CN110358864A - The one-step method multiple real time fluorescence quantifying PCR detection primer and probe of SVA, FMDV, SVDV and VSV - Google Patents

Abstract

The invention discloses the one-step method multiple real time fluorescence quantifying PCR detection primers and probe of a kind of SVA, FMDV, SVDV and VSV, belong to field of biological technology detection.And the primer and probe based on offer has developed for while detecting the kit of SVA, FMDV, SVDV and VSV.Kit provided by the invention and detection method is easy to operate, high specificity, sensibility are high, reproducible, it may be implemented to SVA, FMDV, SVDV and VSV while accurate qualitative and quantitative detection, it in the quality monitoring in the detection of SVA, FMDV, SVDV and VSV and production of vaccine, such as vaccine production process and will rationalize with playing a significant role in seedling, have a extensive future.

Description

The one-step method multiple real time fluorescence quantifying PCR detection of SVA, FMDV, SVDV and VSV are drawn Object and probe

Technical field

The invention belongs to the veterinary animal Pathogen test in technical field of biological, be related to a kind of couple of SVA, FMDV, SVDV and VSV carries out the one-step method multiple real time fluorescence quantifying PCR detection primer and TaqMan probe of qualitative and quantitative analysis, especially It is related to the one-step method multiple real time fluorescence quantifying PCR inspection that a kind of couple of SVA, FMDV, SVDV and VSV carry out qualitative and quantitative analysis Test agent box and its primer special and TaqMan probe.

Background technique

Foot and mouth disease virus (FMDV), Senecan viral (SVA), swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV) microRNA Viraceae is belonged to.Senecan virus is the Typical Representative of Senecan Tobamovirus, mouth closest with cardiovirus Aphtovirus belongs to aphthovirus genus, and swine vesicular disease virus belongs to enterovirus genus, and vesicular stomatitis virus belongs to Vesiculovirus Belong to.Aftosa, pig blisters and vesicular stomatitis are all the important viral infectious of pig, and Senecan disease is discovery recent years A boar infectious disease, four kinds of viral diseases be all with the visible blister symptom of the coronal band portion of nose, oral area and hoof, Its Clinical symptoms is very much like, can not visually identify.These three diseases of aftosa, vesicular stomatitis and swine pox are arranged by OIE For A class infectious disease, the development of animal husbandry has not only been seriously affected, and has been generation to human health there is also greatly threatening The key object of various countries, boundary quarantine and epidemic prevention.Senecan virus is sent out in a company in Maryland, USA in 2002 for the first time It is existing, 2007, the classes such as bubble occurs in a collection of hog snout mirror for transporting Minn. to from Canada, hoof coronary band festers Like blister disease symptoms, aftosa, vesicular stomatitis disease and pig blisters are excluded through detection, is determined as filling in eventually by PCR detection Interior card virus-positive.So far from 2014, it finds the U.S., China, Canada, Brazil, Thailand, Colombia etc. are multinational Clinical case caused by SVA virus.Before 2007, SVA is studied mainly as oncolytic, can effectively treat Neuroendocrine tumor.Aftosa, vesicular stomatitis and swine pox are a kind of urgency of the artiodactyls such as ox, pig and sheep infection Property, hot, highly contagious disease, occur blister and to fester with mucous membrane of mouth, lingual surface, lip, asoscope, hoof and skin of breast It is characterized.The Clinical symptoms of these four infectious diseases and pathological change are all quite similar, are clinically difficult to distinguish, and need to take bubble Liquid, bubble skin, crust and serum etc. are sent to laboratory and carry out antidiastole using conventional aetology and Serology test. But using conventional aetology and Serology test, not only time-consuming, and process is cumbersome, there is presently no to SVA, FMDV, SVDV and VSV carry out the method for quickly identifying detection simultaneously.

Preparing inactivated vaccine is the most effective measure for preventing viral prevalence, and the antigen levels in inactivated vaccine are in infectious disease It is played an important role in prevention.The preparation of inactivated vaccine mainly obtains virus by inactivation after virus is carried out in vitro culture Then liquid is mixed and made into immune vaccine with emulsifier.During preparing vaccine, such as when preparing polyvaccine, accurate, Specifically, rapidly quarantine viral antigen in seedling it is horizontal in production of vaccine monitoring, evaluate vaccine quality and instruct vaccine dosage Etc. be of great significance.

Summary of the invention

One or more aiming at the problems existing in the prior art, one aspect of the invention provide it is a kind of for SVA, FMDV, SVDV and VSV carry out the primer and Taqman probe of multiple real time fluorescence quantifying PCR detection, wherein for detecting SVA's Upstream primer (SVA1-F) and downstream primer (SVA1-R), in the nucleotide sequence of the upstream primer (SVA1-F) such as sequence table Shown in SED ID NO:5, the nucleotide sequence of downstream primer (SVA1-R) is as shown in SEQ ID NO:6 in sequence table；

For carrying out the TaqMan probe (SVA1-P) of real-time fluorescence quantitative PCR detection, the TaqMan probe to SVA (SVA1-P) nucleotide sequence is as shown in SED ID NO:7 in sequence table；

For detecting the upstream primer (FMDV1-F) and downstream primer (FMDV1-R) of FMDV, the upstream primer (FMDV1-F) nucleotide sequence is as shown in SED ID NO:8 in sequence table, the nucleotide sequence of downstream primer (FMDV1-R) As shown in SEQ ID NO:9 in sequence table；

For carrying out the TaqMan probe (FMDV1-P) of real-time fluorescence quantitative PCR detection to FMDV, the TaqMan is visited The nucleotide sequence of needle (FMDV1-P) is as shown in SED ID NO:10 in sequence table；

For detecting the upstream primer (SVDV1-F) and downstream primer (SVDV1-R) of SVDV, the upstream primer (SVDV1-F) nucleotide sequence is as shown in SED ID NO:11 in sequence table, the nucleotide sequence of downstream primer (SVDV1-R) As shown in SEQ ID NO:12 in sequence table；

For carrying out the TaqMan probe (SVDV1-P) of real-time fluorescence quantitative PCR detection, TaqMan probe to SVDV (SVDV1-P) nucleotide sequence is as shown in SED ID NO:13 in sequence table；

For detecting the upstream primer (VSV1-F) and downstream primer (VSV1-R) of VSV, the upstream primer (VSV1-F) Nucleotide sequence as shown in SED ID NO:14 in sequence table, in the nucleotide sequence of downstream primer (VSV1-R) such as sequence table Shown in SEQ ID NO:15；

For carrying out the TaqMan probe (VSV1-P) of real-time fluorescence quantitative PCR detection, TaqMan probe to VSV (VSV1-P) nucleotide sequence is as shown in SED ID NO:16 in sequence table；

The probe is by fluorescent marker, and 5 ' ends are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching Group；And the fluorescent reporter group marked to four kinds of viral diagnosis probes is not identical.

Above-mentioned 5 ' the ends for carrying out the TaqMan probe (SVA1-P) of real-time fluorescence quantitative PCR detection to Senecan virus Reporter fluorescence group is FAM, and 3 ' end fluorescent quenching groups are TAMRA；For carrying out real-time fluorescence quantitative PCR to foot and mouth disease virus 5 ' end reporter fluorescence groups of the TaqMan probe (FMDV1-P) of detection are ROX, and 3 ' end fluorescent quenching groups are BHQ2；For 5 ' end reporter fluorescence groups of the TaqMan probe (SVDV1-P) of real-time fluorescence quantitative PCR detection are carried out to swine vesicular disease virus For CY5,3 ' end fluorescent quenching groups are BHQ2；For carrying out real-time fluorescence quantitative PCR detection to vesicular stomatitis virus 5 ' end reporter fluorescence groups of TaqMan probe (VSV1-P) are HEX, and 3 ' end fluorescent quenching groups are Eclipse.

3 ' the phosphorylated processing in end of above-mentioned probe.

Another aspect of the present invention provides a kind of for carrying out multiple real time fluorescence quantifying to SVA, FMDV, SVDV and VSV PCR detection kit, including above-mentioned primer and TaqMan probe；

Primer described in 50 μ L real-time fluorescence quantitative PCR detection architectures when using the kit and TaqMan probe Dosage is preferred are as follows: 1.5 1.5 1.5 μ L, FMDV1-F (10 μ of μ L, SVA1-P (10 μM) of μ L, SVA1-R (10 μM) of SVA1-F (10 μM) M) 0.5 0.5 1.5 1.5 μ L, SVDV1-R (10 μ of μ L, SVDV1-F (10 μM) of μ L, FMDV1-P (10 μM) of μ L, FMDV1-R (10 μM) M) 1.5 μ L, SVDV1-P (10 μM) 1.5 μ L, VSV1-F (10 μM) 1.5 μ L, VSV1-R (10 μM) 1.5 μ L, VSV1-P (10 μM) 1.5μL。

It further include positive reference substance and negative controls in mentioned reagent box, positive reference substance is Senecan viral RNA, mouth Aphtovirus RNA, swine vesicular disease virus recombinant plasmid PMD-19-SVDV and vesicular stomatitis virus recombinant plasmid PMD-19- VSV, each positive reference substance are unitary package or hybrid packed, and negative controls are without Senecan virus, foot and mouth disease virus, pig The reaction system of hydroa virus and vesicular stomatitis virus, such as H₂O (distilled water, aseptic deionized water etc.)；

It, will be single when carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV using the kit The positive reference substance mixing of one packaging directly uses hybrid packed positive reference substance.

Further include standard items in mentioned reagent box, detects gene, aftosa to carry Senecan viral nucleotide respectively Viral nucleotide detects gene, swine vesicular disease virus nucleotide detection gene and vesicular stomatitis virus nucleotide detection gene Recombinant plasmid pCR-4TOPO-SVA, pCR-4TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV；Each standard items are single Packaging or isoconcentration are hybrid packed；

It, will be single when carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV using the kit The standard items isoconcentration mixing of one packaging directly uses hybrid packed standard items.

