CN109777892A - For detecting real-time fluorescence quantitative PCR-HRM primer of porcine circovirus types 1 and 2 - Google Patents

Abstract

The present invention relates to real-time fluorescence quantitative PCR-HRM primers for detecting porcine circovirus types 1 and 2, belong to epizootiology field.Difference of the primer according to PCV1 and PCV2 genome in the front end sequence area at 5 '-ends of 5 '-end regions and replication-associated protein (Rep) designs, the difference section is that relative to PCV2, in the region, there are sequential nucleotide deletions due to PCV1 genome sequence, causing PCV1 and PCV2 in the region, there are melting temperature (Tm) peak value differences, after real time fluorescence quantifying PCR method amplification by foundation, it may appear that different Tm peak values carries out the differentiation of PCV1 and PCV2.The primer that the present invention designs only needs one group of primer that can carry out antidiastole to PCV1 and PCV2, at the same can also in the clear swinery of specificity PCV1 and PCV2 gradient of infection.Application method of the present invention is simple and effective, low in cost, and accuracy is high.

Description

For detecting real-time fluorescence quantitative PCR-HRM primer of porcine circovirus types 1 and 2

Technical field

The present invention relates to real-time fluorescence quantitative PCR-HRM primers for detecting porcine circovirus types 1 and 2, belong to dynamic Object pestology field.

Background technique

High-resolution melting curve (High Resolution Melt, HRM) is a kind of simple, quick, low cost Detection technique after PCR amplification can be used for high-throughput mutation scanning and Genotyping.The technology is only merely to try in standard PCR Stranded DNA binding dye is added on the basis of agent to carry out, and be not necessarily to sequence-specific probes, directly transported after PCR Row high-resolution melting curve, can be completed the analysis to sample genotype.When use SYBR Green I is fluorescent dye When, a step melting curve response procedures are usually run after real-time fluorescent PCR amplification.I.e. by the temperature of PCR product by Low to high to be stepped up, during heating, double-stranded DNA unwinding is at single strand dna, and originally the fluorescence in conjunction with double-strand contaminated Expect that molecule is just detached from DNA, and no longer generates fluorescence signal.Therefore, as the temperature rises, the fluorescence signal of sample is by turning by force It is weak, to form the melting curve figure that a fluorescence signal increases with temperature and weakened, then with fluorescent value variation with temperature rate For ordinate mapping, the peak shape figure that change in fluorescence rate varies with temperature just is formed, each sample is rendered as one on the figure Peak-shaped curve.We are just referred to as the melting temperature Tm of the sample to specific temperature corresponding to this peak value, and at such a temperature 50% Double-strand unwinding become single-stranded.

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 PCV-1 strain is greater than 99%, PCV-2 Core sequence homology between strain is greater than 96%.ORF1 is the maximum reading frame of PCV1 and PCV2, is located at virus genomic 5 ' End, encodes the replication protein (Rep) of virus, and molecular size range 37KDa is related with the duplication transcription of virus.

Currently, the real time fluorescence quantifying PCR method about PCV1 and PCV2 antidiastole, such as real-time fluorescence quantitative PCR side Method (dye method and sonde method) etc. is reported in succession.The above-mentioned antidiastole about PCV1 and PCV2 is both needed to for PCV1 and PCV2 points The primer (or probe sequence) for not designing specificity is assembled into PCV1 and PCV2 real time fluorescent quantitative after condition optimizing PCR differential diagnostic method.Real time fluorescence quantifying PCR method based on dye method generally requires 4 primers, and is visited based on TaqMan The real time fluorescence quantifying PCR method of the skill of handling needles is also required to 4 primers and 2 probe sequences, and the above method is bothersome laborious, and condition is excellent The relatively complicated complexity of change process, and there are potential cross jammings to influence result judgement for the multiplex PCR of multiple primers.The present invention Creating point is the genome signature design primer based on PCV1 and PCV2, it is thus only necessary to which one group of (two) primer establishes specificity Real-time fluorescence quantitative PCR-HRM method, can specificity antidiastole is carried out to PCV1 and PCV2.Foundation of the invention can Fill up domestic and international related fields blank.

