CN106521030A - Dual fluorescent quantitation RT-PCR detection method for classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) - Google Patents
Dual fluorescent quantitation RT-PCR detection method for classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV) Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
The invention relates to a dual fluorescent quantitation RT-PCR detection method for the classical swine fever virus (CSFV) and the bovine viral diarrhea virus (BVDV) and belongs to the technical field of fluorescent quantitation RT-PCRs. According to the detection method, two groups of primers and probes are used, wherein the sequence of an amplification product of the first primer and probe group is solely consistent with a gene sequence conservation zone of the classical swine fever virus, and the sequence of an amplification product of the second primer and probe group is solely consistent with a gene sequence conservation zone of the BVDV. In addition, the CSFV probe P only appears in the amplification product consistent with the gene sequence conservation zone of the classical swine fever virus, the BVDV probe P only appears in the amplification product consistent with the gene sequence conservation zone of the bovine viral diarrhea virus, and each primer and probe group can eliminate interference of the other primer and probe group. In this way, by means of the detection method, the CSFV can be differentiated from the BVDV, and the classical swine fever virus and the bovine viral diarrhea virus can be detected at the same time.
Description
Technical field
The present invention relates to swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detection method, belong to glimmering
Light quantitative RT-PCR technology field.
Background technology
Swine fever virus（Classical swine fever virus, abbreviation CSFV）With bovine viral diarrhea virus (
Bovine viral diarrhea virus, abbreviation BVDV) flaviviridae, pestivirus are belonged to, for a long time, their institutes
Infectious disease provisions cattle, pig and the sheep husbandry for causing causes huge economic loss.BVDV is worldwide widely distributed,
The animals such as goat, sheep, pig, deer and wallaby are all the host of BVDV and the source of infection.The nucleotide sequence of BVDV and CSFV is about
There is 60% homology, aminoacid there are about 85% homology, and polyclonal antibody has serological cross reaction to two-strain.It is first
First, CSFV and BVDV infected pigs under field conditions (factors), its clinical symptoms are similar with pathological change；Secondly, produce in live vaccine
In, often using cattle source materials such as Ox blood serum, bull testis cell, pancreatin as raw material, once these raw materials have been contaminated BVDV,
The pollution of vaccine product will then be caused, after especially pig is with the contaminated BVDV of vaccine, immune swine can be caused to infect, itself is caused
The clinical symptoms of similar swine fever, bring outside difficulty to the diagnosis of swine fever, will also become polluter.
CN101058830A discloses " one diagnostic kit of pig pestivirus fluorescence quantitative ", and CN101328506A is disclosed
" the fluorescent quantitation quick diagnosis reagent kit and its application process of species specificdetection wild-type classical swine fever virus infection "；Two documents are equal
Independent detection has been carried out to CSFV only.OIE discloses the primer probe sequence of detection BVDV, and mark cannot distinguish between swine fever virus；
CN103397107A discloses " one detection kit of bovine viral diarrhea virus fluorescent quantitation ", but only BVDV is carried out individually
Detection.Clinical detection result shows that existing CSFV or BVDV fluorescence quantitative RT-PCR kits cannot be distinguished by CSFV and BVDV.
At present, it is also imperfect for the associated detecting method of CSFV and BVDV infection, and the detection to pig source BVDV is remained in exploration
Stage；Also not having can be while carries out to swine fever virus and bovine viral diarrhea virus while the related reagent for detecting.
The content of the invention
It is similar to BVDV gene orders based on CSFV, the fact that conventional RT-PCR is difficult to differentiate between both；The purpose of the present invention
It is the detection method that the Multiplex real-time PCR that a kind of tool can distinguish swine fever virus and bovine viral diarrhea virus is provided.
