CN107338331A - The multiple PCR detection primer pair and kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 - Google Patents
The multiple PCR detection primer pair and kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 Download PDFInfo
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Abstract
The present invention provides the multiple PCR detection primer group and kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3, and the primer sets include three pairs of primers.The present invention also provides PRV, porcine circovirus 2 type and the multiple PCR detection kit of the type of pig circular ring virus 3 and the application method of the kit.PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 are detected by multiplex PCR detection using primer sets of the present invention, testing result is all positive, and the testing result of other pig correlated virus is negative, the high specificity of detection.The high sensitivity of multiplex PCR detection is carried out using the primer sets of the present invention, the detection of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 can be used for simultaneously.
Description
Technical field
The invention belongs to biological technical field, is related to PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Multi-PCR detection method, and in particular to the multiplex PCR of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Detection primer pair and kit.
Background technology
Pig circular ring virus is that PCV-2 is main pathogen, and widely distributed in the whole world, to threaten the important of world's pig industry
One of cause of disease.Postweaning multisystemic wasting syndrome (PMWS), pigskin inflammation nephrotic syndrome, pig occurs after PCV-2 infection
Respiratory system mixing disease, breeding difficulty disease.2016, American scholar Phan was using high-throughout method to breaking out dermatitis nephrosis
The sow of syndrome (PDNS) is detected, and finds a kind of new PCV-II hypotype PCV-3.Also find the virus in China afterwards
Presence, and positive rate is higher, is 34.7%.PRV (PRV) can cause pig various acute infectious disease, with heating and
Encephalomyelitis is principal character, and nervous symptoms occurs in newborn piglet, and in-pig shows as miscarriage, stillborn foetus, mummy tire etc..
As aquaculture industry of China is constantly moved towards to modernize the ultra-largeization plant of aquaculture model, the discovery of new virus
And mixed infection, cause huge economic losses to pig industry.And multiplex PCR grows up on the basis of Standard PCR, no
The advantages of only retaining Standard PCR high specificity, high sensitivity, and multipair primer can be added in same system and expands multiple diseases
Former target gene, operating procedure and time can be significantly reduced by having, and save reagent, it is quick, accurate that disease can be accomplished
Diagnosis, it is adapted to the quick detection of multiple pathogens in a large amount of clinical samples, especially mixed infection sample, applied to a variety of
The detection of pathogen.Therefore, the multiplex PCR detection of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 is established
Primer pair and kit are very necessary.The present invention is according to PRV gh genes, porcine circovirus 2 type ORF2 genes and pig
The type ORF2 genes of PCV-II 3 design primer sets, available for establish efficiently, accurately and rapidly diagnostic technique in molecular biology.
The content of the invention
It is an object of the present invention to provide the PRV of a kind of hypersensitivity, specificity and accuracy, pig annulus
The multiple PCR detection primer group and kit of viral 2 types and the type of pig circular ring virus 3.
One of technical scheme is to provide a kind of PRV, porcine circovirus 2 type and pig circular ring virus 3
The multiple PCR detection primer group of type, the primer sets are made up of three pairs of primers,
Wherein, the detection primer pair of PRV, by SEQ ID NO:The forward primer of nucleotide sequence shown in 1 and
SEQ ID NO:The reverse primer composition of nucleotide sequence shown in 2；
The detection primer pair of porcine circovirus 2 type, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 3
ID NO:The reverse primer composition of nucleotide sequence shown in 4；
The detection primer pair of the type of pig circular ring virus 3, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 5
ID NO:The reverse primer composition of nucleotide sequence shown in 6.
In an embodiment of the present invention, the annealing temperature of the primer sets is 52 DEG C.
Primer sets provided by the invention be used for expand PRV gh genes, porcine circovirus 2 type ORF2 genes and
The Partial Fragment of the type ORF2 genes of pig circular ring virus 3, the target gene clip size of amplification be respectively 356bp, 248bp and
408bp。
The two of technical scheme are to provide a kind of PRV, porcine circovirus 2 type and pig circular ring virus
The multiple PCR detection kit of 3 types, include the multiplex PCR of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Detection primer group, the primer sets are made up of three pairs of primers,
Wherein, the detection primer pair of PRV, by SEQ ID NO:The forward primer of nucleotide sequence shown in 1 and
SEQ ID NO:The reverse primer composition of nucleotide sequence shown in 2；
The detection primer pair of porcine circovirus 2 type, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 3
ID NO:The reverse primer composition of nucleotide sequence shown in 4；
The detection primer pair of the type of pig circular ring virus 3, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 5
ID NO:The reverse primer composition of nucleotide sequence shown in 6.
