CN109750125A - One group of primer and kit for quickly identifying PCV1 and PCV2 - Google Patents

Abstract

The present invention relates to one group for detecting the primer of 1 type of pig circular ring virus and porcine circovirus 2 type (porcine circovirus type 1 and type 2, PCV1 and PCV2), belong to epizootiology field.The primer is according to PCV1 and PCV2 genome difference area come design primer.The difference section is located at virus genomic 5 ' end, and in the region, PCV1 ratio PCV2 is compared, and there are the missings of 13 nucleotide, design the specific primer across the missing area, amplifiable to PCV1 and PCV2, but there are length differences to be judged.The present invention only needs one group of primer that can carry out antidiastole to PCV1 and PCV2.

Description

One group of primer and kit for quickly identifying PCV1 and PCV2

Technical field

The invention belongs to epizootiology fields, and in particular to one group of primer and reagent for quickly identifying PCV1 and PCV2 Box.

Background technique

Pig circular ring virus (Porcine circovirus, PCV) is one of the smallest animal virus found so far, when Preceding pig circular ring virus has 3 kinds: PCV1, PCV2 and PCV3.PCV1 is the virus of non-pathogenic；PCV2 is pathogenic virus； The presence or absence of PCV3 pathogenicity needs well further deeply clear.The study found that PCV2 is that postweaning multisystemic failure is comprehensive It levies (Postweaning Multisystemic Wasting Syndrome, PMWS), dermatitis and nephrotic syndrome (PNDS) Main pathogen.

It carries out analysis to the genome of PCV1 and PCV2 to find, 1768 bp of PCV1 full-length genome 1759bp, PCV2 overall length (or 1767 bp), the two sequence homology is less than 80%, but the nucleic acid sequence homology between PCV1 strain is greater than 99%, PCV2 poison Core sequence homology between strain is greater than 96%.ORF1 is the maximum open reading frame of PCV1 and PCV2 (ORF), is located at viral gene 5 ' ends of group, encode the replication protein (Rep) of virus, and molecular size range 37KDa is related with the duplication transcription of virus.

Currently, seen that the research for having PCV1 and PCV2 antidiastole reports that, such as multiplex PCR differential diagnostic method, seeing to have makes PCV1 and PCV2 antidiastole is carried out with 4 primers, and (Chen Juhao waits to identify the duplex PCR of porcine circovirus types 1 and 2 Method establishes China animal doctor's journal, and 2009,11:1390-1394), seeing has 3 primers to carry out PCV1 and PCV2 identification and examine Disconnected (Jiang Yan waits the PCR of pig circular ring virus serum I type and II type to identify animal medicine and is in progress, 2003,24(5): 101- 103), see that (Wang Xin waits application PCR-RFLP method to Sichuan for the identification PCV1 and the PCV2 diagnostic method that have based on PCR-RFLP The detection and parting research HEILONGJIANG ANIMAL SCIENCE AND VETERINARY MEDICINE of pig circular ring virus, 2006,6:7-8).Wherein either use 3 primers Or the equal existence condition optimization of PCV1 and PCV2 is complicated, tests the problems such as primer is more to identify for 4 primers.And PCR-RFLP method To the more demanding of enzyme, if digestion is incomplete, result is easy to cause error result occur.Method of the invention, it is only necessary to one group Primer (2), can carry out scientific differentiation to PCV1 and PCV2, and the process of method for building up facilitates fast as conventional PCR method Victory, without the complex optimization process of similar a plurality of primer, without the digestion process of PCR-RFLP, the present invention can effectively solve mesh The preceding complex cumbersome problem of PCV1 and PCV2 antidiastole.

Summary of the invention

The object of the present invention is to provide primers and kit that one group quickly identifies PCV1 and PCV2.

To achieve the above object, using following technical scheme:

One group of primer for quickly identifying pig circular ring virus 1 type and porcine circovirus 2 type, its nucleotide sequence of the primer sets are as follows It is shown:

Upstream primer P1:5 '-TCGGCAGCGGCAGCACCTC-3 ',

Downstream primer P2:5 '-TCAAAAAGGGAGATTGGAAG-3 '.

