PCV2BH5, PRV, BS2 and SS2 do not present any color, i.e. the reference strains are not amplified, and the cross color reaction between CSFV and PRRSV is not present, thus having good specificity.
6. Dual LAMP sensitivity and interference test
Determining the concentration of the standard template of the lapinized swine fever virus strain and the PRRS CH-1R virus strain prepared in the step 2, converting the concentration into copy number, and then diluting the copy number by 10-fold gradient to be 1 multiplied by 107～100And copying/mu L, mixing two nucleic acids with the same copy concentration in equal volume, testing a mixed template of CSFV and PRRSV by using the double RT-LAMP method established in the step 3 of the experiment, and checking the sensitivity of the method.
As shown in FIG. 2, the concentration of mixed templates of CSFV and PRRSV can be detected by the double fluorescence LAMP method of the invention at least 100 copies.
The CSFV and PRRSV standard templates are combined according to different concentrations to prepare mixed samples with different template concentrations, the double fluorescence LAMP method established by the invention is used for detection, and the interference of different template concentrations on the method is detected. Mixed samples of different template concentrations were as follows: sample 1 (10)7CSFV+100PRRSV), sample 2 (10)6CSFV+101PRRSV), sample 3 (10)5CSFV+102PRRSV) sample 4 (10)4CSFV+103PRRSV), sample 5 (10)3CSFV+104PRRSV), sample 6 (10)2CSFV+105PRRSV), sample 7 (10)1CSFV+106PRRSV)。
As shown in FIG. 3, when the concentrations of the two templates are different, especially when one template is high and the other template is low, the method can still distinguish and detect the two different templates, i.e., the different template concentrations have little interference with the method.
7. Clinical sample validation
Clinical samples of 112 diseased pigs from different pig farms in different areas of Guangxi were collected, these samples included 59 porcine whole blood, 21 lung tissue and 32 lymph nodes. Extracting viral nucleic acid from lung tissue and lymph node tissue: taking a proper amount of lung tissue and lymph nodes, grinding respectively, and mixing the raw materials in a proportion of 1: 5 adding sterilized Phosphate Buffer Solution (PBS) to continue grinding and homogenizing, sucking 1.5mL of homogenate and putting into a 2mL of EP tube, repeatedly freezing and thawing at-70 ℃ for 3 times, centrifuging at 3000r/min for 5min, sucking 250 mu L of supernatant and extracting nucleic acid according to an RNA/DNA co-extraction kit. Nucleic acid extraction of virus from porcine whole blood: taking 100 mu L of pig whole blood, and extracting total viral RNA in the whole blood according to the steps in the whole blood RNA extraction kit specification. And then carrying out amplification detection by using the double fluorescence LAMP established in the experiment step 3, simultaneously carrying out contrast detection by using the established CSFV and PRRSV fluorescence quantitative PCR method, and sending the products with positive amplification to a biological company for sequencing measurement so as to verify the accuracy of the double fluorescence LAMP established by the method.
The double fluorescence RT-LAMP method established by the experiment is used for detecting 112 parts of clinical samples, the detection result is 41 parts of CSFV positive samples, the detection positive rate is 36.6 percent, the PRRSV detection positive samples are 12 parts, and the detection positive rate is 10.7 percent. The detection result is 3 samples with CSFV and PRRSV which are positive at the same time, the sample is a mixed infection sample, the mixed infection rate is 2.68 percent, and the result is consistent with the detection result of the fluorescent quantitative PCR method. All the positive sample products are sent to a biological company for sequencing, and the sequencing results are all corresponding viruses after sequence comparison and analysis.