Technical scheme
One group of fluorescence quantitative RT-RCR detection primer and probe, by first group of primer and probe or/and second group of primer and spy
Pin is constituted；
First group of primer and probe are：
CSFV-F：ATACATAAAGCCCGGCCCTG, as shown in SEQ ID NO.1；
CSFV-R：CTTGCCATCACTACCCGTGA, as shown in SEQ ID NO.2；
CSFV probe P：FAM-CCAGGACTACATGGGCCCAGTCTATCAC-BHQ1, as shown in SEQ ID NO.3；
Second group of primer and probe are：
BVDV-F：AGCAACAGTGGTGAGTTCGT, as shown in SEQ ID NO.4；
BVDV-R：CGTGGCATCTCGAGACCTTT, as shown in SEQ ID NO.5；
BVDV probe P：HEX VIC-ATGGCTTAAGCCCTGAGTACA-BHQ1, as shown in SEQ ID NO.6.
The above-mentioned primer and probe of the present invention：Wherein first group primer and probe are used to expand swine fever virus distinguished sequence,
Second group of primer and probe are used to expand bovine viral diarrhea virus distinguished sequence.The amplified production of first group of primer and probe
Sequence uniquely with swine fever virus gene order conserved region（533bp-671bp）Unanimously；The amplified production of second group of primer and probe
Sequence uniquely with BVDV gene orders conserved region（142bp-237bp）Unanimously.Fluorescence is carried out using the above-mentioned primer of the present invention
Quantitative RT-PCR is detected, CSFV can be made a distinction with BVDV, solved existing CSFV or BVDV fluorescence quantitative RT-RCRs and try
Agent box cannot be distinguished by the technical problem of CSFV and BVDV.
A kind of swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detectable, containing above-mentioned primer
And probe.
Above-mentioned detectable, consisting of：
DEPC H2O： 2.75ul
5×Buffer： 2.5ul
10×Buffer： 1.25ul
2.5mM dNTP： 2ul
CSFV-F: 0.5ul
CSFV-R: 1ul
CSFV probe P： 0.5ul
BVDV-F: 0.5ul
BVDV-R: 1ul
BVDV probe P： 0.5ul
20uM MgCl2： 1.5ul
Enzymatic mixture： 1ul；
The concentration of CSFV-F, CSFV-R, CSFV probe P, BVDV-F, BVDV-R, BVDV probe P is 10 μm of ol/L.
Above-mentioned detectable, is the consumption of a sample of detection.
A kind of swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detection method；
The nucleic acid 10ul of testing sample and the above-mentioned detectable of 15ul are made into into the reaction system of 25ul, fluorescent quantitation is then carried out
RT-PCR reacts, and reads result；
The fluorescence quantitative RT-RCR reaction：
First stage：42 DEG C/30min of reverse transcription；
Second stage：94 DEG C/3min of denaturation；
Phase III：92 DEG C/15sec, 45 DEG C/30sec, 72 DEG C/60sec, totally 5 circulations；
Fourth stage：92 DEG C/10sec, 56 DEG C/60sec, totally 40 circulations；
Last 40 DEG C/10sec；
Fluorescence is collected in fourth stage 56 DEG C of extensions of annealing of circulation every time；
The reading result：
If serpentine amplification curve occur in FAM sense channels, and Ct values are less than or equal to 30, then contain CSFV in sample；
If HEX VIC sense channels there is serpentine amplification curve, and Ct values are less than or equal to 30, then contain BVDV in sample.
Above-mentioned detection method,
Primer and probe for expanding swine fever virus distinguished sequence be：Primer CSFV-F, CSFV-R and CSFV probe P；
Primer and probe for expanding bovine viral diarrhea virus distinguished sequence be：BVDV-F, primer BVDV-R and BVDV
Probe P.
The amplified production that above-mentioned detection method is obtained is sequenced, CSFV probe P are only occurred in and swine fever virus gene
Sequence conservation（533bp-671bp）In consistent amplified production, BVDV probe P are only occurred in and bovine viral diarrhea virus disease
Virus gene sequence conservation（142bp-237bp）In consistent amplified production.Illustrate, every group of primer and probe can exclude other one
The interference of group primer and probe；Therefore, detection method of the invention can detect swine fever virus and bovine viral diarrhea simultaneously
Poison.
