CN106086238A - The PCR detection method of porcine circovirus 2 type, the primer and detection kit - Google Patents

Abstract

The invention provides the PCR detection method of a kind of porcine circovirus 2 type, the primer and detection kit, described PCR detection method is simple to operate, technology requires low, is suitable for Clinical detection.Described PCR detection method, comprises the following steps: (1) carries out PCR amplification with the DNA of extraction in sample for template, and wherein the primer sequence of PCR amplification is: upstream 5 TGGGATGATCTACTGAGAC 3；Downstream 5 ATTTCATATGGAAATTCAG 3；(2) amplified production is carried out electrophoresis；(3) analyze, result of determination, there is 268bp amplified band in sample, and negative control without corresponding amplified band time, result be the positive.

Description

PCR detection method, primers and detection kit for porcine circovirus type 2

technical field

The invention relates to a PCR detection method for porcine circovirus type 2, used primers and a detection kit.

Background technique

Porcine circovirus (Porcine circovirus, PCV) is the smallest animal virus that has been discovered. PCV has two genotypes, type 1 (PCV1) and type 2 (PCV2), among which PCV1 is not pathogenic, while PCV2 is one of the main pathogens causing multisystemic wasting syndrome (PMWS) in weaned piglets. PMWS was first discovered in Canada in 1997. The main manifestations of PMWS are growth retardation, pale skin, dyspnea and death. In my country, Lang Hongwu tested 559 pig serum samples collected from 7 provinces of Beijing, Hebei, Shandong, Tianjin, Jiangxi, Jilin and Henan by ELISA in 2000, and found that the positive rate was 42.9%, and the antibody was positive The rate increases with pig age. Then, from November 2002 to August 2002, Wang Zhongtian used the PCR method to detect PCV2 in 55 tissue samples from 12 large-scale pig farms infected with PCV2 in Beijing, Tianjin, Guangdong and other places, and found that In 12 pig farms, 11 diseased pig herds mainly manifested as multisystemic wasting syndrome in weaned piglets, and 1 had dermatitis and nephrotic syndrome. It can be seen that the disease is prevalent in some large-scale pig farms in my country.

PCV2 mainly damages the immune organs of infected pigs, causing the decline and disorder of the body's immune function. It often cooperates with porcine parvovirus, porcine pneumonia virus, porcine reproductive and respiratory syndrome virus and other microorganisms to exert its pathogenicity. The diseases caused by PCV2 mainly include weaned piglet multisystem failure syndrome, porcine dermatitis and nephrotic syndrome, reproductive and respiratory syndrome, exudative dermatitis, etc. PCV2 often plays a major role in the whole pathogenesis process.

Since there are no effective prevention and control measures for the disease, and the disease often exists in the form of subclinical infection, it is particularly important to do a good job in the diagnosis and monitoring of the disease.

Contents of the invention

purpose of invention

The object of the invention is to provide a PCR detection method for porcine circovirus type 2, which is simple in operation, low in technical requirements and suitable for clinical detection.

Another object of the present invention is to provide primers for the PCR detection method of porcine circovirus type 2.

Another object of the present invention is to provide a detection kit corresponding to the above detection method.

Summary of the invention

According to a first aspect of the present invention, a kind of PCR detection method of porcine circovirus type 2 is provided, comprising the following steps:

(1) Use the DNA extracted from the sample as a template for PCR amplification, where the primer sequence for PCR amplification is:

Upstream 5-TGGGATGATCTACTGAGAC-3;

downstream 5-ATTTCATATGGAAATTCAG-3;

(2) Electrophoresis of the amplified product;

(3) Analyzing and judging the results, if a 268bp amplified band appears in the sample and there is no corresponding amplified band in the negative control, the result is positive.

Preferably, the conditions and procedures for PCR amplification are: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, gradient annealing at 43-56°C for 30 seconds, extension at 72°C for 30 seconds, a total of 36 cycles, and finally extension at 72°C for 6 minutes. Further preferably, the conditions and procedures of PCR amplification are: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 49°C for 30 seconds, extension at 72°C for 30 seconds, a total of 36 cycles, and finally extension at 72°C for 6 minutes.