In an embodiment of the present invention, the SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:Nucleotides shown in 5
The concentration of the forward primer of sequence is 10 μm of ol/L, the SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:Shown in 6
The concentration of the reverse primer of nucleotide sequence is 10 μm of ol/L.
As the technical scheme of optimization, kit provided by the invention also includes TaKaRa Ex Taq, 10 × Ex Taq
Buffer、dNTP Mixture、ddH2O, negative quality-control product and positive quality control product.
Preferably, described negative quality-control product is ddH2O；The positive quality control product is the positive matter of PRV
The positive plasmid of grain, the positive plasmid of porcine circovirus 2 type and the type of pig circular ring virus 3.
The application method of kit provided by the invention comprises the following steps：Using the DNA of testing sample as template, with
Such as the SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward primer of nucleotide sequence shown in 5 and the SEQ ID
NO:2、SEQ ID NO:4、SEQ ID NO:In the presence of the reverse primer of nucleotide sequence shown in 6, detected by multiplex PCR,
PRV, the presence situation of porcine circovirus 2 type and the type of pig circular ring virus 3 and mixed infection are judged according to testing result
Situation.
In an embodiment of the present invention, the SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:Nucleotides shown in 5
The concentration of the forward primer of sequence is 10 μm of ol/L, and the SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6 institutes
The concentration for showing the reverse primer of nucleotide sequence is 10 μm of ol/L.
In embodiments of the present invention, the PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 is multiple
The reaction system of PCR detections is 25 μ L, and the reaction system includes：
(1) 10 μm of ol/L SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward direction of nucleotide sequence shown in 5
Each 1 μ L of primer, and 10 μm of ol/L SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:Nucleotide sequence shown in 6 it is reverse
Each 1 μ L of primer；
(2) the μ L of DNA profiling 2.5 of testing sample；
(3)TaKaRa Ex Taq 0.3μL；
(4)10×Ex Taq Buffer 2.5μL；
(5)dNTP Mixture 1μL；
(6)ddH2O 12.7μL。
In embodiments of the present invention, the PRV, porcine circovirus 2 type and the type multiplex PCR of pig circular ring virus 3
The amplification program of detection is：
(1) 94 DEG C of pre-degeneration 5min；
(2) 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 35s, carry out 36 circulations；
(3) 72 DEG C of extension 7min.
The beneficial effects of the invention are as follows：PRV, porcine circovirus 2 type and pig circular ring virus provided by the invention
The multiple PCR detection primer group and kit of 3 types, the primer sets include three pairs of primers (including SEQ ID NO:1、SEQ ID
NO:3、SEQ ID NO:The forward primer of nucleotide sequence shown in 5, and SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:
The reverse primer of nucleotide sequence shown in 6).The primer sets are used to expand PRV gh genes, porcine circovirus 2 type
The Partial Fragment of ORF2 genes and the type ORF2 genes of pig circular ring virus 3, the target gene clip size of amplification be respectively 356bp,
248bp and 408bp.The present invention also provides the multiplex PCR inspection of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
The application method of test agent box and the kit.Detected using primer sets of the present invention by multiplex PCR to pseudorabies
Virus, porcine circovirus 2 type and the type of pig circular ring virus 3 are detected, and testing result is the positive, and other pig correlated virus are the moon
Property, the high specificity of detection.The high sensitivity of multiplex PCR detection is carried out using the primer sets of the present invention, reaches 1.0 × 103Copy
Shellfish/μ L.The detection of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 can be used for simultaneously.