Beneficial effect
Fluorescence quantitative RT-RCR detection is carried out using the above-mentioned primer of the present invention, CSFV can be made a distinction with BVDV.This
Bright detection method can detect swine fever virus and bovine viral diarrhea virus simultaneously.
In the detection method of the present invention, swine fever virus and bovine viral diarrhea virus Multiplex real-time PCR are detected
Method adopts viral RNA pillar extracts kit, and extraction time is short, and whole detection process takes stopped pipe anti-less than 3 hours
Should, detection finishes directly process, it is to avoid uncaps and causes Aerosol Pollution.Process is simple, detection time is short, sensitivity is high.
Description of the drawings
Amplification figures of the Fig. 1-2 for specificity experiments；Amplification curve of " S " the type curve in Fig. 1 for CSFV, in Fig. 2
" S " type curve for BVDV amplification curve, Fig. 1, the straight line in 2 are PRRS virus, Porcine epidemic diarrhea virus, pig
The negative result of Transmissible gastroenteritis virus and negative control；
Amplification figures of the Fig. 3-4 for repeated experiment；" S " type curve in Fig. 3 is that the amplification of swine fever virus difference strain is bent
Line, " S " the type curve in Fig. 4 are the amplification curve of bovine viral diarrhea virus difference strain, and Fig. 3, the straight line in 4 are feminine gender
The testing result of control；
Amplification figures of the Fig. 5-6 for sensitivity experiment；Amplification of " S " the type curve in Fig. 5 for the swine fever virus of variable concentrations
Curve, wherein, four curves from top to bottom are corresponding in turn to dilution 10-1、10-2、10-3、10-4 The amplification of the CSFV after times is bent
Line, dilution 10-5、10-6Linearly, the testing result of negative control is straight line to the amplification curve of the CSFV after times；" S " in Fig. 6
Amplification curve of the type curve for the BVDV of variable concentrations, wherein, 5 curves from top to bottom are corresponding in turn to dilution 10-1 、10-2、
10-3、10-4 、10-5The amplification curve of the BVDV after times, dilution 10-6The amplification curve of the CSFV after times is linear, negative control
Testing result be straight line；As can be seen here, 10 are diluted-5、10-6The testing result of the CSFV after times is negative, dilution 10-6After times
The testing result of CSFV be negative.
Specific embodiment
In this specific embodiment, involved swine fever virus（Classical swine fever virus）, it is recorded in
In January, 2015 in July, 2016 is obtained from isolation identification in the pig of morbidity；Shandong Province's animal epidemic prevention and control are stored in now
Center processed.
Involved bovine viral diarrhea virus (Bovine viral diarrhea virus), are recorded in 2013 1
The moon in July, 2014 is obtained from isolation identification in the pig of morbidity；Shandong Animal Disease Prevention and Control Center is stored in now.
Involved PRRS virus（Porcine reproductive and respiratory syndrome
virus ）, it is recorded in what in January, 2015 in July, 2016 was obtained from isolation identification in the pig of morbidity；Now it is stored in Shandong Province to move
Thing Epidemic disease prevention and control centre.
Involved Porcine epidemic diarrhea virus（Porcine epidemic diarrhea virus）It is recorded in 2015
January in July, 2016 is obtained from isolation identification in the pig of morbidity；In being now stored in Shandong Province's animal epidemic prevention and controlling
The heart.
Involved transmissible gastro-enteritis viruss（Transmissible gastroenteritis virus）It is recorded in
In January, 2015 in July, 2016 is obtained from isolation identification in the pig of morbidity；Shandong Province's animal epidemic prevention and control are stored in now
Center processed.
Design primer and probe
For expanding the primer and probe of swine fever virus distinguished sequence：
Forward primer CSFV-F：ATACATAAAGCCCGGCCCTG, as shown in SEQ ID NO.1；
Downstream primer CSFV-R：CTTGCCATCACTACCCGTGA, as shown in SEQ ID NO.2；
CSFV probe P：FAM-CCAGGACTACATGGGCCCAGTCTATCAC-BHQ1, as shown in SEQ ID NO.3；
For expanding the primer and probe of bovine viral diarrhea virus distinguished sequence：
Forward primer BVDV-F：AGCAACAGTGGTGAGTTCGT, as shown in SEQ ID NO.4；
Downstream primer BVDV-R：CGTGGCATCTCGAGACCTTT, as shown in SEQ ID NO.5；
BVDV probe P：HEX VIC-ATGGCTTAAGCCCTGAGTACA-BHQ1, as shown in SEQ ID NO.6.