Application of the above-mentioned kit in the detection for carrying out non-disease diagnostic purpose to SVA, FMDV, SVDV and VSV Belong to the contents of the present invention.

Above-mentioned detection apply for it is a kind of with multiple real time fluorescence quantifying PCR technology to SVA, FMDV, SVDV and VSV into The qualitative and quantitative analysis method of row non-disease diagnostic purpose, comprising the following steps:

1) it establishes standard curve: the recombinant plasmid pCR4- of the detection gene of SVA, FMDV, SVDV and VSV will be carried respectively The standard items isoconcentration of the unitary package of TOPO-SVA, pCR4-TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV mix or Directly using the hybrid packed standard items of isoconcentration, standard concentration each in mixed liquor is diluted to 1 according to 10 times of gradients × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹copies/μL；With the standard items of various concentration It is fixed to carry out multiple real time fluorescence as template under the guidance of primer of any of claims 1 or 2 and TaqMan probe for mixed liquor PCR detection is measured, after detection, is mapped with the concentration Log value (X-axis) of each standard items to its corresponding Ct value (Y axis), draws mark Directrix curve；

2) geneome RNA for extracting sample to be tested, using the geneome RNA of extraction as template, as claimed in claim 1 or 2 Primer and TaqMan probe guidance under carry out multiple real time fluorescence quantifying PCR detection；

3) qualitative detection to SVA, FMDV, SVDV and VSV, then root are realized with the variation of obtained CT value or fluorescence signal According to the standard curve in the intensity and step 1) of fluorescence signal, contained SVA, FMDV, SVDV and VSV in sample to be tested are obtained Copy number, realize quantitative detection.

Above-mentioned steps 2) in sample to be tested include being given for the raw material, vaccine semi-finished product and finished product, pig farm of production of vaccine Sample product.

Above-mentioned steps 1) and step 2) in 50 μ L real-time fluorescence quantitative PCR detection architectures include: 2 μ L of template, in real time it is glimmering Light quantifies 25 μ L (being purchased from TakaRa company) of one-step method PCR reaction solution 2 × One Step RT-PCR Buffer III, 1 μ L of TaKaRa Ex Taq HS 1 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II (is purchased from TakaRa company), 1.5 1.5 1.5 μ L, FMDV1-F (10 of μ L, SVA1-P (10 μM) of μ L, SVA1-R (10 μM) of SVA1-F (10 μM) μM) 0.5 μ L, FMDV1-R (10 μM) 0.5 μ L, FMDV1-P (10 μM) 1.5 μ L, SVDV1-F (10 μM) 1.5 μ L, SVDV1-R 1.5 1.5 1.5 μ L, VSV1-P (10 of μ L, VSV1-R (10 μM) of μ L, VSV1-F (10 μM) of (10 μM) 1.5 μ L, SVDV1-P (10 μM) μM) 1.5 μ L, 4.0 μ L, RNA-free H of template ribonucleic acid₂O 3.0μL；And/or

Multiple real time fluorescence quantifying PCR testing conditions in the step 1) and step 2) are as follows: first 42 DEG C of reverse transcriptions 20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).

Above-mentioned steps 3) in determination method are as follows:

If sample channel corresponding to the marked fluorescent reporter group in the 5 ' ends of SVA1-P (is not wrapped in 36 circulations Include the 36th circulation) there is " S " type amplification curve, then it is positive (containing SVA in sample) to be confirmed as SVA；If sample is in FMDV1- There is " S " in channel corresponding to the marked fluorescent reporter group in the 5 ' ends of P (not including the 36th circulation) in 36 circulations It is positive (containing FMDV in sample) to be then confirmed as FMDV for type amplification curve；If the sample fluorescence marked at the 5 ' ends of SVDV1-P There is " S " type amplification curve in channel corresponding to reporter group (not including the 30th circulation) in 30 circulations, then is confirmed as The SVDV positive (containing SVDV in sample)；If sample channel corresponding to the marked fluorescent reporter group in the 5 ' ends of VSV1-P There is " S " type amplification curve in (not including the 33rd circulation) in 33 circulations, then is confirmed as the VSV positive and (contains in sample VSV)；If sample is in any channel, more than 38 circulations (including the 38th circulation) does not occur " S " type amplification curve, confirms It is negative (SVA or FMDV or SVDV and/or VSV is not contained in sample) for corresponding virus；If sample holds institute the 5 ' of SVA1-P Channel corresponding to the marked fluorescent reporter group in the 5 ' ends of channel corresponding to the fluorescent reporter group of label or FMDV1-P 36-38 circulation between (including the 36th circulation and do not include the 38th circulation) occur " S " type amplification curve, be determined as It is suspicious, it need to examine again；If sample channel corresponding to the marked fluorescent reporter group in the 5 ' ends of SVDV1-P is followed at 30-38 There is " S " type amplification curve in (including the 30th circulation and do not include the 38th circulation) between ring, is determined as suspicious, need to examine again； If sample channel corresponding to 5 ' the marked fluorescent reporter groups in end of VSV1-P recycled at 33-38 between (including the 33 circulations and do not include the 38th circulation) there is " S " type amplification curve, be determined as suspicious, need to examine again；

Preferably, if there is " S " type amplification curve in the circulation of 36, the channel FAM in sample, it is confirmed as the SVA positive；If There is " S " type amplification curve in the circulation of 36, the channel ROX in sample, is then confirmed as the FMDV positive；If 30, the channel CY5 in sample Occur " S " type amplification curve in circulation, is then confirmed as the SVDV positive；If there is " S " type in the circulation of 33, the channel HEX in sample to expand Increase curve, is then confirmed as the VSV positive；If the channel FAM or the channel ROX or the channel CY5 and/or 38, the channel HEX are followed in sample More than ring do not occur " S " type amplification curve, is then confirmed as SVA or FMDV or SVDV and/or VSV is negative；If the channel FAM and There is " S " type amplification curve between 36-38 circulation in the channel ROX and be then determined as suspicious, need to examine again；If the channel CY5 is Appearance " S " type curve is then determined as suspicious between 30-38 circulation, need to examine again；If the channel HEX is between the 33-38 circulation There is " S " type amplification curve and be then determined as suspicious, need to examine again.

Multiple real time fluorescence quantifying PCR inspection based on identification SVA, FMDV, SVDV and VSV that above technical scheme provides Test agent box and its primer special and TaqMan probe can implement simultaneously quickly detection to SVA, FMDV, SVDV and VSV, can be epidemic disease Quality monitoring and rationalization in seedling production process (if the exact level of vaccine antigen is prepared in assessment, contain with seedling for vaccine antigen Amount provides data basis) strong foundation is provided, it is ensured that the safety and reasonability of vaccine inoculation, to Senecan virus, aftosa The production of viral vaccine, swine vesicular disease virus vaccine and vesicular stomatitis virus vaccine has directive function.Reagent of the invention Box and detection method is easy to operate, high specificity, sensibility are high, reproducible, may be implemented to SVA, FMDV, SVDV and VSV Accurate qualitative and quantitative detection, will be in the detection of SVA, FMDV, SVDV and VSV (including clinical pathological material of disease or training in clinical diagnosis Support object in Senecan virus and foot and mouth disease virus accurate detection) and production of vaccine in play a significant role, have a extensive future.

Detailed description of the invention

Fig. 1 is the present invention for carrying out the primer screening of real-time fluorescence quantitative PCR detection to SVA, FMDV, SVDV and VSV Amplification curve；

Fig. 2 is the present invention for carrying out the standard of multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV The amplification curve of product；

Fig. 3 is the present invention for carrying out the standard of multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV Curve；

Fig. 4 is the specificity that SVA, FMDV, SVDV and VSV of the present invention carry out multiple real time fluorescence quantifying PCR detection method Testing result；

Fig. 5 is that SVA, FMDV, SVDV and VSV of the present invention carry out multiple real time fluorescence quantifying PCR detection method repeatability Amplification curve testing result.

Specific embodiment

Biological genome is a direct most objective index for reacting biological essential information, contained by different virus Genomic information is different, can be mutual using genome base by different virus taxis to different groups by genomic information The principle that recruits pair, it can be achieved that specific site gene order massive amplification.

The present invention is based on the above basic principles to devise 4 pairs of specific primers and 4 oligonucleotide probes, mutual using base The principle recruited pair establishes the multiple real time fluorescence quantifying PCR detection of a kind of specific detection SVA, FMDV, SVDV and VSV Method, and based on the obtained specific primer of design and probe provide it is a kind of can detect and identify simultaneously SVA, FMDV, SVDV and The multiple real time fluorescence quantifying PCR detection kit of VSV.

By following specific embodiments, the present invention will be described in detail.

Method therefor is conventional method unless otherwise instructed in following embodiments, and specific steps can be found in: " molecular cloning Experiment guide " (" Molecular Cloning:A Laboratory Manual " Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor)。

The percent concentration is mass/mass (W/W, unit g/100g) percent concentration, matter unless otherwise instructed Amount/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percent concentration.

The acquirement approach of various biomaterials described in embodiment is to provide a kind of approach of experiment acquisition only to reach To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to mentioning in embodiment Show and is replaced.

The primer is synthesized by Beijing Hua Da gene Co., Ltd；Probe used is synthesized by TAKARA genome company.

Embodiment is implemented under the premise of the technical scheme of the present invention, gives detailed embodiment and specific Operating process, embodiment will be helpful to understand the present invention, but should not be taken as limiting the invention.