Brief description of the drawings
Fig. 1 is the multiplexed PCR amplification result electrophoresis of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Figure；Wherein, 1：PRV substance pcr amplification products；2：PCV-2 substance pcr amplification products；3：PCV-3 substance pcr amplification products；4：
PRV, PCV-2 and PCV-3 multiplexed PCR amplification product；5：Negative control；M：DNA Marker DL 2000；
Fig. 2 is the multiplex PCR specific test result of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Electrophoretogram；Wherein, 1：PRV substance pcr amplification products；2：PCV-2 substance pcr amplification products；3：PCV-3 substances PCR amplification productions
Thing；4：PRV, PCV-2 and PCV-3 multiplexed PCR amplification product；5-9 is followed successively by from left to right：PDCoV、PEDV、TGEV、
PRRSV, CSFV amplified production；10：Negative control；M：DNA Marker DL 2000；
Fig. 3 is the multiplex PCR sensitivity tests result of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Electrophoretogram；Wherein, 1-8 is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0×105、1.0×104、1.0
×103、1.0×102、1.0×101The pcr amplification product of copy/μ L detectable concentrations；9：Negative control；M：DNA Marker DL
2000；
Fig. 4 is the multiplex PCR replica test result of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Electrophoretogram；Wherein A, B and C represent first week, second week and the PCR amplification electrophoretograms of the 3rd week respectively；1：PRV substances
Pcr amplification product；2：PCV-2 substance pcr amplification products；3：PCV-3 substance pcr amplification products；4：PRV, PCV-2 and PCV-3
Multiplexed PCR amplification product；M：DNA Marker DL 2000.
Embodiment
It is to preferably further understand the present invention to provide following embodiments, it is not limited to the optimal embodiment party
Formula, most preferred embodiment are not construed as limiting to present disclosure and protection domain, and anyone is under the enlightenment of the present invention or incites somebody to action
Method and production as the present invention and any and present invention that the features of other prior arts are combined and drawn are same or like
Product, it is within the scope of the present invention.
Experiment material source involved in the present invention is as follows, and other unaccounted materials are common commercially available product, can be led to
Market purchase is crossed to obtain：
1st, bacterial strain and plasmid
Escherichia coli Top10 competent cells are purchased from Beijing Tiangeng biochemical technology Co., Ltd.
PCV-3 (type of pig circular ring virus 3), PCV-2 (porcine circovirus 2 type), PRV (PRV), CSFV (swine fevers
Virus), PRRSV (porcine reproductive and respiratory syndrome virus), PEDV (Porcine epidemic diarrhea virus), PDCoV (the coronal diseases of pig fourth type
Poison) and TGEV (transmissible gastro-enteritis virus) separated from each pig farm clinical sample in Jiangxi and obtain and protected by this laboratory
Deposit.
2nd, reagent
Yeast extract, tryptone, ampicillin are purchased from Beijing Suo Laibao Science and Technology Ltd；
Viral RNA/DNA extraction kit (centrifugation column type), TaKaRa Ex Taq, 10 × Ex Taq Buffer, dNTP
Mixture, DL2000DNA marker, pMD18-T carriers purchase Takara (Dalian) company；
The small extraction reagent kit of plasmid is purchased from Beijing Tiangeng biochemical technology Co., Ltd；
Glue reclaim kit is purchased from OMEGA companies.
3rd, equipment
SorvallST16R high speed freezing centrifuges, Legend Micro 21R high speed freezing centrifuges and Nano
Drop2000 is purchased from Thermo companies of the U.S.；
Applied Biosystems 7500PCR instrument is purchased from Applied biosystems；
Agarose horizontal electrophoresis tank is purchased from the bio tech ltd of Beijing 61；
Gel imaging system is purchased from Shanghai Peiqing Science Co., Ltd.
Embodiment 1：PRV, porcine circovirus 2 type and the type multi-PCR detection method of pig circular ring virus 3 are established
1st, design of primers and synthesis
According to the gh conservative regions, PCV-2 of PRV (KU057086) the HB1201 strains logged in GenBank
(KX814348) ORF2 of the ORF2 conservative regions of AY4844 strains and PCV-3 (KX966193) PCV3-US/SD2016 strains is protected
Defending zone domain, work bioengineering skill is given birth to by Shanghai to specific primer, primer using the Software for Design three of Primer Premier 5.0
Art service company synthesizes, and (wherein, " F " represents forward primer to the sequence of the primer sets of gained, and " R ", which is represented, reversely to be drawn as shown in table 1
Thing)：
Table 1：Primer information
2nd, viral DNA extracts
According to the MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0 Viral extractions of TaKaRa companies
The operational manual of kit extracts the RNA/DNA of PRV, PCV-2 and PCV-3 virus respectively, and operating procedure is as follows：
(1) after mill ground sample, centrifuging and taking supernatant；
(2) supernatant, 200 μ L Buffer VGB, 20 μ L Proteinase Ks and 1 μ L Carrier RNA are mixed after taking 200 μ L centrifugations
It is even, in 56 DEG C of water-bath 10min；
(3) liquid after cracking adds 200 μ L absolute ethyl alcohols and mixed, and is transferred to Spin Columns, adds step by step after centrifugation
Enter Buffer RWA/RWB, centrifuge.