Specific test
1.2.1
CSFV and BVDV original venom is chosen, 200ul is respectively taken in 1.5ml centrifuge tubes.Had using the gloomy health biotechnology exploitation in Beijing
The viral RNA pillar extracts kit of limit company, extracts the nucleic acid of virus liquid respectively with reference to description.
3 plants of the PRRS virus that simultaneously laboratory is preserved, 3 plants of Porcine epidemic diarrhea virus, transmissible gastroenteritis of swine
3 plants of virus, totally 9 strain virus liquid.The each 200ul of this 9 strain virus liquid is taken in 1.5ml centrifuge tubes, using the gloomy health biotechnology in Beijing
The test kit that the viral RNA pillar of development corporation, Ltd. is extracted, extracts the nucleic acid of 9 parts of virus liquids with reference to description.
By above-mentioned 3 plants of PRRS virus, 3 plants of Porcine epidemic diarrhea virus, 3 plants of transmissible gastro-enteritis viruss nucleic acid
And the nucleic acid of swine fever virus and bovine viral diarrhea virus carries out following detections：
The reactant liquor 15ul for preparing is taken, the above-mentioned nucleic acid of 10ul is separately added into, is finally made into the reaction system of 25ul.Reaction formula of liquid
As shown in table 1：
Table 1
| Composition | Amount（uL） |
| The CSFV-F of 10 μm of ol/L | 0.5 |
| The CSFV-R of 10 μm of ol/L | 1 |
| The CSFV probe P of 10 μm of ol/L | 0.5 |
| The BVDV-F of 10 μm of ol/L | 0.5 |
| The BVDV-R of 10 μm of ol/L | 1 |
| The BVDV probe P of 10 μm of ol/L | 0.5 |
| DEPC H2O | 2.75 |
| 10×Buffer | 1.25 |
| 5×Buffer | 2.5 |
| MgCl2（20mmol/L） | 1.5 |
| dNTP（25mmol/L） | 2 |
| Enzymatic mixture | 1 |
Machine in reaction system is carried out into fluorescence quantitative RT-RCR reaction, result is then read.The response parameter of quantitative fluorescent PCR is such as
Lower setting：
First stage：42 DEG C/30min of reverse transcription；
Second stage：94 DEG C/3min of denaturation；
Phase III：92 DEG C/15sec, 45 DEG C/30sec, 72 DEG C/60sec, totally 5 circulations；
Fourth stage：92 DEG C/10sec, 56 DEG C/60sec, totally 40 circulations；
Last 40 DEG C of 10sec；
Fluorescence is collected in fourth stage 56 DEG C of extensions of annealing of circulation every time.
As a result read：For multichannel PCR instrument, FAM is utilized respectively（465-510）Passage and HEX VIC（533-580）It is logical
Read result in road；If using ABI instrument, the corresponding passage of FAM fluoresceins and the corresponding passage unstressed configuration of HEX fluoresceins are selected respectively
Quenching group, reads result.
The present invention uses multichannel PCR instrument（Including ABI instrument）：In FAM（465-510）There is one " S " in passage
Type amplification curve, as shown in Figure 1；Because the probe of detection CSFV is fluorescein-labeled with FAM, so using FAM（465-510）It is logical
Amplification curve of the fluorescence signal that road is received for the amplified production of flag F AM fluorescein probe, is the amplification curve of CSFV.