Preferably, the PCR reaction system is:

PCR buffer;

_The final concentration of MgCl 2.5mmol/L;

dNTP final concentration 0.2mmol/L;

TaqDNA polymerase 1 unit;

The final concentration of both upstream and downstream primers is 0.5 μmol/l;

Template 2.0 μl;

Add ddH ₂ O (double distilled water) to a final volume of 50 μl.

Wherein, the final concentration of TaqDNA polymerase is a conventional parameter in the field, and the final concentration of the template can be determined by those skilled in the art through experiments, with the goal of obtaining an effective PCR amplification reaction. The effective PCR amplification reaction means that when the DNA template contains enough target DNA sequence to carry out the PCR amplification reaction, the amount of the amplified product is enough to be detected, and the result is positive.

The negative control can be selected by those skilled in the art. Preferably, the PCR detection method uses porcine pseudorabies virus as a negative control.

According to the second aspect of the present invention, primers for the PCR detection method of porcine circovirus type 2 are also provided, and its sequence is:

Upstream 5-TGGGATGATCTACTGAGAC-3;

Downstream 5-ATTTCATATGGAAATTCAG-3.

According to a third aspect of the present invention, there is also provided a PCR detection kit for porcine circovirus type 2, said kit comprising:

PCR buffer;

Primer pair, the sequence is:

Upstream 5-TGGGATGATCTACTGAGAC-3,

downstream 5-ATTTCATATGGAAATTCAG-3;

_MgCl2 ;

dNTP;

Taq DNA polymerase;

ddH ₂ O;

negative control;

DNA Ladder.

Preferably, the negative control is porcine pseudorabies virus.

The PCR detection method of porcine circovirus type 2 described in the present invention is simple to operate, and technical requirement is lower, is convenient for clinical detection or epidemiological investigation, and 67 pieces of suspected PMWS disease materials in Taizhou area detected, wherein 42 pieces show as positive , the positive rate can reach 62.6%.

Description of drawings

Fig. 1 is the PCR amplification electrophoresis figure of embodiment 1, wherein, M: 100bp DNA Ladder; 1,2: PCV2 amplification product;

Figure 2 is the PCR amplification electrophoresis diagram of the sensitivity test, in which, M: 100bp DNA Ladder; 1, 2, 3, 4, 5, 6, and 7 represent dilutions of 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 ^-6 , 10 ^-7 multiples of PCV2 amplification products.

detailed description

Below in conjunction with embodiment do specific explanation.

1.1 Main reagents and instruments

Genomic DNA Rapid Extraction Kit, 100bp DNA Ladder, 6× DNA Loading Dye, and dNTP Mix are all products of Shanghai Sangon Bioengineering Co., Ltd.; TaqDNA polymerase is Fermentas product, model EP0404, the concentration is 1u/μl; 25mM MgCl ₂ and 10×Buffer are Fermentas products; PCR amplification instrument (PTC-200rev), JS-380A automatic gel image analyzer, Mikro200R refrigerated high-speed centrifuge, DYY-7C electrophoresis instrument, DYCP-31DN agarose horizontal electrophoresis tank, Thermostat, micropipette, etc.

1.2 Design and synthesis of primers

Referring to the full PCV2 gene sequence JX682407 published on GenBank and the five PCV2 sequences isolated and identified by the inventor (GenBank accession numbers: KC788750, KF039888, KF039889, KF039890, KF039891), a pair of specific primers for amplifying the PCV2 fragment was designed and amplified. The augmented fragment is located at 717-985bp, the length is 268bp, and the sequence of the primer is:

Upstream 5-TGGGATGATCTACTGAGAC-3;

Downstream 5-ATTTCATATGGAAATTCAG-3.

The primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd., and stored in a -20°C refrigerator for later use, with a concentration of 25 μmol/L.

1.3 Extraction of DNA template

Spleen, lymph nodes, lungs and other diseased tissues of pigs with typical symptoms of PMWS were collected and operated according to the instructions of the rapid genomic DNA extraction kit. The extracted DNA templates were stored at -20°C for future use.