Summary of the invention

The present invention is provided to detect real-time fluorescence quantitative PCR-HRM primer of porcine circovirus types 1 and 2, it is only necessary to One group of (two) primer, establish specificity real-time fluorescence quantitative PCR-HRM method, can specificity to PCV1 and PCV2 into Row antidiastole.Foundation of the invention can fill up domestic and international related fields blank.

To achieve the above object, using following technical scheme:

For detecting real-time fluorescence quantitative PCR-HRM primer of porcine circovirus types 1 and 2, its nucleotides sequence of the primer sets Column are as follows:

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

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

For detecting the real-time fluorescence quantitative PCR-HRM method of porcine circovirus types 1 and 2 comprising the steps of:

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

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

(3), different using melting temperature (Tm) value difference caused by high-resolution melting curve method, to porcine circovirus types 1 and 2 (PCV1 and PCV2) carries out antidiastole.

Application of the PCR-HRM primer on preparation antidiastole porcine circovirus types 1 and 2 kit.

The present invention has the advantages that

The present invention is based on the genome signature design primers of PCV1 and PCV2, it is thus only necessary to which one group of (two) primer is established special Property real-time fluorescence quantitative PCR-HRM method, can specificity antidiastole is carried out to PCV1 and PCV2.It is of the present invention to draw Object foundation of the invention can fill up domestic and international related fields blank.Primer specificity provided by the present invention is strong, high sensitivity, Identification method is simple, and efficiency and accuracy rate are higher.

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.

The design of primers region of Fig. 3 real time fluorescence quantifying PCR method.

Fig. 4 melting curve analysis.

The analysis of Fig. 5 amplification curve.

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), pig parvoviral (PPV), classical porcine pseudorabies virus (PRV), variant porcine Pseudorabies virus (V-PRV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine Epidemic Diarrhea Malicious (PEDV) is separated identification by Fujian Province Academy Of Agricultural Sciences Animal Husbandry And Veterinary Medicine Institute and is saved.

3 type of pig circular ring virus (PCV3), the thin circovirus virus of pig (PTTV) single positive nucleic acid are raiseeed by Fujian Academy of Agricultural Sciences It herds veterinary institute identification and saves.

2, design of primers and synthesis

2.1 PCV1 and PCV2 genome sequences compare

With PCV1 representative strains (PK plants, the GenBank number of logging in DG650650；BJ-1 plants, the GenBank number of logging in FJ475129) and 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 difference at the 5 '-ends of white (Rep), but exist before and after difference section The conserved region of PCV1 and PCV2 specificity (see box in figure).

2.2 design of primers

It is analyzed based on what PCV1 and PCV2 genome sequence compared as a result, in conserved region --- the front end of PCV1 and PCV2 specificity Upstream primer P1(Fig. 3 of conserved regions design specificity), in conserved region --- the downstream primer of rear end conserved regions design specificity P2(Fig. 3), which can simultaneously expand PCV1 and PCV2,

Wherein primer P1 and P2 sequence are as follows:

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

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

3, real-time fluorescence quantitative PCR expands

The genomic DNA of PCV1 and PCV2 is extracted in conventional manner.With designed specific real-time fluorescence quantitative PCR primer P1 Real-time fluorescence quantitative PCR amplification is carried out with P2.

20 μ L optimal reaction systems of optimization are system: 10 μ L of SYBR Premix Ex TaqTM, up/down trip are drawn Each 0.5 μ L of object (5 μm of ol/L), 1 μ L of template, water complement to 20 μ L.Optimum reaction condition are as follows: 95 DEG C of 1min initial denaturations； 95 DEG C of 10 s, 58 DEG C of 10 s, 72 DEG C of 10 s, totally 40 circulations, after circulation terminates, make melting curve.