(4) 30 μ L are added without RNAase ddH after centrifuging2O dissolving DNAs/RNA, -80 DEG C save backup.
3rd, PCR is expanded
PRV genomic DNA, PCV-2 genomic DNA, the PCV-3 genomic DNA of above onestep extraction respectively, with
And PRV, PCV-2 and PCV-3 the mixed genomic DNA (are 1 by PRV, PCV-2 and PCV-3 volume ratio:1:1 prepares) it is mould
Plate, expanded respectively by following reaction system and response procedures：
The reaction system of PCR detections is 25 μ L, including：
(1) 10 μm of ol/L SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward direction of nucleotide sequence shown in 5
Each 1 μ L of primer, and 10 μm of ol/L SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:Nucleotide sequence shown in 6 it is reverse
Each 1 μ L of primer；
(2) the μ L of DNA profiling 2.5 of testing sample；
(3)TaKaRa Ex Taq 0.3μL；
(4)10×Ex Taq Buffer 2.5μL；
(5)dNTP Mixture 1μL；
(6)ddH2O 12.7μL。
In embodiments of the present invention, the type of pig circular ring virus 3, porcine circovirus 2 type and the PRV multiplex PCR
The amplification program of detection is：
(1) 94 DEG C of pre-degeneration 5min；
(2) 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 35s, carry out 36 circulations；
(3) 72 DEG C re-extend 7min.
The agarose gel electrophoresis of PCR primer 2% observes electrophoresis result, and electrophoresis result is shown in Fig. 1, in figure, 1：PRV substances PCR
Amplified production；2：PCV-2 substance pcr amplification products；3：PCV-3 substance pcr amplification products；4：PRV, PCV-2 and PCV-3's is more
Weight pcr amplification product；5：Negative control；M：DNA Marker DL 2000.
As a result illustrate：It will be seen from figure 1 that using PRV, PCV-2 and PCV-3 the mixed genomic DNA as the multiple of template
The target gene clip size of pcr amplification product is respectively 248bp, 356bp and 408bp, with PRV, pig circular ring virus 2
Malicious 2 types are consistent with the length of the type purpose fragment of pig circular ring virus 3.
Embodiment 2：The multi-PCR detection method of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 is excellent
Change
Using 50 DEG C, 52 DEG C, 54 DEG C, 56 DEG C of thermograde optimizes to reaction, by optimizing reaction condition, confirms
Following reaction system and amplification program are optimum condition：
Optimum response program：25 μ L reaction system, including：
(1) 10 μm of ol/L SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward direction of nucleotide sequence shown in 5
Each 1 μ L of primer, and 10 μm of ol/L SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:Nucleotide sequence shown in 6 it is reverse
Each 1 μ L of primer；
(2) the μ L of DNA profiling 2.5 of testing sample；
(3)TaKaRa Ex Taq 0.3μL；
(4)10×Ex Taq Buffer 2.5μL；
(5)dNTP Mixture 1μL；
(6)ddH2O 12.7μL。
Optimal amplification program is：
(1) 94 DEG C of pre-degeneration 5min；
(2) 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 35s, carry out 36 circulations；
(3) 72 DEG C re-extend 7min.
Embodiment 3：The multiple PCR detection kit of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Prepare
The multiplex PCR detection of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 provided by the invention tries
Agent box, including：TaKaRa Ex Taq、10×Ex Taq Buffer、dNTP Mixture、ddH2O, primer sets are (including such as SEQ
ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward primer of nucleotide sequence shown in 5, and SEQ ID NO:2、SEQ ID
NO:4、SEQ ID NO:The reverse primer of nucleotide sequence shown in 6), negative quality-control product (ddH2O), positive quality control product (preparation
Standard plasmid).