HEX\VIC（533-580）There is " S " type amplification curve in passage, as shown in Figure 2；Because the probe of inspection BVDV is with HEX fluorescence
Plain labelling, so using HEX（533-580）The fluorescence signal of channel reception is the amplified production of labelling HEX fluorescein probes
Amplification curve, is the amplification curve of BVDV.Amplified production in reaction system is sequenced；Wherein, CSFV probe P are marked with
Amplified production, its sequence is：atac ataaagcccg gccctgtcta ctaccaggac
tacatgggcccagtctatca cagagctcct ttagagttct ttgatgaggc ccagttctgc gaggtgacta
Agagaatagg cagggtcacg ggtagtgatg gcaag, as shown in SEQ ID NO.7, the sequence is uniquely and swine fever virus
Gene order conserved region（533bp-671bp）Unanimously；The amplified production of BVDV probe P is marked with, its sequence is：agcaacagtg
gtgagttcgt tggatggctt aagccctgag tacagggtag tcgtcagtgg ttcgacgcct tggaataaag
Gtctcgagat gccacg, as shown in SEQ ID NO.8, the sequence uniquely with BVDV gene orders conserved region（142bp-
237bp）Unanimously.Illustrate, primer CSFV-F, CSFV-R and CSFV probe P energy specific amplification CSFV gene orders conserved region, figure
Change curve of 1 " S " the type curve for the amount of CSFV gene orders conserved region amplified production.Primer BVDV-F, BVDV-R and
BVDV probes P energy specific amplification BVDV gene orders conserved region, " S " the type curve of Fig. 2 is that BVDV gene orders conserved region is expanded
The change curve of the amount of volume increase thing.And primer CSFV-F, CSFV-R and CSFV probe P, primer BVDV-F, BVDV-R and BVDV are visited
PRRS virus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis viruss will not be expanded by pin P.Swine fever virus
Can not detect with bovine viral diarrhea virus Multiplex real-time PCR degree is obscured than larger PRRS virus, pig
Epidemic diarrhea virus and transmissible gastro-enteritis viruss, with good specificity.
In addition, CSFV probe P are only occurred in and swine fever virus gene order conserved region（533bp-671bp）Consistent amplification
In product, BVDV probe P are only occurred in and bovine viral diarrhea virus virus gene sequence conserved region（142bp-237bp）Unanimously
Amplified production in, illustrate, every group of primer and probe can exclude the interference of another set primer and probe.
Replica test
Identified 10 plants of swine fever virus strain is chosen, 3 plants of identified pig source bovine viral diarrhea virus strain is chosen.Take 10
The each 200ul of strain CSFV virus liquids is in 10 different 1.5ml centrifuge tubes；Take 3 plants of BVDV（2 plants of BVDV-1 types, BVDV-2 types 1
Strain）200ul is in 1.5ml centrifuge tubes；The examination extracted using the viral RNA pillar of Beijing Sen Kang biotechnology development corporation, Ltd.
Agent box, extracts the nucleic acid of 13 parts of virus liquids respectively with reference to description.By the nucleic acid of 10 plants of swine fever virus and 3 plants of bovine viral diarrhoeas
It is divided into 30 parts after the nucleic acid mixing of virus, eventually forms the nucleic acid of 30 parts of two-strain liquid mixing；The mixing of two-strain liquid
The template that nucleic acid is reacted as fluorescence quantitative RT-RCR.Using 15ul reactant liquors, 10ul templates are common for fluorescence quantitative RT-RCR reaction
The system of 25ul；Reaction formula of liquid is as shown in table 1.
The reactant liquor 15ul for preparing is taken, the nucleic acid 10ul of the two-strain liquid for mixing is added, is finally made into the anti-of 25ul
System is answered, negative control is concurrently set（Reactant liquor 15ul+DEPC water 10ul）, fluorescent quantitation RT- is carried out using multichannel PCR instrument
PCR reacts, and then reads result.The response parameter of fluorescence quantitative RT-RCR is arranged as follows：
First stage：42 DEG C/30min of reverse transcription；
Second stage：94 DEG C/3min of denaturation；
Phase III：92 DEG C/15sec, 45 DEG C/30sec, 72 DEG C/60sec, totally 5 circulations,
Fourth stage：92 DEG C/10sec, 56 DEG C/60sec, totally 40 circulations；
Last 40 DEG C of 10sec；
Fluorescence is collected in fourth stage 56 DEG C of extensions of annealing of circulation every time.