Embodiment 1, primer and TaqMan probe design

1.1, primer and TaqMan probe design

Different plugs are obtained from the nucleic acid database GenBank (http://www.ncbi.nlm.nih.gov) of NCBI retrieval Interior card virus stain whole genome sequence (GeneBank serial number: NC_011349, KT321458, KX173339, KX173338, KX173340、KX751943、KX751944、KY747510、KY038016、KY747511、KY747512、KX751945、 KX751946、KX377924、KY419132、DQ641257、KU051392、KT757280、KU051391、KY486158、 KY486165,KC667560,KR063109,KR063107,KY368743)；The full-length genome sequence of different mouth disease virus strains Column (GeneBank serial number: AF506822.2, KX712091.1, HQ412603.1, AJ539141.1, AY390432.1, AY304994.1,GQ406249.1,KT968663.1,HQ632773.1)；The full-length genome sequence of different swine vesicular disease virus strains Column (GeneBank serial number: AF121268, D16364.1, KT284994, KT284979, KT284999, KT284980, KT284983、KT284985、KT285001、KT285002、KT284986、KT284984、AY429470、FJ409085、 AJ004636,EU151450)；Different vesicular stomatitis virus strains whole genome sequence (GeneBank serial number: AY383716.1、K02379.1、JX121110.1、MF196238.1、MF196237.1、MF196237.1、KC905171.1、 KP688353.1)。

After the whole genome sequence of strains different in above-mentioned different virus is compared respectively with DNA Star software, root According to custom primer, TaqMan probe design principle, nucleotide sequence relatively conservative between the different strains of Senecan virus is chosen Nucleotide sequence (region 3C) relatively conservative, swine vesicular disease virus are different between (region 3C), foot and mouth disease virus difference strain Core relatively conservative between nucleotide sequence (region VP1) relatively conservative, vesicular stomatitis virus difference strain between strain Nucleotide sequence (n-quadrant) is as detection gene, and specific conservative's segment preferably wherein is as detection target sequence.

About the 3C gene of Senecan virus, Hales L M etc., Complete genome sequence analysis of Seneca Valley virus-001,a novel oncolytic picornavirus[J].Journal of General Virology, 2008,89 (5): the 3C gene that Senecan virus has been illustrated in 1265-1275. is conservative area Domain；The separation identification and Study on Pathogenicity of Zhao Xiaoya, pig Sai Neijia paddy viral (SVA), (Agriculture In South China is big for master thesis Learn, 2016 master's thesis (24-33)) in the 3C gene choose 4763-4879 bit base section as target sequence foundation Seneca Valleyvirus Taq Man fluorescent quantitative PCR detection method, but the primer and probe that this method is established is It is no can be used for identifying and detect SVA, FMDV, SVDV and VSV simultaneously to those skilled in the art be it is unknown, in addition Primer and probe disclosed in the document is served only for detecting individual Senecan virus, when it is used for multiple fluorescence quantitative PCR detection When, sensibility can reduce, if be enough can be used for detect SVA, FMDV, SVDV and VSV simultaneously for those skilled in the art For be also it is unknown.The present inventor then finds detection sequence of specific conservative's segment as Senecan virus in the 3C gene Column, by making great efforts finally to determine that the detection sequence is Senecan virus from 5 ' end 6564-6820 bit bases (SED in sequence table Shown in ID NO:1), and multiple groups are designed based on the detection sequence, real-time fluorescence quantitative PCR detection is carried out to Senecan virus Primer and TaqMan probe, as follows, two groups be shown in which are (in group 1 in SVA1-F, SVA1-R, SVA1-P and group 2 SVA2-F, SVA2-R,SVA2-P)；

Gene is detected about foot and mouth disease virus, lot of domestic and international research and utilization 3D gene carries out fluorescence as target sequence at present Quantitative RT-PCR method detects foot and mouth disease virus, and a few studies make target sequence using foot and mouth disease virus 2B gene and 5 ' UTR genes It carries out fluorescent quantitative RT-PCR method and detects foot and mouth disease virus, but these documents are only limitted to detect a kind of individually virus.This hair Bright people then has found highly conserved and special nucleotide region as detection in foot and mouth disease virus 3C gene region by effort Sequence, and finally determine that the detection sequence is foot and mouth disease virus from 5 ' end 5986-6553 bit bases (SED ID in sequence table NO:2), and based on the detection sequence design multiple groups to foot and mouth disease virus carry out real-time fluorescence quantitative PCR detection primer and TaqMan probe, two groups be shown in which as follows (group 1 in FMDV1-F, FMDV1-R, FMDV1-P and group 2 in FMDV2-F, FMDV2-R,FMDV2-P)；

About pig blisters poison and vesicular stomatitis virus gene is detected, at present lot of domestic and international research and utilization SVDV The N gene of VP1 gene and VSV carry out regular-PCR as target sequence and fluorescent quantitative RT-PCR method is detected, but these texts It offers and is only limitted to detect a kind of individually virus.The present inventor is by effort, in VP1 gene and the vesicular stomatitis disease of pig blisters poison The N gene region of poison finds highly conserved and special nucleotide region as detection sequence respectively, and finally determines pig blister Virus holds 52- from 5 ' from 5 ' ends 2448-3288 bit base (SED ID NO:3 in sequence table), vesicular stomatitis virus Detection of 1380 bit bases (SED ID NO:4 in sequence table) respectively as detection pig blisters poison and vesicular stomatitis virus Sequence, and multiple groups are designed based on the detection sequence, real time fluorescent quantitative is carried out to pig blisters poison and vesicular stomatitis virus The primer and TaqMan probe of PCR detection respectively illustrate two groups therein (for pig blisters poison in group 1 as follows SVDV2-F, SVDV2-R, SVDV2-P in SVDV1-F, SVDV1-R, SVDV1-P and group 2 are for vesicular stomatitis virus VSV2-F, VSV2-R, VSV2-P in VSV1-F, VSV1-R, VSV1-P and group 2 in group 1)；

Group 1:

SVA1-F (upstream primer): 5 '-TATCTCAGATCCCTGGCTGTC-3 ' (sequence location: Senecan virus is from 5 ' 6634-6654 bit base is held, SED ID NO:5 in sequence table)；

SVA1-R (downstream primer): 5 '-CCTGATGATCACATTGTTGAGC-3 ' (sequence location: Senecan virus is from 5 ' 6741-6762 bit base is held, SED ID NO:6 in sequence table)；

SVA1-P (TaqMan probe): 5 '-FAM-CACGCTTACGGCGAGCGTCGCATCAAG-TAMRA-3 ' (sequence Position: Senecan virus holds 6661-6687 bit bases, SED ID NO:7 in sequence table from 5 ')；

FMDV1-F (upstream primer): 5 '-YGCCTMCCTHGTDCCTCGTCAYCTYT-3 ' (sequence locations: hoof-and-mouth disease Poison holds 6158-6179 bit bases, SED ID NO:8 in sequence table, wherein Y=C, T, M=A, C, H=A, T, C, D from 5 ' =G, A, T)；

FMDV1-R (downstream primer): 5 '-GAGAGCATGTCCTGTCCTYTYACTTT-3 ' (sequence locations: hoof-and-mouth disease Poison holds 6260-6281 bit bases, SED ID NO:9 in sequence table, wherein Y=C, T from 5 ')；

(the sequence positions FMDV1-P (TaqMan probe): 5 '-ROX-CNGAGAAGTAYGACAAGATCATGYT-BHQ2-3 ' Set: foot and mouth disease virus holds 6183-6207 bit bases, SED ID NO:10 in sequence table, wherein N=A, G, C, T, Y from 5 ' =C, T)；

SVDV1-F (upstream primer): 5 '-taggcatgtgaagaattaccac-3 ' (sequence location: pig blisters poison from 5 ' hold 2597-2618 bit bases, SED ID NO:11 in sequence table)；

SVDV1-R (downstream primer): 5 '-atcagagtcgtggttcttgta-3 ' (sequence location: pig blisters poison is from 5 ' 2682-2700 bit base is held, SED ID NO:12 in sequence table)；

(the sequence positions SVDV1-P (TaqMan probe): 5 '-CY5-tcagagtcaaccgtggagaacttcct-BHQ2-3 ' Set: pig blisters poison holds 2625-2651 bit bases, SED ID NO:13 in sequence table from 5 ')；

VSV1-F (upstream primer): 5 '-CCRGGYCAAGAAATTGACAA-3 ' (sequence location: vesicular stomatitis virus from 5 ' hold 854-873 bit bases, SED ID NO:14 in sequence table, wherein R=A, G, Y=C, T)；

VSV1-R (downstream primer): 5 '-TAYTCGATGTCGTCRGGCTGTC-3 ' (sequence location vesicular stomatitis virus From SED ID NO:15 in 5 ' end 1014-1035 bit bases, sequence table, wherein Y=C, T；R=A, G)；

VSV1-P (TaqMan probe): 5 '-HEX-ATTCATACATGCCGTATATGATTGACTT-Eclipse-3 ' (sequence Column position: vesicular stomatitis virus holds 880-906 bit bases, SED ID NO:16 in sequence table from 5 ')；

Group 2:

SVA2-F (upstream primer): 5 '-tataagatgactcctgccaac-3 ' (sequence location: Senecan virus is from 5 ' 6898-6918 bit base is held, SED ID NO:17 in sequence table)；

SVA2-R (downstream primer): 5 '-agaatttggaagccatgctctc-3 ' (sequence location: Senecan virus is from 5 ' 7025-7046 bit base is held, SED ID NO:18 in sequence table)；

(the sequence positions SVA2-P (TaqMan probe): 5 '-FAM-ttctgtcttccctccgacttcctctc-TAMRA-3 ' Set: Senecan virus holds 6924-6949 bit bases, SED ID NO:19 in sequence table from 5 ').