Reagent used is mainly purchased from precious bioengineering (Dalian) Co., Ltd (Takara) in this kit preparation process,
Remaining is purchased from TIANGEN Biotech (Beijing) Co., Ltd., Beijing Chemical Plant.The preparation process of the kit is as follows：
1st, prepared by PCR reaction solutions
(1) design of primers and synthesis
The PRV gh gene orders, PCV-2 ORF2 gene orders and the PCV-3 that have been delivered according to GenBank ORF2 bases
Because of sequence, carry out sequence alignment with the softwares of MEGA 6.0 and find out conserved sequence.According to conserved sequence Primer Premier
5.0 Software for Design multiple PCR detection primer groups, the primer sets include three pairs of primers.Primer is limited by Shanghai life work bioengineering
Company synthesizes, and obtains SEQ ID NO:The primer sets of nucleotide sequence shown in 1-6.
(2) dissolving of primer
Synthetic primer sets are taken (to there is such as SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:Nucleotides shown in 5
The forward primer of sequence, and SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:The reverse primer of nucleotide sequence shown in 6)
Dry powder pipe, 12,000rpm centrifugation 1min, ddH is added according to primer dissolving specification2O dissolves dry powder, makes above-mentioned 3 pairs of primers,
The concentration of totally 6 kinds of primers is respectively 10 μM, and vibration mixes centrifugation, standby；
(3) PCR reaction solutions are prepared
PCR reaction solutions are prepared in following ratios：10 μm of ol/L SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:5
Each 1 μ L of forward primer of shown nucleotide sequence, and 10 μm of ol/L SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:6
Each μ L of 1 μ L, TaKaRa Ex Taq 0.3 of reverse primer of shown nucleotide sequence, μ L of 10 × Ex Taq Buffer 2.5,
dNTP Mixture 1μL。
2nd, prepared by negative quality-control product
The effect of negative quality-control product be for prevent because of pollution and caused by false positive.The present invention uses ddH2O is as cloudy
Property quality-control product, is monitored to whole experiment process.
3rd, prepared by positive quality control product
The effect of positive quality control product is that whether monitoring reagent fails and whether performance declines.The present invention uses positive restructuring matter
Grain is used as positive quality control product.According to molecule clone technology conventional in the art, the structure of positive recombinant plasmid and extraction
Method is as follows：
First with SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward primer of nucleotide sequence shown in 5,
With SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:Genome of the reverse primer of nucleotide sequence shown in 6 to extraction
DNA enters performing PCR amplification；Then PCR primer is reclaimed as purpose fragment using omega Gel Extraction Kit；Using big
The even pMD18-T Cloning Vector kits of precious biology, above-mentioned purpose fragment is inserted in pMD18-T carriers, structure weight
Group plasmid, by converting and screening, obtain the positive colony bacterium containing the recombinant plasmid inserted with above-mentioned purpose fragment；The positive gram
Grand bacterium extracts the plasmid in culture bacterium solution with Tiangeng plasmid extraction kit and uses ultraviolet specrophotometer after expanding and cultivating
Quantified.
4th, quality control standard：
Negative quality-control product：It is negative；
Positive quality control product：It is positive.
Conditions above should meet that otherwise, this time experiment is considered as invalid, and total Test should re-start simultaneously.
5th, analysis judges：
PCR primer is identified through 2% agarose gel electrophoresis, the band for having size to be consistent, is then the positive, two or three
Meet the electrophoretic band of size, be then judged as mixed infection, no band then illustrates no PRV, pig circular ring virus
The infection of 2 types and the type of pig circular ring virus 3.
Embodiment 4：The multi-PCR detection method of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 is special
Specific assay
The multiplex PCR of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 established using the present invention is examined
Survey method is respectively with PRV, PCV-2, PCV-3, PDCoV, PEDV, TGEV, PRRSV, CSFV genomic DNA and PRV, PCV-
2 and PCV-3 the mixed genomic DNA is that template enters performing PCR amplification, as a result sees Fig. 2；In figure, 1：PRV substance pcr amplification products；
2：PCV-2 substance pcr amplification products；3：PCV-3 substance pcr amplification products；4：PRV, PCV-2 and PCV-3 multiplexed PCR amplification
Product；5-9 is followed successively by from left to right：PDCoV, PEDV, TGEV, PRRSV, CSFV amplified production, 10：Negative control；M：DNA
Marker DL 2000。
As a result illustrate：PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 provided using the application
Multi-PCR detection method is feminine gender to the testing result of PDCoV, PEDV, TGEV, PRRSV, CSFV virus, and only pig puppet is mad
The PCR testing results of dog disease poison, porcine circovirus 2 type and the type of pig circular ring virus 3 are positive, and show the multi-PCR detection method
Specificity it is good.