Example 1

Porcine pseudorabies virus was used as negative control, DNA extracted in 1.3 was used as template, and the above-mentioned specific primers were used for amplification. The reaction system was: 5 μl each of 10×Buffer and 25mM MgCl ₂ , 4 μl of 2.5mmol/L dNTP, and TaqDNA polymerized 1 μl of enzyme, 1 μl of upstream and downstream primers, 2.0 μl of template, add ddH ₂ O to a final volume of 50 μl, mix well and put into PCR amplification instrument. The reaction program is: pre-denaturation at 95°C for 5 minutes; denaturation at 94°C for 30s, annealing at different temperatures in the range of 43-56°C for 30s, extension at 72°C for 30s, a total of 41 cycles, and finally extension at 72°C for 6 minutes, and the annealing temperature was found to be 49°C When the amplification effect is the best. After the reaction, the PCR products were electrophoresed on 1.5% agarose and observed with a gel imaging system. A specific band was seen at about 268bp, consistent with the expected size. The results are shown in Figure 1 (1 in Figure 1 is the result of the amplification product when the annealing temperature is 49°C, and 2 is the result of the amplification product of the negative control).

The PCR product was recovered according to the requirements of the DNA gel recovery kit, and the recovered product was sent to the biological company for sequencing. The sequencing result was compared with the PCV2 sequence in GenBank, and the result showed that the amplified band was the PCV2 sequence.

Example 2 Specificity Determination

With the DNA extracted in 1.3, the DNA of classical swine fever splenic drenching vaccine, the DNA of porcine pseudorabies virus, and the DNA of porcine Escherichia coli as templates, use the above-mentioned designed PCV2 specific primers to amplify according to the method of Example 1, electrophoresis, observation result. The results showed that the designed primers could only amplify the DNA fragment of PCV2, about 268bp, while the other amplification results showed no bands. Among them, the commercial vaccine of swine fever, spleen and drenched seedlings of Weida Beinong Technology Group Co., Ltd., the DNA of porcine pseudorabies virus and the DNA of porcine Escherichia coli were isolated, identified and preserved by the applicant according to the existing technology.

Example 3 Sensitivity Determination

Dilute the DNA extracted in 1.3 to different degrees, and perform PCR amplification using the method in Example 1. After the amplification, 10 μL of the PCR product is used for electrophoresis to test the sensitivity of the PCR method. The results are shown in Figure 2, which shows that after the DNA template is diluted from 10 ^-1 to 10 ^-7 , the optimized conditions can still amplify fragments at 10 ^-4 , indicating that the sensitivity is high.

Example 4 Validation test of PCR detection method

Adopt the method for embodiment 1, 5 PCV2 strains of inventor's separation and identification (inventor isolates the PCV2 virus strain of many strains different biological characteristics from Jiangsu central area (Taizhou city and surrounding areas) diseased pig farm in recent years, and The PK15 cell cultures that were subjected to Cap gene sequence determination (GenBank accession numbers: KC788750, KF039888, KF039889, KF039890, KF039891) and phylogenetic tree analysis were tested for PCV2, and a negative control (porcine pseudorabies virus PK15 cells culture), and calculate the coincidence rate with the test results of the virus isolation and identification method. The results showed that the two methods detected 5 strains of PCV2 and the negative control, and the negative and positive results were completely consistent, with a coincidence rate of 100%.

The application of embodiment 5 PCR detection method

Using the method of Example 1, the PCV2 detection was carried out on the suspected diseased pigs collected by the Teaching Animal Hospital of Jiangsu Agriculture and Animal Husbandry Science and Technology Vocational College in the surrounding areas of Taizhou.

Using the amplification conditions of Example 1, PCV2 was detected on 67 suspected PMWS samples collected. The results showed that 42 of them were positive, and the positive rate could reach 62.6%.