FMDV2-F (upstream primer): 5 '-aagatcatgttggacggcagagccat-3 ' (sequence locations: hoof-and-mouth disease Poison holds 6198-6223 bit bases, SED ID NO:20 in sequence table from 5 ')；

FMDV2-R (downstream primer): 5 '-atgtcccgcacgcgattcccacggt-3 ' (sequence locations: foot and mouth disease virus From SED ID NO:21 in 5 ' end 6310-6334 bit bases, sequence table)；

FMDV2-P (TaqMan probe): 5 '-ROX-cagtgactacagagtgtttgagtttgag-BHQ2-3 ' (sequence Position: foot and mouth disease virus holds 6230-6257 bit bases, SED ID NO:22 in sequence table from 5 ').

SVDV2-F1 (upstream primer): 5 '-actcaccaaataatgtacgtac-3 ' (sequence locations: swine vesicular disease virus From SED ID NO:23 in SED ID NO:1 in 5 ' end 2865-2886 bit bases, sequence table, sequence table)；

SVDV2-R (downstream primer): 5 '-aatctggcccacccgtcatag-3 ' (sequence location: swine vesicular disease virus from 5 ' end 3029-3049 bit bases, SED ID NO:2 in sequence table, SED ID NO:24 in sequence table)；

(the sequence positions SVDV2-P (TaqMan probe): 5 '-CY5-tggcccagtacccacaaaggtgaatag-BHQ2-3 ' Set: swine vesicular disease virus holds 2894-2920 bit bases, SED ID NO:25 in sequence table from 5 ')；

VSV2-F (upstream primer): 5 '-ttggaaagagagaggagaat-3 ' (sequence location: vesicular stomatitis virus from 5 ' hold 348-367 bit bases, SED ID NO:26 in sequence table)；

VSV2-R (downstream primer): 5 '-aactttagatctgcccactct-3 ' (sequence locations: vesicular stomatitis virus From SED ID NO:27 in 5 ' end 492-510 bit bases, sequence table)；

VSV2-P (TaqMan probe): 5 '-HEX-attttcgatctagtgaaagttgaagaact-Eclipse-3 ' (sequence Column position: vesicular stomatitis virus holds 375-402 bit bases, SED ID NO:28 in sequence table from 5 ')；

The reporter fluorescence group of above-mentioned TaqMan probe be FAM, ROX, CY5 and HEX, fluorescent quenching group TAMRA, BHQ2, BHQ2 and Eclipse.It is extended when to prevent PCR amplification, 3 ' the phosphorylated processing in end of above-mentioned TaqMan probe.

As shown in Figure 1, showing above-mentioned two groups of primer and probes combination carries out multiple reality to SVA, FMDV, SVDV and VSV When quantitative fluorescent PCR testing result, it is seen that organize 1 primer and probe combination sensibility be apparently higher than group 2 primer and spy Needle combination, determine group 1 primer and probe group be combined into preferred primer and probe group for SVA, FMDV, SVDV and VSV into The detection of row multiple real time fluorescence quantifying PCR.

The multiple real time fluorescence quantifying PCR of embodiment 2:SVA, FMDV, SVDV and VSV detect

One, extract SVA, FMDV geneome RNA and carry swine vesicular disease virus target gene (VP1 gene) and The recombinant plasmid of vesicular stomatitis virus target gene (N gene)

According to Axyprep^TMBody Fluid Viral DNA/RNA Miniprep Kit (AXYGEN company) specification from Senecan virocyte culture (Jin Yu Bao Ling company vaccine strain, for obtaining standard items and positive reference substance) and aftosa Virocyte culture (Jin Yu Bao Ling company vaccine strain, for obtaining standard items and positive reference substance) extracts Senecan respectively Virus genome RNA and foot-and-mouth disease virus genome RNA；Specifically includes the following steps:

(1) press kit specification, prepare isopropanol containing 1% glacial acetic acid in advance and in reagent Buffer W1A and The dehydrated alcohol of prescribed concentration is added in Buffer W2；

(1) press kit specification, prepare isopropanol containing 1% glacial acetic acid in advance and in reagent Buffer W1A and The dehydrated alcohol of prescribed concentration is added in Buffer W2；

(2) sample to be extracted of 200 μ L is added in 1.5mL centrifuge tube, and 200 μ L Buffer V-L are added, whirlpool shake After swinging mixing, 5min is stood；

(3) add 75 μ L Buffer V-N, whirlpool shake in the 1.5mL centrifuge tube for being mixed with sample and reagent of step (2) Mixing is swung, 12000g is centrifuged 5min；

(4) supernatant is transferred in 2mL centrifuge tube (providing in kit), adds 300 μ L isopropanols (1% glacial acetic acid), on It is lower to be inverted 6-8 times, it is uniformly mixed；

(5) preparation pipe (providing in kit) is placed in another 2mL centrifuge tube, the mixed liquor of step (4) is taken to move into system In standby pipe, 6000g is centrifuged 1min；

(6) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 500 μ L Buffer W1A, room temperature are added 1min is stood, 12000g is centrifuged 1min；

(7) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 800 μ L Buffer W2,12000g are added It is centrifuged 1min；

(8) filtrate is abandoned, pipe will be prepared and put back into the 2mL centrifuge tube of step (5), 1min is directly centrifuged with 12000g；

(9) pipe will be prepared to be placed in the 1.5mL centrifuge tube (providing in kit) of another cleaning, is added preparing periosteum center 40 μ L are stored at room temperature 1min without enzyme water, and 12000g is centrifuged 1min, affords RNA.

Swine vesicular disease virus target gene (VP1 gene) and vesicular stomatitis virus target gene (N gene) are sent to Hai Shenggong bioengineering Co., Ltd carries out target gene synthesis, engineering bacteria is obtained, according to GeneJET Plasmid Miniprep Kit (Thermo company) specification, which extracts to obtain from the culture bacterium solution of the engineering bacteria, carries pig blisters disease Recombinant plasmid (the also referred to as pig blisters disease of malicious target gene (VP1 gene) and vesicular stomatitis virus target gene (N gene) Malicious recombinant plasmid and vesicular stomatitis virus recombinant plasmid), it is respectively designated as PMD-19-SVDV and PMD-19-VSV.

Using the RNA/DNA extracted from sample to be tested using above method as detection sample；It is trained from Senecan virocyte The RNA extracted in supporting is as Senecan virus positive control product；The RNA extracted from foot and mouth disease virus cell culture is as mouth hoof Epidemic disease poison positive reference substance；Carry swine vesicular disease virus target gene (VP1 gene) and vesicular stomatitis virus target gene Positive reference substance of the recombinant plasmid of (N gene) respectively as swine vesicular disease virus and vesicular stomatitis virus；According to following two Method will be prepared respectively from Senecan virus, the RNA (i.e. positive reference substance) extracted in foot and mouth disease virus cell culture Standard items.

Two, the standard curve of the multiple real time fluorescence quantifying PCR detection of SVA, FMDV, SVDV and VSV is established

1, PCR amplification Senecan virus and foot and mouth disease virus detect geneome RNA

The geneome RNA for the Senecan virus extracted using step 1 is template, in primer SVA-STANDARD-F (5 '- TGGCTCCTTCGAGGCTCTCATC-3 ', SEQ ID NO:29) and SVA-STANDARD-R (5 '- AGGCGATGATATCAACCATGTC-3 ', SED ID NO:30) guidance under according to PCR amplification system (25 shown in the following table 1 μ L) amplification Senecan virus target gene (3C gene) sequence, PCR amplification condition are as follows: 42 DEG C of reverse transcription 20min of elder generation, 95 DEG C Initial denaturation 30s；Then 94 DEG C of denaturation 10s, 58 DEG C of annealing 30s, 72 DEG C of extension 45s, 30 circulations (PCR amplification), 72 DEG C of extensions 10min.After amplification, recycling, purifying pcr amplification product obtain Senecan virus 3C gene order.

The foot-and-mouth disease virus genome RNA extracted using step 1 is template, in primers F MDV-STANDARD-F (5 '- GAAGAAACCTGTCGCTTTGAAAG-3 ', SED ID NO:31) and FMDV-STANDARD-R (5 '- TCCGTCCTTTGCAAGAACGGCTC-3 ', SED ID NO:32) guidance under according to PCR amplification system (25 shown in the following table 2 μ L) amplification foot and mouth disease virus target gene (3C gene) sequence, PCR amplification condition are as follows: first 42 DEG C of reverse transcription 20min, 95 DEG C Initial denaturation 30s；Then 94 DEG C of denaturation 10s, 58 DEG C of annealing 30s, 72 DEG C of extension 45s, 30 circulations (PCR amplification), 72 DEG C of extensions 10min.After amplification, recycling, purifying pcr amplification product obtain foot and mouth disease virus 3C gene order.

The PCR amplification system of 1 Senecan virus target gene of table

The PCR amplification system of 2 foot and mouth disease virus target gene of table

2, standard items are prepared

Senecan virus target gene and foot and mouth disease virus target gene that above-mentioned steps 1 obtain are cloned into pCR- respectively In 4 TOPO carriers (being purchased from Invitrogen company), screening positive recombinant plasmid send Huada gene company to be sequenced.Sequencing result Show that obtain sequence correctly carries Senecan virus target gene (3C gene) and foot and mouth disease virus target gene (3C respectively Gene) recombinant plasmid, be respectively designated as pCR-4TOPO-SVA and pCR-4TOPO-FMDV, i.e. SVA and FMDV standard items.It will What step 1 obtained carries swine vesicular disease virus target gene (VP1 gene) and vesicular stomatitis virus target gene (N base Cause) recombinant plasmid, be respectively designated as PMD-19-SVDV and PMD-19-VSV, i.e. SVDV and VSV standard items.Use Qubit3.0 The concentration of above-mentioned each standard items is measured respectively, and calculates copy (copies) number of each standard items.