4, real-time fluorescence quantitative PCR melting curve

After reaction, according to the melting curve made, according to PCV1 and PCV2, there are specific nucleotides for real-time fluorescence quantitative PCR Acid missing leads to the G/C content difference of amplification region, and G/C content difference will lead to the melting curve peak value Tm value for specificity occur and deposit PCV1 and PCV2 infection is judged in difference, as a result are as follows:

It can be seen that from the melting curve (Fig. 4) of generation, if occurring single specificity in Tm=Tm=(87.73 ± 0.15) DEG C Peak is judged to the PCV1 positive；If single specific peak occur in Tm=(88.27 ± 0.15) DEG C is judged to the PCV2 positive.

5, specificity experiments

To optimize postcondition and temperature to swinery common transmittable disease, such as pig parvoviral (PPV), classical porcine pseudorabies virus (PRV), variant porcine Pseudorabies virus (V-PRV), porcine reproductive and respiratory syndrome virus (PRRSV), Porcine Epidemic Diarrhea Malicious (PEDV), 3 type of pig circular ring virus (PCV3), the thin circovirus virus of pig (PTTV) are expanded, and melting curve and expansion are observed after amplification Increase curve, judges the specificity of this research.

From melting curve (Fig. 4) analyze as it can be seen that only PCV1 and PCV2 may occur in which specificity melting curve peak value, and other (PPV, PRV, V-PRV, PRRSV, PEDV, PCV3 and PTTV are showed no the melting curve peak value of specificity to swinery common transmittable disease (see the experiment contrast of Fig. 4).

It is analyzed from amplification curve (Fig. 5) as it can be seen that only PCV1 and PCV2 may occur in which the amplification curve of specificity, and other swinerys (PPV, PRV, V-PRV, PRRSV, PEDV, PCV3 and PTTV are showed no the amplification curve of specificity (see Fig. 5's to common transmittable disease Experiment contrast).

From melting curve and amplification curve as it can be seen that primer specificity of the invention is strong.

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 real-time fluorescence quantitative PCR inspection using the method after optimization It surveys, and the method for foundation and the virus purification carried out using PK15 cell experiment is compared.As a result as it can be seen that real-time fluorescence is fixed Amount PCR method detects that PCV1 is 2 parts positive, positive rate 10.53%；It is separated to 1 plant of virus using PK-15, positive rate is 5.26%, and the strain pathological material of disease it is optimized after method carry out real time fluorescence quantifying PCR method test positive.Real-time fluorescence Quantifying PCR method detects that PCV2 is 15 parts positive, positive rate 78.95%；It is separated to 11 plants of virus using PK-15, positive rate is 57.89%, and 11 strains pathological material of disease it is optimized after method carry out real time fluorescence quantifying PCR method detection be the positive.It is above-mentioned The result shows that the method that this research is established is higher than the sensitivity of classical way, and the pathological material of disease for going out virus through virus purification is corresponding The coincidence rate of testing result be 100%, show the method that the present invention establishes, can be used for the clinical PCV1 and PCV2 that carries out and identify Diagnosis 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>for detecting real-time fluorescence quantitative PCR-HRM primer of porcine circovirus types 1 and 2

<130> 2

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<400> 1

ttcggcagcg gcagcacctc 20

<210> 2

<211> 23

<212> DNA

<213>artificial sequence

<400> 2

aataatcaaa aagggagatt gga 23

Claims (3)

1. real-time fluorescence quantitative PCR-HRM primer for detecting porcine circovirus types 1 and 2, it is characterised in that: the primer Its nucleotide sequence of group is as follows:

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

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

2. the side real-time fluorescence quantitative PCR-HRM that primer as described in claim 1 is used to detect porcine circovirus types 1 and 2 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, real-time fluorescence quantitative PCR amplification is carried out with primer P1 and P2 described in claim 1；

(3), different using melting temperature (Tm) value difference caused by high-resolution melting curve method, to porcine circovirus types 1 and 2 Carry out antidiastole.

3. PCR-HRM primer as described in claim 1 is on preparation antidiastole porcine circovirus types 1 and 2 kit Using.