The primer is for quickly identifying the detection method of pig circular ring virus 1 type and porcine circovirus 2 type, comprising following Step:

(1) genomic DNA is extracted from doubtful clinical sample；

(2) using the DNA of extraction as template, PCR amplification is carried out with the primer P1 and P2；

(3) the purpose band size of this group of primer pair PCV1 amplification is 148bp, and the purpose band size to PCV1 amplification is 161bp。

(4) 3% conventional agarose gel electroresis appraisal, the molecular weight standard used are DL500, are based on one standard Band (for 150bp) carrys out judging result.

Application of the primer in preparation 1 type of antidiastole pig circular ring virus and porcine circovirus 2 type kit.

The present invention has the advantages that

Present invention only requires one group of primer (2), scientific differentiation can be carried out to PCV1 and PCV2, the process of method for building up and often Rule PCR method is equally convenient and efficient, without the complex optimization process of similar a plurality of primer, without the digestion of PCR-RFLP Journey, the present invention can effectively solve the problems, such as that current PCV1 and PCV2 antidiastole is complex cumbersome.

Detailed description of the invention

Fig. 1 PCV1 and PCV2 nucleotide homology compares.

The analysis of PCV1 and PCV2 the nucleotide difference area Fig. 2.

Fig. 3 agarose electrophoretic analysis, wherein 1-10 swimming lane be respectively PCV1, PCV2, PCV1 and PCV2 mixed infection, PPV, PRV, V-PRV, PRRSV, PEDV, PCV3 and PTTV.

Specific embodiment

The present invention will be further described for following example.

Embodiment 1

1, strain:

1 type of pig circular ring virus (PCV1) and porcine circovirus 2 type (PCV2), 3 type of pig circular ring virus (PCV3), pig parvoviral (PPV), classical porcine pseudorabies virus (PRV), variant porcine Pseudorabies virus (V-PRV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV) is separated identification by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute and is protected It deposits.

2, PCV1 and PCV2 sequence alignment analysis

2.1 PCV1 and PCV2 genome sequences compare

Selection PCV1 representative strains (PK plants, the GenBank number of logging in DG650650；BJ-1 plants, the GenBank number of logging in FJ475129) PCV2 representative strains (JZ-9 plants, the GenBank number of logging in MH059556；PCV-45 plants, the GenBank number of logging in KC514989) for, DNAStar biological software is selected to carry out nucleotide homology comparison, it is as a result visible: PCV1 mutual nucleotide homology In 99.7% or more, the PCV2 mutual nucleotide homology nucleosides mutual in 99.3% or more, PCV1 and PCV2 Acid homology between 77.7%-77.8% (Fig. 1), showing PCV1 and PCV2, there are the nucleotide homology of partial region is higher.

Discovery (Fig. 2), PCV1 and PCV2 are compared in the 5 '-end regions egg related to duplication of genome through nucleotide homology The front end sequence area existing characteristics nucleotide deletion area (see figure 2) at the 5 '-ends of white (Rep).

3 design of primers

Analysis based on 2 is as a result, design specific primer in each up and down of PCV1 and PCV2 missing area, which can be same When PCV1 and PCV2 are expanded, wherein primer P1 and P2 sequence are as follows:

Upstream primer P1:5 '-TCGGCAGCGGCAGCACCTC-3 '

Downstream primer P2:5 '-TCAAAAAGGGAGATTGGAAG-3 '.

The primer sets (P1 and P2) are 148bp to the amplification size of PCV1, and the amplification size to PCV2 is 161bp.

3, the foundation of PCR method

The genomic DNA of PCV1 and PCV2 is extracted in conventional manner.It is carried out with designed Specific PCR primers P1 and P2 conventional PCR amplification, and annealing temperature (50-60) DEG C is optimized.Recommend according to Quick Taq HS DyeMix amplification kit 50 μ L configure PCR reaction solution: 25 μ L of Quick Taq HS DyeMix, each 1 μ L of up/down trip primer (10 μM of each), mould 1 μ L of plate, water complement to 50 μ L.Optimum reaction condition after optimization are as follows: 94 DEG C of 2min initial denaturations；94 ℃ 20 s,56 ℃ 20 s, 72 DEG C of 10 s, totally 30 recycle, 72 DEG C of overall elongation 7min.