As a result read：Using FAM（465-510）Passage reads result, as shown in Figure 3；Using HEX VIC（533-580）
Result is read, as shown in Figure 4.From the figure 3, it may be seen that 10 plants of different swine fever virus can detect the positive, as a result in controllable model
Within enclosing；It can be seen that, this Multiplex real-time PCR is more stable for the detection of swine fever virus.As shown in Figure 4：3 parts of cattle diseases
Viral diarrhea virus can detect the positive, as a result within the scope of controllable, it is seen that this Multiplex real-time PCR for
The detection of bovine viral diarrhea virus is more stable.
Sensitivity test
Swine fever virus and the pig source bovine viral diarrhea virus strain for choosing identified purification is each 1 plant, carries using viral RNA pillar
Take test kit operating process and extract nucleic acid, all-wave length microplate reader (SpectraMax i3x) determines OD260, OD260/280 meters
Calculation nucleic acid concentration is 40.748ng/ul (CSFV), 39.803ng/ul（BVDV）, do 10-1, 10-2, 10-3, 10-4 ,10-5 , 10-6
Decade doubling dilution.The nucleic acid of diluted concentration identical virus liquid is respectively taken after 10ul and is sufficiently mixed, eventually form 6 parts it is different
The nucleic acid of dilution two-strain liquid mixing, the mixing nucleic acid of different dilution two-strain liquid as fluorescent quantitation
The template of RT-PCR reactions.Fluorescence quantitative RT-RCR reaction is using 15ul reactant liquors, 10ul templates, the system of common 25ul；Reaction
Formula of liquid is as shown in table 1.
The reactant liquor 15ul for preparing is taken, the nucleic acid 10ul of the three kinds of antibacterials for mixing is added, is finally made into the reaction of 25ul
System；Concurrently set negative control（Reactant liquor 15ul+DEPC water 10ul）, quantitative fluorescent PCR is carried out using multichannel PCR instrument anti-
Should, then read result.The response parameter of quantitative fluorescent PCR is arranged as follows：
First stage：42 DEG C/30min of reverse transcription；
Second stage：94 DEG C/3min of denaturation；
Phase III：92 DEG C/15sec, 45 DEG C/30sec, 72 DEG C/60sec, totally 5 circulations；
Fourth stage：92 DEG C/10sec, 56 DEG C/60sec, totally 40 circulations；
Last 40 DEG C of 10sec；
Fluorescence is collected in fourth stage 56 DEG C of extensions of annealing of circulation every time.
As a result read：Using FAM（465-510）Passage reads result, as shown in Figure 5；Fig. 5 shows, the detection pole of CSFV
Limit is 40.748pg/ul.Using HEX VIC（533-580）Result is read, as shown in Figure 6；Fig. 6 shows, the detectable limit of BVDV
It is 3.9803pg/ul.With the existing CSFV that cannot be distinguished by compared with the fluorescence quantitative RT-PCR detecting agent of BVDV, the present invention's is double
The susceptiveness of weight fluorescence RT-PCR test kit is not reduced.