Embodiment 5：The multi-PCR detection method of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 is quick
Perception experiment
(there is such as SEQ ID NO first with primer sets:1、SEQ ID NO:3、SEQ ID NO:Nucleotides sequence shown in 5
The forward primer of row, and SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:The reverse primer of nucleotide sequence shown in 6) it is right
PRV, PCV-2 and PCV-3 of extraction the mixed genomic DNA are that template enters performing PCR amplification；Then omega Gel are used
Extraction Kit reclaim PCR primer as purpose fragment；Using the pMD18-T Cloning Vector of the precious biology in Dalian
Kit, above-mentioned purpose fragment is inserted in pMD18-T carriers, construction recombination plasmid, by converting and screening, obtained containing insertion
There is the positive colony bacterium of the recombinant plasmid of above-mentioned purpose fragment；The positive colony bacterium is carried after expanding and cultivating with Tiangeng plasmid
The plasmid in kit extraction culture bacterium solution is taken, extracting method is：It is correct that 200 μ L PCR are accredited as positive and sequencing result
Bacterium solution is inoculated in LB fluid nutrient mediums of the 20mL added with ampicillin, after cultivating 8h, 12000rpm centrifugation 2min, is abandoned
Clearly；Add 2mL PBS to be resuspended, 12000rpm centrifugation 2min, abandon after supernatant with reference to small extraction reagent kit specification (Beijing day of plasmid
Root) extraction plasmid, the concentration of the recombinant plasmid extracted after extraction by the measure of ultramicrospectrophotometer Nanodrop 2000,
Plasmid concentration is converted into unit volume molecule copy number further according to recombinant plasmid molecular mass and Avgadro constant, and will
Recombinant plasmid is with 1.0 × 108Copy/μ L are 10 times of gradient dilutions of progress after original concentration, respectively with 1.0 × 108、1.0×107、
1.0×106、1.0×105、1.0×104、1.0×103、1.0×102、1.0×101Copy/μ L are that template carries out multiplex PCR expansion
Increase to examine PRV provided by the invention, porcine circovirus 2 type and the multiplex PCR detection side of the type of pig circular ring virus 3
The sensitivity of method, testing result are shown in Fig. 3, and in figure, 1-8 is followed successively by from left to right：1.0×108、1.0×107、1.0×106、1.0
×105、1.0×104、1.0×103、1.0×102、1.0×101The pcr amplification product of copy/μ L detectable concentrations；9：It is negative right
According to；M：DNA Marker DL 2000.
As a result show：PRV provided by the invention, porcine circovirus 2 type and the type of pig circular ring virus 3 it is multiple
The detection limit of PCR detection method is 1.0 × 103Copy/μ L, show that established PCR method sensitiveness is good.
Embodiment 6：The multi-PCR detection method weight of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
Renaturation is tested
Examined using the multiplex PCR of PRV of the present invention, porcine circovirus 2 type and the type of pig circular ring virus 3
Survey method, respectively with the PRV of extraction genomic DNA, PCV-2 genomic DNA, PCV-3 genomic DNA, and PRV,
PCV-2 and PCV-3 the mixed genomic DNA (volume ratio 1:1:1) it is template, amplification three times (interval one week) is carried out, with true
Determine the repeatability of testing result, as a result see Fig. 4, wherein A, B and C represent first week, second week and the amplifications of the PCR of the 3rd week respectively
As a result electrophoretogram；1：PRV substance pcr amplification products；2：PCV-2 substance pcr amplification products；3：PCV-3 substances PCR amplification productions
Thing；4：PRV, PCV-2 and PCV-3 multiplexed PCR amplification product；M：DNA Marker DL 2000.