3, real-time fluorescence quantitative PCR standard curve is established

PCR-4TOPO-SVA, pCR-4TOPO-FMDV, PMD- is prepared using each standard items that above-mentioned steps 2 obtain 19-SVDV and PMD-19-VSV recombinant plasmid concentration is 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、 1 ×10²、1×10¹10 times of gradient standard items mixed liquors of copies/ μ L, using the standard items mixed liquor of various concentration as template, Multiple real time fluorescence quantifying PCR detection, 50 μ L real-time fluorescences are carried out under the guidance for organizing primer and probe shown in 1 (embodiment 1) The detection architecture of quantitative PCR is as shown in table 3 below, real-time fluorescence quantitative PCR testing conditions be (quantitative PCR apparatus, model C FX96, Purchased from U.S. Bole): first 42 DEG C of reverse transcriptions 20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 A circulation (PCR amplification).

3 SVA, FMDV, SVDV and VSV multiple real time fluorescence quantifying PCR detection architecture of table

The real-time fluorescence quantitative PCR amplification curve of each standard items is as shown in Fig. 2, standard items amplification curve is smooth " S " Shape curve (positive), it is SVA standard items (pCR-4TOPO-SVA), concentration that eight groups of zero lines of band are corresponding from left to right in Fig. 2 It is respectively as follows: 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹copies/μL；Eight in Fig. 2 Group band × lines corresponding from left to right are FMDV standard items (pCR-4TOPO-FMDV), and concentration is respectively as follows: 1 × 10⁸、 1× 10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹copies/μL；Eight groups of band △ lines are from left-hand in Fig. 2 The corresponding right side is SVDV standard items (PMD-19-SVDV), and concentration is respectively as follows: 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、 1 ×10⁴、1×10³、1×10²、1×10¹copies/μL；In Fig. 2 eight groups tape symbol lines it is not corresponding from left to right be VSV mark Quasi- product (PMD-19-VSV), concentration is respectively as follows: 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、 1×10¹copies/μL。

After detection, using the Log value of the concentration of each standard items as X-axis, mapped using recurring number (Ct value) as Y-axis, Standard curve is drawn, standard curve is as shown in figure 3, wherein the related coefficient of SVA, FMDV, SVDV, VSV are respectively R²= 0.994(SVA)、R²=0.992 (FMDV), R²=0.998 (SVDV) and R²=0.993 (VSV), therefore error is smaller, draws To standard curve can be used for to SVA, FMDV, SVDV and VSV carry out multiple real time fluorescence quantifying PCR detection, by standard song The linear equation that line obtains is respectively as follows: y=-3.583x+45.717 (SVA)；Y=-3.328x+45.667 (FMDV)；Y=- 3.261x+41.768(SVDV)；Y=-3.295x+42.692 (VSV).

Three, multiple real time fluorescence quantifying PCR detection is carried out to SVA, FMDV, SVDV and VSV

With multiple real time fluorescence quantifying PCR detection method of the invention to from vaccine strain, (sample to be tested, such as vaccine are raw Raw material, semi-finished product and the finished product of production) in geneome RNA/DNA (detection sample) for extracting detected, with what is extracted in embodiment 2 Senecan viral RNA, foot and mouth disease virus RNA, the recombinant plasmid for carrying swine vesicular disease virus target gene and the vesiculovirus mouth of carrying The recombinant plasmid of scorching virus target gene is as positive reference substance (step 1), using no enzyme water as negative controls, according to real-time Fluorescence quantitative PCR detection as a result, in sample to be tested whether containing Senecan virus, foot and mouth disease virus, swine vesicular disease virus and Vesicular stomatitis virus is qualitatively judged, and establishing criteria curve quantifies the copy number of virus.

It is specific that detection method includes the following steps:

1) using the geneome RNA/DNA extracted from sample to be tested as template, in embodiment 1 organize 1 shown in primer and Multiple real time fluorescence quantifying PCR detection is carried out under the guidance of probe, the detection architecture of 50 μ L real-time fluorescence quantitative PCRs includes: mould 25 μ L of 4 μ L of plate, real time fluorescent quantitative one-step method PCR reaction solution 2 × One Step RT-PCR Buffer III (is purchased from TakaRa company), TaKaRa Ex Taq HS 1 μ L (being purchased from TakaRa company), PrimeScript RT Enzyme Mix 1.5 1.5 1.5 μ L of μ L, SVA-P (10 μM) of μ L, SVA-R (10 μM) of 1 μ L of II (being purchased from TakaRa company), SVA-F (10 μM), 0.5 0.5 1.5 1.5 μ L of μ L, SVDV-F (10 μM) of μ L, FMDV-P (10 μM) of μ L, FMDV-R (10 μM) of FMDV-F (10 μM), SVDV-R (10 μM) 1.5 μ L, SVDV-P (10 μM) 1.5 μ L, VSV-F (10 μM) 1.5 μ L, VSV-R (10 μM) 1.5 μ L, VSV-P (10 μM) 1.5 μ L, RNA-free H₂O 3μL.Real-time fluorescence quantitative PCR reaction condition are as follows: first 42 DEG C of reverse transcriptions 20min, 95 DEG C 30s initial denaturation；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).In the annealing knot of each circulation Fluorescence signal detection is carried out when beam.

2) the qualitative and quantitative inspection to SVA, FMDV, SVDV and VSV is realized with the variation of obtained CT value or fluorescence signal It surveys, if " S " type amplification curve occurs in (not including the 36th circulation) in 36 circulations in the channel FAM, is confirmed as Senecan disease Malicious positive (containing Senecan virus in sample to be tested), if (not including the 36th circulation) occurs in 36 circulations in the channel ROX " S " type amplification curve is then confirmed as the foot and mouth disease virus positive (containing foot and mouth disease virus in sample to be tested), if 30 in the channel CY5 There is " S " type amplification curve in (not including the 30th circulation) in a circulation, then is confirmed as the positive (sample to be tested of swine vesicular disease virus In contain swine vesicular disease virus), if in the channel HEX in 33 circulations (do not include the 33rd circulation) amplification of " S " type occur bent It is positive (containing vesicular stomatitis virus in sample to be tested) to be then confirmed as vesicular stomatitis virus for line.

3) to the sample to be tested for being determined as certain virus-positive in step 2), intensity further according to the fluorescence signal and before really Fixed respective standard curve obtains the copy number of corresponding virus contained in sample to be tested, realizes that the Viral Quantification detects.

If if sample to be tested follows for 38 in the channel FAM and/or the channel/ROX or the channel/CY5 or channel HEX in step 2) More than ring (including 38 circulations) do not occur " S " type amplification curve, then are confirmed as corresponding virus feminine gender and (do not contain in sample to be tested Senecan virus or foot and mouth disease virus or swine vesicular disease virus or vesicular stomatitis virus)；If in the channel FAM and the channel ROX Between 36-38 circulation (including the 36th circulation, do not include the 38th circulation) " S " type amplification curve occur, be then determined as can It doubts, need to examine again；There is " S " in the channel CY5 between the 30-38 circulation (including the 30th circulation, do not include the 38th circulation) Type curve is then determined as suspicious, need to examine again；The channel HEX includes the 33rd circulation between the 33-38 circulation, does not include the 38 circulations) there is " S " type amplification curve and is then determined as suspicious, it need to examine again.

Testing result is as shown in table 4 below, it is seen that and the testing result of 1-4 sample is SVA positive (infection Senecan virus), Show Senecan virus in 1-4 sample；The testing result of 5-10 sample is FMDV positive (infection foot and mouth disease virus), is shown Contain foot and mouth disease virus in 5-10 sample.And the Senecan virus or aftosa of detection can be obtained according to fluorescence signal intensity The copy number of virus.

4 10 parts of cell cultures of table carry out SVA, FMDV, SVDV and VSV multiple real time fluorescence quantifying PCR testing result

It is embodiment 3, the sensibility that multiple real time fluorescence quantifying PCR detection is carried out to SVA, FMDV, SVDV and VSV, special Property and repeatability

3.1, sensitivity Detection

It is combined using the primer and probe of present invention group 1 to SVA, FMDV, SVDV and VSV as shown in Fig. 2, being also shown Carry out the sensibility of multiple real time fluorescence quantifying PCR detection.According to fig. 2 result as it can be seen that the present invention to SVA, FMDV, SVDV and The detection method that VSV carries out multiple real time fluorescence quantifying PCR can detecte to 1 × 10³copies/μL (SVA/FMDV/SVDV) With 1 × 10⁴Copies/ μ L (VSV), and amplification curve is specific " S " type curve, it is seen that primer and TaqMan of the invention Probe is suitable for sensibility with higher in conjunction with SVA, FMDV, SVDV and VSV RNA.The method that the present invention uses is multiple Real time fluorescence quantifying PCR method, the primer and probe combination for substance PCR detection is for carrying out multiple real time fluorescence quantifying When PCR, can interfere with each other its detection sensitivity is caused to decline due to different primers and probe, in other words can be used in list The primer and probe combination of weight real-time fluorescence quantitative PCR detection not necessarily can be used in multiplex PCR detection.