1, specificity experiments

To optimize postcondition to swinery common transmittable disease, such as pig parvoviral (PPV), classical porcine pseudorabies virus (PRV), variation Type porcine pseudorabies virus (V-PRV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine epidemic diarrhea virus (PEDV), pig 3 type of circovirus (PCV3), the thin circovirus virus of pig (PTTV) are expanded, and judge the specificity of this research.

5, agarose electrophoresis is identified

After PCR amplification, the agarose 3% carries out conventional agarose gel electrophoresis, as a result as it can be seen that PCV1 positive size is 148bp, it is about parallel with the molecular weight standard of 150bp (see the 1st swimming lane of Fig. 3)；PCV2 size is 161bp, positioned at 150bp's Above molecular weight standard (see the 2nd swimming lane of Fig. 3)；If occurring two band of 148bp and 161bp simultaneously, be judged as PCV1 and PCV2 mixed infection (see the 3rd swimming lane of Fig. 3).

Specificity experiments show that only PCV1 and PCV2 may occur in which the band of specificity, and other swinery common transmittables are sick (PPV, PRV, V-PRV, PRRSV, PEDV, PCV3 and PTTV are showed no the band of specificity (see the 4-10 swimming lane of Fig. 3).

6, clinical application

19 parts of pig PMWS pathological material of diseases of clinical inspection are detected using this method, after pathological material of disease conventionally milled processed, The genomic DNA that virus is extracted using nucleic acid extraction kit carries out PCR detection using the method after optimization, and by foundation Method and the virus purification carried out using PK-15 cell experiment are compared.As a result as it can be seen that PCR method detects the PCV1 positive 2 Part, positive rate 10.53%；Using PK-15 be separated to virus 1 plant, positive rate 5.26%, and the strain pathological material of disease it is optimized after Method carry out PCR method test positive.The PCR method that the present invention establishes detects that PCV2 is 15 parts positive, and positive rate is 78.95%；It is using the pathological material of disease PCR method detection that PK-15 is separated to 11 plants of virus, positive rate 57.89%, and 11 strains It is positive.Wherein PCV1 and PCV2 mixed infection, positive rate 5.26% are detected there are also 1 part.The result of pathological material of disease separation is carried out , there are two purpose bands in PCR detection, is PCV1 and PCV2 mixed infection.The above results show this research establish method and The sensitivity of classical Virus Isolation is higher, and goes out meeting for the corresponding PCR testing result of pathological material of disease of virus through virus purification Rate is 100%, shows the method that the present invention establishes, and can be used for clinical development PCV1 and PCV2 antidiastole research.

The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

SEQUENCE LISTING

<110>Fujian Academy animal and veterinary research institute

<120>one groups of primers and kit for quickly identifying PCV1 and PCV2

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213>artificial sequence

<400> 1

tcggcagcgg cagcacctc 19

<210> 2

<211> 20

<212> DNA

<213>artificial sequence

<400> 2

tcaaaaaggg agattggaag 20

Claims (3)

1. one group of primer for quickly identifying pig circular ring virus 1 type and porcine circovirus 2 type, it is characterised in that: the primer sets its Nucleotide sequence is as follows:

Upstream primer P1:5 '-TCGGCAGCGGCAGCACCTC-3 ',

Downstream primer P2:5 '-TCAAAAAGGGAGATTGGAAG-3 '.

2. primer as described in claim 1 is for quickly identifying the detection side of pig circular ring virus 1 type and porcine circovirus 2 type Method, which is characterized in that comprise the steps of:

(1) genomic DNA is extracted from doubtful clinical sample；

(2) using the DNA of extraction as template, PCR amplification is carried out with primer P1 and P2 described in claim 1；

(3) the purpose band size of this group of primer pair PCV1 amplification is 148bp, and the purpose band size to PCV2 amplification is 161bp；

(4) 3% conventional agarose gel electroresis appraisal, the molecular weight standard used are DL500, are based on one standard band (for 150bp) carrys out judging result.

3. primer as described in claim 1 is in preparation 1 type of antidiastole pig circular ring virus and porcine circovirus 2 type kit Application.