The full genome length of PCV2 is 1767bp or 1768bp. After PCV2 invades the host, it can promote the apoptosis of lymphocytes and inhibit the proliferation of lymphocytes, resulting in the loss of lymphocytes and changes in subgroups, so that the immune system of the body is low, and it cannot respond effectively to the immunogen in time, which is easy to cause Secondary infection. Therefore, it is often co-infected with porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), porcine parvovirus (PPV), mycoplasma hyopneumoniae, Haemophilus parasuis, etc., which aggravates its harm. So far, there is still a lack of effective measures for the control of PCV2 infection, and there are no better vaccines and drugs on the market to prevent and treat the disease. Therefore, establishing a fast and simple diagnostic method is of great significance to strengthen the monitoring and prevention of the disease.

PCR technology has been widely used in many fields. It has strong sensitivity, accuracy and specificity, and can quickly diagnose, making up for the time-consuming and labor-intensive shortcomings of traditional detection methods. The present invention designs a pair of specific primers according to the PCV2 sequence published in GenBank, and establishes a PCR detection method for PCV2 by optimizing its reaction conditions, which has good specificity and sensitivity, and is effective for Taizhou and surrounding areas. 67 clinical materials in the area were tested, and the positive rate was as high as 62.6%, indicating that PCV2 is harmful to the area. The establishment of this method is beneficial to the monitoring of PCV2 in large-scale pig farms, and provides a basis for early prevention of PCV2 pathogenesis.

sequence listing

<110> Jiangsu Vocational College of Agriculture and Animal Husbandry Science and Technology

<120> PCR detection method, primers and detection kit for porcine circovirus type 2

<160>2

<170>PatentIn version 3.4

<210> 1

<211>19

<212>DNA

<213> Artificial sequence

<400> 1

tgggatgatc tactgagac 19

<210> 2

<211>19

<212>DNA

<213> Artificial sequence

<400> 2

atttcatatg gaaattcag 19

Claims (8)

1. the PCR detection method of a porcine circovirus 2 type, it is characterised in that order comprises the following steps:

(1) carrying out PCR amplification with the DNA of extraction in sample for template, wherein the primer sequence of PCR amplification is:

Upstream 5-TGGGATGATCTACTGAGAC-3；

Downstream 5-ATTTCATATGGAAATTCAG-3；

(2) amplified production is carried out electrophoresis；

(3) analyze, result of determination, there is 268bp amplified band in sample, and negative control without corresponding amplified band time, result For the positive.

2. PCR detection method as claimed in claim 1, it is characterised in that described PCR detection method is with porcine pseudorabies virus As negative control.

3. PCR detection method as claimed in claim 1, it is characterised in that condition and the program of PCR amplification be: 95 DEG C of pre-changes Property 5min;94 DEG C of degeneration 30s, 43-56 DEG C of Gradient annealing 30s, 72 DEG C extend 30s, totally 36 circulations, last 72 DEG C of extensions 6min。

4. PCR detection method as claimed in claim 3, it is characterised in that condition and the program of PCR amplification be: 95 DEG C of pre-changes Property 5min;94 DEG C of degeneration 30s, 49 DEG C of annealing 30s, 72 DEG C extend 30s, totally 36 circulations, and last 72 DEG C extend 6min.

5. the PCR detection method as according to any one of claim 1-4, it is characterised in that PCR reaction system is:

PCR buffer；

MgCl₂Final concentration 2.5mmol/L；

DNTP final concentration 0.2mmol/L；

Taq DNA polymerase 1 unit；

Upstream and downstream primer final concentration is 0.5 μm ol/l；

Template 2.0 μ l；

Add ddH₂O to final volume 50 μ l.

6., for a primer for porcine circovirus 2 type PCR detection method, its sequence is:

Upstream 5-TGGGATGATCTACTGAGAC-3；

Downstream 5-ATTTCATATGGAAATTCAG-3.

7. the PCR detection kit for porcine circovirus 2 type, it is characterised in that described test kit includes:

PCR buffer；

Primer pair, sequence is:

Upstream 5-TGGGATGATCTACTGAGAC-3,

Downstream 5-ATTTCATATGGAAATTCAG-3；

MgCl₂；

dNTP；

Taq archaeal dna polymerase；

ddH₂O；

Negative control；

DNA Ladder。

8. PCR detection kit as claimed in claim 7, it is characterised in that described negative control is porcine pseudorabies virus.