3.2, specific detection

According to the method in embodiment 2 to bovine viral diarrhoea bovine diarrhoea virus (BVDV), Porcine epidemic diarrhea virus (PEDV), swine fever virus (CSFV), transmissible gastro-enteritis virus (TGEV), bovine parainfluenza virus (BPIV) and Bovine Ephemeral Fever Viral (BEFV), normal PK-15 cell extraction RNA are template, to pseudorabies (PRV), rhinotracheitis virus (IBRV), pig circle 2 type of circovirus virus (PCV2), parvovirus (PPV), brucella (Rev.1) extract DNA as template, with normal PK-15 cell RNA be negative controls, while Senecan viral RNA to extract in embodiment 2 and foot and mouth disease virus RNA, carrying pig water The recombinant plasmid of the viral target gene of blister disease, the recombinant plasmid for carrying vesicular stomatitis virus target gene are positive reference substance, Using no enzyme water as blank control.Multiple real time fluorescence quantifying is carried out under the guidance of primer and probe shown in group 1 in embodiment 1 PCR detection, shown in the detection architecture table 3 as above of 50 μ L real-time fluorescence quantitative PCRs, real-time fluorescence quantitative PCR testing conditions are (fixed PCR instrument is measured, model C FX96 is purchased from U.S. Bole): first 42 DEG C of reverse transcriptions 20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).

Testing result is as shown in Figure 4, it is seen that only as Senecan virus (SVA), foot and mouth disease virus of positive control (FMDV), there is specific " S " type amplification curve (result sun in swine vesicular disease virus (SVDV) and vesicular stomatitis virus (VSV) Property), other samples do not occur specific " S " type amplification curve (result is negative), it is seen that the method for the present invention can be examined specifically Measure SVA, FMDV, SVDV and VSV.

3.3, repeatability detection

Using above-described embodiment 2 obtain each standard items be prepared pCR-4TOPO-SVA, pCR-4TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV recombinant plasmid concentration is 1 × 10⁸、1×10⁷、1×10⁶、1×10⁵、1×10⁴、1× 10³、 1×10²、1×10¹10 times of gradient standard items mixed liquors of copies/ μ L, each gradient in triplicate, with various concentration Standard items mixed liquor as template, organize that multiple real time fluorescence is carried out under the guidance of primer and probe shown in 1 is fixed in embodiment 1 PCR detection is measured, shown in the detection architecture table 3 as above of 50 μ L real-time fluorescence quantitative PCRs, real-time fluorescence quantitative PCR testing conditions are (quantitative PCR apparatus, model C FX96 are purchased from U.S. Bole): first 42 DEG C of reverse transcriptions 20min, 95 DEG C of initial denaturation 30s；Then 94 DEG C It is denaturalized 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).

Testing result as can be seen from Fig. 5 repeats three times as shown in Fig. 5 and table 5, and SVA, FMDV, SVDV and VSV are respectively In the amplification curve of each concentration gradient relatively gather, will also realize that the recurring number of each concentration gradient without obvious poor from the following table 5 result Away from recurring number standard deviation difference only up to 0.67, it is seen that the present invention carries out SVA, FMDV, SVDV and VSV multiple real-time The repeatability of fluorescent quantitative PCR detection method is preferably.

The repeated result of 5 multiple real time fluorescence quantifying PCR detection method of table

Embodiment 4, the multiple real time fluorescence quantifying PCR detection kit of SVA, FMDV, SVDV and VSV

The multiple real time fluorescence quantifying PCR detection kit of SVA, FMDV, SVDV and VSV provided by the invention includes:

For carrying out multiple real time fluorescence quantifying PCR inspection to SVA, FMDV, SVDV and VSV shown in group 1 in embodiment 1 The primer (SVA1-F, SVA1-R, FMDV1-F, FMDV1-R, SVDV1-F, SVDV1-R, VSV1-F and VSV1-R) of survey and TaqMan probe (SVA1-P, FMDV1-P, SVDV1-P and VSV1-P).

Specifically, the multiple real time fluorescence quantifying PCR detection kit of SVA, FMDV, SVDV and VSV provided by the invention Reagent including being used for 50 μ L multiple real time fluorescence quantifying PCR detection architectures below: real time fluorescent quantitative one-step method PCR reaction Liquid 2 × One Step RT-PCR Buffer III 25 μ L (being purchased from TakaRa company), 1 μ L of TaKaRa Ex Taq HS (purchase In TakaRa company), PrimeScript RT Enzyme Mix II 1 μ L (being purchased from TakaRa company), SVA-F (10 μM) 1.5 μ L, SVA-R (10 μM) 1.5 μ L, SVA-P (10 μM) 1.5 μ L, FMDV-F (10 μM) 0.5 μ L, FMDV-R (10 μM) 0.5 μ L, FMDV-P (10 μM) 1.5 μ L, SVDV-F (10 μM) 1.5 μ L, SVDV-R (10 μM) 1.5 μ L, SVDV-P (10 μM) 1.5 μ L, VSV- F (10 μM) 1.5 μ L, VSV-R (10 μM) 1.5 μ L, VSV-P (10 μM) 1.5 μ L, 4.0 μ L, RNA-free H of template ribonucleic acid₂O 3.0 μL。

For convenience of detection, it may also include positive reference substance and negative controls in kit, positive reference substance is Senecan Viral RNA, foot and mouth disease virus RNA, swine vesicular disease virus recombinant plasmid and vesicular stomatitis virus recombinant plasmid, each positive are right It can be with unitary package according to product, or hybrid packed, negative controls are without Senecan virus, foot and mouth disease virus, pig water The reaction system of blister disease virus and vesicular stomatitis virus, such as H₂O (distilled water, aseptic deionized water etc.).Use the kit When carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV, the positive reference substance of unitary package is mixed Or directly use hybrid packed positive reference substance.

It for convenience of detection, may also include standard items in kit, detect base to carry Senecan viral nucleotide respectively Cause, foot and mouth disease virus nucleotide detection gene, swine vesicular disease virus nucleotide detection gene and vesicular stomatitis virus nucleotide Recombinant plasmid pCR-4TOPO-SVA, pCR-4TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV of gene are detected (referring to reality Apply example 2), each standard items can for individually packaging, can also isoconcentration it is hybrid packed；Using the kit to SVA, FMDV, SVDV and When VSV carries out multiple real time fluorescence quantifying PCR detection, the standard items isoconcentration of unitary package is mixed or directly using mixing packet The standard items of dress.

For convenience of detection, it may also include the standard curve and specification of the acquisition of embodiment 2, the description in kit Including PCR reaction condition: first 42 DEG C of reverse transcription 20min, 95 DEG C of 30s initial denaturations；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification)；And to the judgement explanation of testing result in embodiment 2.

The multiple real time fluorescence quantifying PCR detection of embodiment 5, doubtful SVA, FMDV, SVDV and VSV pathological material of disease in pig farm

The detection kit that the embodiment is provided with embodiment 5 is to 14 parts of doubtful SVA, FMDV, the SVDV collected from pig farm With VSV samples (serum, sample to be tested) in extract geneome RNA detected, with Senecan virus genome RNA, Foot-and-mouth disease virus genome RNA, swine vesicular disease virus recombinant plasmid and vesicular stomatitis virus recombinant plasmid are positive reference substance, Using no enzyme water as negative controls, sample to be tested is carried out according to the 50 μ L reaction system and reaction conditions provided in kit more Weight real-time fluorescence quantitative PCR detection.Whether contain according to the specification provided in kit in sample to be tested according to testing result There are SVA, FMDV, SVDV and VSV to be qualitatively judged, and the copy number of virus is quantified.

Testing result is as shown in table 6 below, it is seen that 1,2,5,8,10, No. 12 sample detection result, which is that SVA is positive, (to infect in plug Card virus), No. 6 sample detection results are SVA negative (being uninfected by Senecan virus), and No. 13 sample detection results are that SVA can It doubts, shows containing Senecan virus in 1,2,5,8,10, No. 12 sample, and calculated according to fluorescence signal intensity and standard curve The copy number of Senecan virus in sample to be tested, without Senecan virus in No. 6 samples, No. 13 samples need to be examined again；3,4,7, No. 14 sample detection results are FMDV positive (infection foot and mouth disease virus), and No. 9 sample detection results are that FMDV is suspicious, No. 11 samples Testing result is FMDV negative (being uninfected by foot and mouth disease virus), is shown containing foot and mouth disease virus in 3,4,7, No. 14 samples, and according to Fluorescence signal intensity and standard curve have calculated the copy number of the foot and mouth disease virus in sample to be tested, and No. 9 samples need to be examined again, and 11 Foot and mouth disease virus is free of in sample；1-14 sample SVDV and VSV testing result is feminine gender, i.e., is free of in 1-14 sample SVDV and VSV.

Doubtful SVA, FMDV, SVDV and VSV sample the multiple real time fluorescence quantifying PCR testing result in 6 14 parts of pig farms of table

Finally, it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not intended to restrict the invention, Although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still may be used To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features. All within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should be included in of the invention Within protection scope.