Claims (9)
1. A primer composition for detecting CSFV and PRRSV is characterized in that the primer composition comprises 2 groups of specific primers and probes, wherein the first group of specific primers and probes comprises CSFV-F3, CSFV-B3, CSFV-FIP and CSFV-BIP, and CSFV-Probe, the second group of specific primers and probes comprises PRRSV-F3, PRRSV-B3, PRRSV-FIP, PRRSV-BIP, and PRRSV-probe, the first group and the second group of specific primers and probes are nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the sequence table in sequence, or the nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the nucleotide sequences shown in SEQ ID No.1 to SEQ ID No.10 in the sequence list and has the same function with the nucleotide sequence shown in the sequence list; the 5 'end of the CSFV-probe is marked with FAM fluorescent group, the 3' end is marked with BHQ1 quenching group, and the FAM fluorescent group is green under the wavelength of 520 nm; the 5 'end of the PRRSV-probe is marked with a CY5.5 fluorescent group, the 3' end is marked with a BHQ2 quenching group, and the CY5.5 fluorescent group is red at the wavelength of 690 nm.
2. A kit for detecting CSFV and PRRSV, comprising the primer composition of claim 1.
3. The kit for detecting CSFV and PRRSV virus according to claim 2, characterized in that the concentration ratio of the inner primer CSFV-FIP, CSFV-BIP, PRRSV-FIP, PRRSV-BIP, the outer primer CSFV-F3, CSFV-B3, PRRSV-B3, PRRSV-F3 and the probes CSFV-Probe, PRRSV-Probe in the kit is 80: 10: 1.
4. the kit for detecting CSFV and PRRSV according to claim 2 or 3, characterized in that each 25ul of the reaction system of the kit comprises 2 ul of template RNA, 12.5 ul of 2 Xbuffer, 320U of Bst3.0DNA polymerase, 40pmol each of inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP, 5pmol each of outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3, 0.5pmol each of probes CSFV-Probe and PRRSV-Probe, and double distilled water to 25 ul; the 2 Xbuffer solution has a composition of 100mM KCl, 200mM Tris-HCl, pH8.8, 100mM (NH)4)2SO4，80mMMgSO4，8Mbetaine，1％Tween-20，14mMdNTPs。
5. A method for detecting CSFV and PRRSV in pigs, characterized in that a loop-mediated isothermal amplification reaction is performed using the primer composition or kit according to any of claims 1-4.
6. The method for detecting CSFV and PRRSV of claim 5, wherein the concentration ratio of the inner primer CSFV-FIP, CSFV-BIP, PRRSV-FIP, PRRSV-BIP, the outer primer CSFV-F3, CSFV-B3, PRRSV-B3, PRRSV-F3 and the probes CSFV-Probe, PRRSV-Probe in the reaction system of the loop-mediated isothermal amplification reaction is 80: 10: 1.
7. the method for detecting CSFV and PRRSV according to claim 5 or 6, wherein the reaction system of the loop-mediated isothermal amplification reaction comprises 2 μ L of template RNA, 12.5 μ L of 2 Xbuffer, 320U of Bst3.0DNA polymerase, 40pmol each of inner primers CSFV-FIP, CSFV-BIP, PRRSV-FIP and PRRSV-BIP, 5pmol each of outer primers CSFV-F3, CSFV-B3, PRRSV-B3 and PRRSV-F3, 0.5pmol each of probes CSFV-Probe and PRRSV-Probe, and double distilled water to 25 μ L per 25 μ L of reaction system; the 2 Xbuffer solution has the composition of 100 MKCl, 200 MpH8.8 Tris-HCl, 100mM (NH)4)2SO4，80mMMgSO4，8Mbetaine，1％Tween-20，14mMdNTPs。
8. The method for detecting CSFV and PRRSV according to claim 5 or 6, wherein the reaction process of the loop-mediated isothermal amplification reaction comprises 90 minutes at 62 ℃ and 5 minutes at 85 ℃.
9. The primer composition for detecting CSFV and PRRSV in claim 1, the kit for detecting CSFV and PRRSV in any one of claims 2 to 4, and the method for detecting CSFV and PRRSV in any one of claims 5 to 8 are applied to the simultaneous detection of classical swine fever and porcine reproductive and respiratory syndrome.
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