<110>Shandong Animal Disease Prevention and Control Center
<120>Swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detection method
<160>8
<210>1
<211>20
<212>DNA
<213>Synthetic
<400>1
ATACATAAAG CCCGGCCCTG 20
<210>2
<211>20
<212>DNA
<213>Artificial sequence
<400>2
CTTGCCATCA CTACCCGTGA 20
<210>3
<211>28
<212>DNA
<213>Artificial sequence
<400>3
CCAGGACTAC ATGGGCCCAG TCTATCAC 28
<210>4
<211>20
<212>DNA
<213>Artificial sequence
<400>4
AGCAACAGTG GTGAGTTCGT 20
<210>5
<211>20
<212>DNA
<213>Artificial sequence
<400>5
CGTGGCATCT CGAGACCTTT 20
<210>6
<211>21
<212>DNA
<213>Artificial sequence
<400>6
ATGGCTTAAG CCCTGAGTAC A 21
<210>7
<211>139
<212>DNA
<213>Artificial sequence
<400>7
atacataaag cccggccctg tctactacca ggactacatg ggcccagtct atcacagagc 60
tcctttagag ttctttgatg aggcccagtt ctgcgaggtg actaagagaa taggcagggt 120
cacgggtagt gatggcaag 139
<210>8
<211>96
<212>DNA
<213>Artificial sequence
<400>8
agcaacagtg gtgagttcgt tggatggctt aagccctgag tacagggtag tcgtcagtgg 60
ttcgacgcct tggaataaag gtctcgagat gccacg 96
Claims (6)
1. one group of fluorescence quantitative RT-RCR detection primer and probe, it is characterised in that by first group of primer and probe or/and
Two groups of primers and probe composition；
First group of primer and probe are：
CSFV-F：ATACATAAAGCCCGGCCCTG, as shown in SEQ ID NO.1；
CSFV-R：CTTGCCATCACTACCCGTGA, as shown in SEQ ID NO.2；
CSFV probe P：FAM-CCAGGACTACATGGGCCCAGTCTATCAC-BHQ1, as shown in SEQ ID NO.3；
Second group of primer and probe are：
BVDV-F：AGCAACAGTGGTGAGTTCGT, as shown in SEQ ID NO.4；
BVDV-R：CGTGGCATCTCGAGACCTTT, as shown in SEQ ID NO.5；
BVDV probe P：HEX VIC-ATGGCTTAAGCCCTGAGTACA-BHQ1, as shown in SEQ ID NO.6.
2. primer according to claim 1 and probe, it is characterised in that drawn by first group of primer and probe and second group
Thing and probe composition.
3. a kind of swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detectable, it is characterised in that contain
Primer and probe described in claim 2.
4. detectable according to claim 3, it is characterised in that consisting of：
DEPC H2O： 2.75ul
5×Buffer： 2.5ul
10×Buffer： 1.25ul
2.5mM dNTP： 2ul
CSFV-F: 0.5ul
CSFV-R: 1ul
CSFV probe P： 0.5ul
BVDV-F: 0.5ul
BVDV-R: 1ul
BVDV probe P： 0.5ul
20uM MgCl2： 1.5ul
Enzymatic mixture： 1ul；
The concentration of CSFV-F, CSFV-R, CSFV probe P, BVDV-F, BVDV-R, BVDV probe P is 10 μm of ol/L.
5. a kind of swine fever virus, bovine viral diarrhea virus Multiplex real-time PCR detection method, it is characterised in that adopt
Detectable described in claim 3 or 4；
The nucleic acid 10ul of testing sample and 15ul detectable are made into into the reaction system of 25ul, fluorescent quantitation RT- is then carried out
PCR reacts, and reads result；
The fluorescence quantitative RT-RCR reaction：
First stage：42 DEG C/30min of reverse transcription；
Second stage：94 DEG C/3min of denaturation；
Phase III：92 DEG C/15sec, 45 DEG C/30sec, 72 DEG C/60sec, totally 5 circulations；
Fourth stage：92 DEG C/10sec, 56 DEG C/60sec, totally 40 circulations；
Last 40 DEG C/10sec；
Fluorescence is collected in fourth stage 56 DEG C of extensions of annealing of circulation every time；
The reading result：
If serpentine amplification curve occur in FAM sense channels, and Ct values are less than or equal to 30, then contain CSFV in sample；
If HEX VIC sense channels there is serpentine amplification curve, and Ct values are less than or equal to 30, then contain BVDV in sample.
6. detection method according to claim 5, it is characterised in that
Primer and probe for expanding swine fever virus distinguished sequence be：Primer CSFV-F, CSFV-R and CSFV probe P；
Primer and probe for expanding bovine viral diarrhea virus distinguished sequence be：BVDV-F, primer BVDV-R and BVDV
Probe P.
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