Sequence table

<110>Jinyu Baoling Biology Drugs Co., Ltd

<120>the one-step method multiple real time fluorescence quantifying PCR detection primer and probe of SVA, FMDV, SVDV and VSV

<160> 32

<170> SIPOSequenceListing 1.0

<210> 1

<211> 257

<212> DNA

<213>Senecan is viral (Seneca virus A)

<400> 1

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gctgggaccg tatctcagat ccctggctgt ctcggtgcac gcttacggcg agcgtcgcat 120

caagattacc ggtggcctcc cctccggttg tgccgcgacc agcctgctga acacagtgct 180

caacaatgtg atcatcagga ctgctctggc attgacttac aaggaatttg aatatgacat 240

ggttgatatc atcgcct 257

<210> 2

<211> 568

<212> DNA

<213>foot and mouth disease virus (Foot and Mouth Disease Virus)

<400> 2

gaagaaacct gtcgctttga aagtgaaagc aaagaacttg atcgtcactg agagtggtgc 60

tcccccgact gacttgcaaa agatggtcat gggtaacacc aagcctgttg agctcatcct 120

cgacgggaag acggtggcca tctgctgcgc cactggagtg tttggtactg cctaccttgt 180

tcctcgtcat cttttcgcag agaagtatga caagatcatg ttggacggca gagccatgac 240

agacagtgac tacagagtgt ttgagtttga gattaaagtg aaaggacagg acatgctctc 300

agacgccgcg ctcatggtgc ttcaccgtgg gaatcgcgtg cgggacatca cgaagcactt 360

ccgtgatgtg gcaagaatga agaaaggcac ccccgtcgtc ggcgtgatca acaacgctga 420

tgttgggaga ctgatcttct ctggtgaggc ccttacctac aaggacattg tagtgtgcat 480

ggacggagac accatgcccg gtctcttcgc ctacaaagcc gccaccaagg cgggttactg 540

tggaggagcc gttcttgcaa aggacgga 568

<210> 3

<211> 849

<212> DNA

<213>pig blisters is malicious (Swine Vesicular Disease Virus)

<400> 3

gggcccccag gaggagtgac ggaaggaatc attgcccgcg tcgctgatac cgtagggagt 60

ggaccagtca actcggagtc catcccggct ctaaccgccg cagaaacagg gcacacgtca 120

caggttgtgc catcagacac aatgcaaact aggcatgtga agaattacca ctcaaggtca 180

gagtcaaccg tggagaactt cctatgcaga tccgcatgcg tattctatac cacttacaag 240

aaccacgact ctgatggtga caacttcgca tattgggtca tcaacacacg gcaagttgct 300

caactacgcc ggaagctcga aatgttcacg tatgcaagat ttgatctgga gttaaccttc 360

gtgatcacca gcactcagga gcaacccacc actcaaggcc aagacacacc agtactcact 420

caccaaataa tgtacgtacc gccaggtggc ccagtaccca caaaggtgaa tagttacagc 480

tggcaaacgt ccaccaaccc gagtgtgttc tggacagaag gaaacgctcc acctcggatg 540

tcgataccat tcattggcat cggcaatgca tacagcatgt tctatgacgg gtgggccaga 600

ttcgacaagc aaggaacata tggcattagt acactaaata acatggggac actatatatg 660

agacatgtga atagtggggg ccccggtccc attgtgagca cagtgcgaat atatttcaaa 720

ccaaagcatg tcaaaacatg ggtcccacga ccacccaggc tatgccaata tgaaaaagct 780

ggcaatgtga attttgtacc cacgagcgtg acagagggca ggacagatat aacaaccatg 840

aggaccacc 849

<210> 4

<211> 1269

<212> DNA

<213>vesicular stomatitis virus (Vesicular stomatitis virus)

<400> 4

atggctccta cagttaagag aatcattaac gactcaatta ttcagccgaa attaccggcc 60

aacgaggatc cggtcgaata cccggctgat tacttcaaaa ataataccaa tatagtattg 120

tatgtgagca ccaaagtggc actaaatgat ttgagagcat acgtatacca gggtatcaag 180

tccggtaatc catccatcct ccacataaat gctcatctct atgctgcatt aaagggagtg 240

gaaggaactt tagacagaga ctggattagc tttggaagaa caattggaaa gagagaggag 300

aatgtgaaaa ttttcgatct agtgaaagtt gaagaactga agacagcact tcctgatggg 360

aaatcagacc ctgaccgttc tgctgaggat gataaatggc ttcccatcta catcctaggt 420

ctttacagag tgggcagatc taaagttacg gattacagaa agaaactact ggacgggctt 480

gaaaatcagt gcaaagtggc gtcaaccaga tttgagagtc tagtcgagga tggtctcgac 540

ttctttaaca tatgggagaa tgatccaaat ttcaccaaga tagttgctgc agtggatatg 600

ttcttccaca tgctcaaaaa gcatgaacgt gctccaatca gatacggaac catagtctca 660

agattcaagg actgtgcagc acttgcaaca tttgggcatc tcagcaaagt cagtggactc 720

tcaattgagg aactcacaac atgggtcctg aatagggaag ttgcagacga gctatgccag 780

atgatgtatc cgggtcaaga aattgacaaa gcagattcat acatgccgta tatgattgac 840

tttgggttat ctcagaaatc cccatattca tcagtgaaga atccagcttt tcatttctgg 900

ggacaacttg ctgcactctt gctaagatca actcgggcaa aaaatgctag acagcctgac 960

gacatcgaat acacttcact aacttgcgca agtttactgc tgtcatttgc tgttgggtcc 1020

tcagcagaca ttgaacagca gttctatatt ggagaagaca aatacacaac agaaaaagat 1080

gatggtctga agaaatcaga tgtcccacca aaaggaagaa atgtcgtgga ctggcttggc 1140

tggtatgatg acaatggggg aaaacccaca ccagatatgc tcaacttcgc aagaagagca 1200

gtcaactctc tgcagtcact tcgtgaaaag acaattggca aatacgcaaa agtagaattt 1260

gacaaatag 1269

<210> 5

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tatctcagat ccctggctgt c 21

<210> 6

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

cctgatgatc acattgttga gc 22

<210> 7

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

cacgcttacg gcgagcgtcg catcaag 27

<210> 8

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

ygcctmccth gtdcctcgtc ayctyt 26

<210> 9

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gagagcatgt cctgtcctyt yacttt 26

<210> 10

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

cngagaagta ygacaagatc atgyt 25

<210> 11

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

taggcatgtg aagaattacc ac 22

<210> 12

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

atcagagtcg tggttcttgt a 21

<210> 13

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

tcagagtcaa ccgtggagaa cttcct 26

<210> 14

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 14

ccrggycaag aaattgacaa 20

<210> 15

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 15

taytcgatgt cgtcrggctg tc 22

<210> 16

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 16

attcatacat gccgtatatg attgactt 28

<210> 17

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

tataagatga ctcctgccaa c 21

<210> 18

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

agaatttgga agccatgctc tc 22

<210> 19

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

ttctgtcttc cctccgactt cctctc 26

<210> 20

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 20

aagatcatgt tggacggcag agccat 26

<210> 21

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 21

atgtcccgca cgcgattccc acggt 25

<210> 22

<211> 28

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 22

cagtgactac agagtgtttg agtttgag 28

<210> 23

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 23

actcaccaaa taatgtacgt ac 22

<210> 24

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 24

aatctggccc acccgtcata g 21

<210> 25

<211> 27

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 25

tggcccagta cccacaaagg tgaatag 27

<210> 26

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 26

ttggaaagag agaggagaat 20

<210> 27

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 27

aactttagat ctgcccactc t 21

<210> 28

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 28

attttcgatc tagtgaaagt tgaagaact 29

<210> 29

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 29

tggctccttc gaggctctca tc 22

<210> 30

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 30

aggcgatgat atcaaccatg tc 22

<210> 31

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 31

gaagaaacct gtcgctttga aag 23

<210> 32

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 32

gaagaaacct gtcgctttga aag 23

Claims (10)

1. primer and Taqman probe for carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV, It is characterized by:

For detecting the upstream primer (SVA1-F) and downstream primer (SVA1-R) of SVA, the core of the upstream primer (SVA1-F) Nucleotide sequence is as shown in SED ID NO:5 in sequence table, SEQ in the nucleotide sequence of downstream primer (SVA1-R) such as sequence table Shown in ID NO:6；

For carrying out the TaqMan probe (SVA1-P) of real-time fluorescence quantitative PCR detection, the TaqMan probe (SVA1- to SVA P nucleotide sequence) is as shown in SED ID NO:7 in sequence table；

For detecting the upstream primer (FMDV1-F) and downstream primer (FMDV1-R) of FMDV, the upstream primer (FMDV1-F) Nucleotide sequence as shown in SED ID NO:8 in sequence table, in the nucleotide sequence of downstream primer (FMDV1-R) such as sequence table Shown in SEQ ID NO:9；

For carrying out the TaqMan probe (FMDV1-P) of real-time fluorescence quantitative PCR detection, the TaqMan probe to FMDV (FMDV1-P) nucleotide sequence is as shown in SED ID NO:10 in sequence table；

For detecting the upstream primer (SVDV1-F) and downstream primer (SVDV1-R) of SVDV, the upstream primer (SVDV1-F) Nucleotide sequence as shown in SED ID NO:11 in sequence table, the nucleotide sequence such as sequence table of downstream primer (SVDV1-R) Shown in middle SEQ ID NO:12；

For carrying out the TaqMan probe (SVDV1-P) of real-time fluorescence quantitative PCR detection, the TaqMan probe to SVDV (SVDV1-P) nucleotide sequence is as shown in SED ID NO:13 in sequence table；

For detecting the upstream primer (VSV1-F) and downstream primer (VSV1-R) of VSV, the core of the upstream primer (VSV1-F) Nucleotide sequence is as shown in SED ID NO:14 in sequence table, SEQ in the nucleotide sequence of downstream primer (VSV1-R) such as sequence table Shown in ID NO:15；

For carrying out the TaqMan probe (VSV1-P) of real-time fluorescence quantitative PCR detection, the TaqMan probe (VSV1- to VSV P nucleotide sequence) is as shown in SED ID NO:16 in sequence table；

The probe is by fluorescent marker, and 5 ' ends are marked with fluorescent reporter group, and 3 ' ends are marked with fluorescent quenching group； And the fluorescent reporter group marked to four kinds of viral diagnosis probes is not identical.