As a result illustrate：Utilize PRV of the present invention, porcine circovirus 2 type and the type of pig circular ring virus 3
Multi-PCR detection method is measured in different time points, and measurement result is consistent, shows that method repeatability of the present invention is good
It is good.
Embodiment 7：The multi-PCR detection method pair of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
The detection of clinical sample
Examined using the multiplex PCR of PRV of the present invention, porcine circovirus 2 type and the type of pig circular ring virus 3
Survey method detects to picking up from 32, Jiangxi, 256 parts of pig farm sample (lung, lymph node, spleen), to verify the practicality of this method
Property.As a result PRV has 104 parts for the positive, and PCV-2 has 212 parts for the positive, and PCV-3 has 2 parts of detection samples to be positive.
As a result show：PRV of the present invention, porcine circovirus 2 type and the type of pig circular ring virus 3 it is multiple
PCR detection method has feasibility in clinical practice.
SEQUENCE LISTING
<110>Agricultural University Of Jiangxi
<120>A kind of multiple PCR detection primer pair of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3
And kit
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gcgtgtactg cgactgcgtg t 21
<210> 2
<211> 23
<212> DNA
<213>Artificial sequence
<400> 2
cgacctggcg tttattaacc gag 23
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
atggtatggc gggaggagta 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
gcggtggaca tgatgagatt 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
tggtgccgta gaaatctgtc 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
gcctaaacga atgggaaact 20
Claims (10)
1. the multiple PCR detection primer group of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3, its feature
It is, the primer sets are made up of three pairs of primers,
Wherein, the detection primer pair of PRV, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 1
ID NO:The reverse primer composition of nucleotide sequence shown in 2；
The detection primer pair of porcine circovirus 2 type, by SEQ ID NO:The forward primer and SEQ ID of nucleotide sequence shown in 3
NO:The reverse primer composition of nucleotide sequence shown in 4；
The detection primer pair of the type of pig circular ring virus 3, by SEQ ID NO:The forward primer and SEQ ID of nucleotide sequence shown in 5
NO:The reverse primer composition of nucleotide sequence shown in 6.
2. multiple PCR detection primer group according to claim 1, it is characterised in that the annealing temperature of the primer sets is
52℃。
3. multiple PCR detection primer group according to claim 1, it is characterised in that the primer sets are used to expand pig puppet
The Partial Fragment of rabies viruses gh genes, porcine circovirus 2 type ORF2 genes and the type ORF2 genes of pig circular ring virus 3, amplification
Target gene clip size is respectively 356bp, 248bp and 408bp.
4. the multiple PCR detection kit of a kind of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3, its feature
It is, includes the multiple PCR detection primer group of PRV, porcine circovirus 2 type and the type of pig circular ring virus 3, it is described to draw
Thing group is made up of three pairs of primers,
Wherein, the detection primer pair of PRV, by SEQ ID NO:The forward primer and SEQ of nucleotide sequence shown in 1
ID NO:The reverse primer composition of nucleotide sequence shown in 2；
The detection primer pair of porcine circovirus 2 type, by SEQ ID NO:The forward primer and SEQ ID of nucleotide sequence shown in 3
NO:The reverse primer composition of nucleotide sequence shown in 4；
The detection primer pair of the type of pig circular ring virus 3, by SEQ ID NO:The forward primer and SEQ ID of nucleotide sequence shown in 5
NO:The reverse primer composition of nucleotide sequence shown in 6.
5. kit according to claim 4, it is characterised in that the SEQ ID NO:1、SEQ ID NO:3 and SEQ
ID NO:The concentration of the forward primer of nucleotide sequence shown in 5 is 10 μm of ol/L, the SEQ ID NO:2、SEQ ID NO:4
With SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 6 is 10 μm of ol/L.
6. kit according to claim 4, it is characterised in that also including TaKaRa Ex Taq, 10 × Ex Taq
Buffer、dNTP Mixture、ddH2O, negative quality-control product and positive quality control product；Preferably, described negative quality-control product is
ddH2O；The positive quality control product is positive plasmid, the positive plasmid and pig annulus of porcine circovirus 2 type of PRV
The positive plasmid of viral 3 types.