2. primer according to claim 1 and TaqMan probe, it is characterised in that:

It reports at the 5 ' ends for carrying out the TaqMan probe (SVA1-P) of real-time fluorescence quantitative PCR detection to Senecan virus Fluorophor is FAM, and 3 ' end fluorescent quenching groups are TAMRA；

5 ' the end reports for carrying out the TaqMan probe (FMDV1-P) of real-time fluorescence quantitative PCR detection to foot and mouth disease virus are glimmering Light group is ROX, and 3 ' end fluorescent quenching groups are BHQ2；

It reports at the 5 ' ends for carrying out the TaqMan probe (SVDV1-P) of real-time fluorescence quantitative PCR detection to swine vesicular disease virus Fluorophor is CY5, and 3 ' end fluorescent quenching groups are BHQ2；

It reports at the 5 ' ends for carrying out the TaqMan probe (VSV1-P) of real-time fluorescence quantitative PCR detection to vesicular stomatitis virus Fluorophor is HEX, and 3 ' end fluorescent quenching groups are Eclipse；

3 ' the phosphorylated processing in end of the probe.

3. for carrying out multiple real time fluorescence quantifying PCR detection kit to SVA, FMDV, SVDV and VSV, it is characterised in that:

Including primer of any of claims 1 or 2 and TaqMan probe；

The dosage of primer and TaqMan probe described in 50 μ L real-time fluorescence quantitative PCR detection architectures when using the kit It is preferred that are as follows: 1.5 1.5 1.5 μ L, FMDV1-F (10 μM) 0.5 of μ L, SVA1-P (10 μM) of μ L, SVA1-R (10 μM) of SVA1-F (10 μM) μ L, FMDV1-R (10 μM) 0.5 μ L, FMDV1-P (10 μM) 1.5 μ L, SVDV1-F (10 μM) 1.5 μ L, SVDV1-R (10 μM) 1.5 μ 1.5 1.5 1.5 1.5 μ L of μ L, VSV1-P (10 μM) of μ L, VSV1-R (10 μM) of μ L, VSV1-F (10 μM) of L, SVDV1-P (10 μM).

4. kit according to claim 3, it is characterised in that: further include positive reference substance and feminine gender in the kit Reference substance, positive reference substance are Senecan viral RNA, foot and mouth disease virus RNA, swine vesicular disease virus recombinant plasmid PMD-19- SVDV and vesicular stomatitis virus recombinant plasmid PMD-19-VSV, each positive reference substance is unitary package or hybrid packed, negative Reference substance be without Senecan virus, foot and mouth disease virus, swine vesicular disease virus and vesicular stomatitis virus reaction system, such as H₂O (distilled water, aseptic deionized water etc.)；

When carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV using the kit, by single packet The positive reference substance of dress mixes or directly uses hybrid packed positive reference substance.

5. kit according to claim 3 or 4, it is characterised in that: further include standard items in the kit, to divide It Xie Dai not Senecan viral nucleotide detection gene, foot and mouth disease virus nucleotide detection gene, the inspection of swine vesicular disease virus nucleotide Cls gene and the vesicular stomatitis virus nucleotide detection recombinant plasmid pCR-4 TOPO-SVA of gene, pCR-4 TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV；Each standard items are that unitary package or isoconcentration are hybrid packed；

When carrying out multiple real time fluorescence quantifying PCR detection to SVA, FMDV, SVDV and VSV using the kit, by single packet The standard items isoconcentration of dress mixes or directly uses hybrid packed standard items.

6. kit described in claim 3 or 4 or 5 is in the inspection for carrying out non-disease diagnostic purpose to SVA, FMDV, SVDV and VSV Application in survey.

7. application according to claim 6, it is characterised in that:

Non-disease diagnostic purpose is carried out to SVA, FMDV, SVDV and VSV with multiple real time fluorescence quantifying PCR technology determine to be a kind of Property, quantitative detecting method, comprising the following steps:

1) it establishes standard curve: the recombinant plasmid pCR4-TOPO- of the detection gene of SVA, FMDV, SVDV and VSV will be carried respectively The standard items isoconcentration mixing or direct of the unitary package of SVA, pCR4-TOPO-FMDV, PMD-19-SVDV and PMD-19-VSV Using the hybrid packed standard items of isoconcentration, standard concentration each in mixed liquor is diluted to 1 × 10 according to 10 times of gradients⁸、1× 10⁷、1×10⁶、1×10⁵、1×10⁴、1×10³、1×10²、1×10¹copies/μL；With the standard items mixed liquor of various concentration As template, multiple real time fluorescence quantifying PCR is carried out under the guidance of primer of any of claims 1 or 2 and TaqMan probe Detection after detection, maps to its corresponding Ct value (Y-axis) with the concentration Log value (X-axis) of each standard items, draws standard curve；

2) geneome RNA for extracting sample to be tested draws using the geneome RNA of extraction as template of any of claims 1 or 2 Multiple real time fluorescence quantifying PCR detection is carried out under the guidance of object and TaqMan probe；

3) qualitative detection to SVA, FMDV, SVDV and VSV is realized with the variation of obtained CT value or fluorescence signal, further according to glimmering Standard curve in the intensity and step 1) of optical signal obtains the copy of contained SVA, FMDV, SVDV and VSV in sample to be tested Number realizes quantitative detection.

8. application according to claim 7, it is characterised in that:

Sample to be tested in the step 2) includes the raw material for production of vaccine, vaccine semi-finished product and finished product, pig farm inspection sample Product.

9. application according to claim 7 or 8, it is characterised in that:

50 μ L real-time fluorescence quantitative PCR detection architectures in the step 1) and step 2) include: 2 μ L of template, and real-time fluorescence is fixed Measure 2 × One of one-step method PCR reaction solution Step RT-PCR Buffer III, 25 μ L (being purchased from TakaRa company), TaKaRa Ex 1 μ L (being purchased from TakaRa company) of 1 μ L of Taq HS (being purchased from TakaRa company), PrimeScript RT Enzyme Mix II, 1.5 1.5 1.5 0.5 μ L of μ L, FMDV1-F (10 μM) of μ L, SVA1-P (10 μM) of μ L, SVA1-R (10 μM) of SVA1-F (10 μM), 0.5 1.5 1.5 1.5 μ L of μ L, SVDV1-R (10 μM) of μ L, SVDV1-F (10 μM) of μ L, FMDV1-P (10 μM) of FMDV1-R (10 μM), 1.5 1.5 1.5 1.5 μ L of μ L, VSV1-P (10 μM) of μ L, VSV1-R (10 μM) of μ L, VSV1-F (10 μM) of SVDV1-P (10 μM), template RNA4.0 μ L, RNA-free H₂O3.0μL；And/or

Multiple real time fluorescence quantifying PCR testing conditions in the step 1) and step 2) are as follows: first 42 DEG C of reverse transcriptions 20min, 95 DEG C initial denaturation 30s；Then 94 DEG C of denaturation 10s, 55 DEG C of annealing 30s, 45 circulations (PCR amplification).

10. the application according to any one of claim 7-9, it is characterised in that:

Determination method in the step 3) are as follows:

If sample channel corresponding to 5 ' the marked fluorescent reporter groups in end of SVA1-P (does not include the in 36 circulations 36 circulations) there is " S " type amplification curve, then it is positive (containing SVA in sample) to be confirmed as SVA；If sample in FMDV1-P 5 ' Channel corresponding to marked fluorescent reporter group (not including the 36th circulation) in 36 circulations is held the amplification of " S " type occur It is positive (containing FMDV in sample) to be then confirmed as FMDV for curve；If the sample fluorescence report base marked at the 5 ' ends of SVDV1-P There is " S " type amplification curve in the corresponding channel of group (not including the 30th circulation) in 30 circulations, then is confirmed as SVDV sun Property (containing SVDV in sample)；If sample channel corresponding to the marked fluorescent reporter group in the 5 ' ends of VSV1-P is at 33 There is " S " type amplification curve in (not including the 33rd circulation) in circulation, then it is positive (containing VSV in sample) to be confirmed as VSV；If sample In any channel, more than 38 circulations (including the 38th circulation) " S " type amplification curve to product does not occur, then is confirmed as corresponding disease Malicious negative (SVA or FMDV or SVDV and/or VSV is not contained in sample)；If the sample fluorescence marked at the 5 ' ends of SVA1-P Channel corresponding to the marked fluorescent reporter group in the 5 ' ends of channel corresponding to reporter group or FMDV1-P is at 36-38 Between circulation (including the 36th circulation and do not include the 38th circulation) occur " S " type amplification curve, be determined as it is suspicious, need weight Inspection；If sample channel corresponding to the marked fluorescent reporter group in the 5 ' ends of SVDV1-P (packet between 30-38 circulation Include the 30th circulation and do not include the 38th circulation) there is " S " type amplification curve, it is determined as suspicious, need to examines again；If sample exists (including the 33rd recycle between 33-38 circulation in channel corresponding to 5 ' the marked fluorescent reporter groups in end of VSV1-P And do not include the 38th circulation) there is " S " type amplification curve, it is determined as suspicious, need to examines again；

Preferably, if there is " S " type amplification curve in the circulation of 36, the channel FAM in sample, it is confirmed as the SVA positive；If sample There is " S " type amplification curve in the circulation of 36, the middle channel ROX, is then confirmed as the FMDV positive；If 30, the channel CY5 circulation in sample Interior appearance " S " type amplification curve is then confirmed as the SVDV positive；If it is bent the amplification of " S " type occur in the circulation of 33, the channel HEX in sample Line is then confirmed as the VSV positive；If in sample the channel FAM or 38, the channel ROX or the channel CY5 and/or the channel HEX circulation with On do not occur " S " type amplification curve, then it is negative to be confirmed as SVA or FMDV or SVDV and/or VSV；If the channel FAM and the channel ROX In there is " S " type amplification curve between 36-38 circulation and be then determined as suspicious, need to examine again；If the channel CY5 is at 30-38 Appearance " S " type curve is then determined as suspicious between circulation, need to examine again；If there is " S " between the 33-38 circulation in the channel HEX Type amplification curve is then determined as suspicious, need to examine again.