7. the application method of the kit according to claim any one of 4-6, it is characterised in that comprise the following steps：With
The DNA of testing sample is template, in the SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:Nucleotide sequence shown in 5
Forward primer, and SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:The presence of the reverse primer of nucleotide sequence shown in 6
Under, detected by multiplex PCR, PRV, porcine circovirus 2 type and the type of pig circular ring virus 3 are judged according to testing result
Presence situation and mixed infection situation.
8. application method according to claim 7, it is characterised in that the SEQ ID NO:1、SEQ ID NO:3、SEQ
ID NO:The concentration of the forward primer of nucleotide sequence shown in 5 is 10 μm of ol/L, and SEQ ID NO:2、SEQ ID NO:4、
SEQ ID NO:The concentration of the reverse primer of nucleotide sequence shown in 6 is 10 μm of ol/L.
9. application method according to claim 7, it is characterised in that the PRV, porcine circovirus 2 type and
The reaction system of the multiplex PCR detection of the type of pig circular ring virus 3 is 25 μ L, and the reaction system includes：
(1) 10 μm of ol/L SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:The forward primer of nucleotide sequence shown in 5
Each 1 μ L, and 10 μm of ol/L SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:The reverse primer of nucleotide sequence shown in 6
Each 1 μ L；
(2) the μ L of DNA profiling 2.5 of testing sample；
(3)TaKaRa Ex Taq 0.3μL；
(4)10×Ex Taq Buffer 2.5μL；
(5)dNTP Mixture 1μL；
(6)ddH2O 12.7μL。
10. application method according to claim 7, the PRV, porcine circovirus 2 type and pig circular ring virus
3 types multiplex PCR detection amplification program be：
(1) 94 DEG C of pre-degeneration 5min；
(2) 94 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 35s, carry out 36 circulations；
(3) 72 DEG C of extension 7min.
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| CN107828916A (en) * | 2017-11-30 | 2018-03-23 | 山东新希望六和集团有限公司 | For detecting primer, PCR kit and the application of the type of pig circular ring virus 3 |
| CN107955839A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of 3 type of porcine circovirus 2 type and circovirus |
| CN108265126A (en) * | 2018-01-12 | 2018-07-10 | 温州大学 | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method |
| CN108456747A (en) * | 2018-05-14 | 2018-08-28 | 湖北省农业科学院畜牧兽医研究所 | A kind of multiple PCR detection kit differentiating pig circular ring virus |
| CN108531656A (en) * | 2018-05-03 | 2018-09-14 | 广西壮族自治区兽医研究所 | A kind of the duplex PCR detection primer and kit of quick differentiation porcine circovirus 2 type and 3 types |
| CN108950085A (en) * | 2018-08-27 | 2018-12-07 | 河南省农业科学院畜牧兽医研究所 | It is a kind of for detecting the primer sets and kit of 3 type of pig circular ring virus and porcine pseudorabies virus street strain |
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| CN107955839A (en) * | 2017-12-13 | 2018-04-24 | 华南农业大学 | For detecting double PCR primer, detection method and the kit of 3 type of porcine circovirus 2 type and circovirus |
| CN108265126A (en) * | 2018-01-12 | 2018-07-10 | 温州大学 | The foundation of 3 type of pig circular ring virus and porcine circovirus 2 type duplex PCR detection method |
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| CN108950085A (en) * | 2018-08-27 | 2018-12-07 | 河南省农业科学院畜牧兽医研究所 | It is a kind of for detecting the primer sets and kit of 3 type of pig circular ring virus and porcine pseudorabies virus street strain |
| KR102208001B1 (en) * | 2020-01-31 | 2021-01-27 | 대한민국 | Composition for simultaneous detection of porcine circovirus type 2 and type 3 and use thereof |
| CN112553372A (en) * | 2020-11-09 | 2021-03-26 | 浙江省动物疫病预防控制中心 | Porcine pseudorabies virus and porcine circovirus type 3 dual-fluorescence quantitative PCR detection primer, probe, kit and method |
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| CN113373119A (en) * | 2021-06-02 | 2021-09-10 | 江西农业大学 | Three-gene deletion recombinant pseudorabies virus strain for expressing African swine fever virus, construction method and application thereof |
| WO2024066083A1 (en) * | 2022-09-29 | 2024-04-04 | 福州奥吉芯生物科技有限公司 | Constant-temperature detection kit and detection method for detecting pathogens |
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Application publication date: 20171110 |