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Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain

A technology for porcine pseudorabies and detection reagents, which is applied in the field of animal disease inspection and detection, can solve the problems of low sensitivity, long time consumption, disadvantage, etc., and achieves the effects of good repeatability, simple operation and improved positive rate.

Inactive Publication Date: 2015-04-29
HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the existing PRV detection methods based on ELISA, PCR and virus isolation generally suffer from low sensitivity and long time consumption, which are very unfavorable for the development of the prevention and control of the disease

Method used

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  • Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain
  • Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain
  • Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Differentiate the design of specific primers and probes of porcine pseudorabies virus vaccine strain gD gene and wild strain gE gene

[0040] When designing primers and probes in the present invention, attention should be paid to avoiding the mutation point of the sequence. After repeated screening and verification, and a large number of comparisons of the PRV gD gene and PRV gE gene sequences in GenBank, the PRV gD gene and PRV gE gene were selected to be highly conserved and have Using the type-specific gene sequence as a template, the gD / gE-specific primer pair and TaqMan MGB probe were designed and named as PRV-F(P1), PRV-R(P2), PRV-F(P3), PRV-R( P4), PRV-MGB-FAM-probe (Probe-P5), PRV-MGB-VIC-probe (Probe-P6), synthesized by Bao Biological Engineering (Dalian) Co., Ltd., used for gD / gE double TaqMan The primer and probe sequences for MGB FQ-PCR amplification are as follows:

[0041] Primer P1: GGTTCAACGAGGGCCAGTA (SEQ ID NO.1)

[0042] Primer P2: ATGA...

Embodiment 2

[0047] Example 2 Establishment of a double fluorescent PCR detection method for distinguishing the gD gene of the porcine pseudorabies virus vaccine strain and the gE gene of the wild strain

[0048] 1. Preparation of template RNA: total RNA was extracted by Trizol method. The specific operation is as follows: take 200 μL of each sample to be tested in a 1.5ml centrifuge tube, then add 600 μL of Trizol and shake in a vortex for 2-3 minutes, add 200 μL of chloroform, after centrifugation, take the supernatant and transfer it to another 1.5ml centrifuge tube , add 200 μL isopropanol to precipitate, wash the precipitate with 75% ethanol by mass percentage, dry it, and finally dissolve the precipitate with 20 μL DEPC (diethylpyrophosphate) water, take 10 μL for reverse transcription, and store the rest at -20 °C.

[0049] 2. Preparation of template DNA: take PRV porcine PK-15 cell culture as a positive control, and normal porcine PK-15 cells as a negative control, and extract tota...

Embodiment 3

[0059]Example 3 Characteristic Evaluation of Double Fluorescent PCR Detection Method for Distinguishing Porcine Pseudorabies Virus Vaccine Strain gD Gene and Wild Strain gE Gene

[0060] 1. Establishment of sensitivity test and standard curve

[0061] The pGEM-T / gD and pGEM-T / gE recombinant plasmids obtained in Example 2 were serially diluted 10 times respectively, and the two plasmids were mixed in a 1:1 ratio, and the final concentration of each plasmid was adjusted to 1.0×10 7 ~1.0×10 0 copy / μL, a total of 8 dilutions, the double FQ-PCR sensitivity test of gD / gE gene was carried out according to the FQ-PCR reaction conditions optimized in Example 2. Take the logarithm of the initial template number of pGEM-T / gD and pGEM-T / gE recombinant plasmids as the X-axis, and the FQ-PCR cycle times C t Regression curve was made on the Y axis to establish a standard curve for the double FQ-PCR method.

[0062] The results showed that the minimum detection limit of gD and gE detected ...

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Abstract

The invention provides a detection reagent and a method for identifying a porcine pseudorabies virus vaccine strains and a wild strain, and belongs to the field of animal pathogen detection. The detection reagent comprises two primer pairs as shown in SEQ ID NO.1-4 and probes as shown in SEQ ID NO.5-6. The invention further provides a method for identifying a porcine pseudorabies virus vaccine strain gD gene and a wild strain gE gene by using the detection reagent. The porcine pseudorabies virus vaccine strains and the wild strain can be simultaneously identified through dual fluorogenic quantitative PCR, high-flux detection on large-scale samples can be achieved, the detection reagent has the characteristics of rapidness, specificity, sensitivity, accuracy, simplicity and convenience, provides an effective tool for surveying porcine pseudorabies infection sources and tracing infection environments and disease sources, and has relatively good application value in prevention and control on porcine pseudorabies.

Description

technical field [0001] The invention relates to the field of animal disease inspection and detection, in particular to double fluorescent quantitative PCR primers and probes for distinguishing porcine pseudorabies virus vaccine strains and wild strains, and the invention also relates to using the primers and probes to detect porcine pseudorabies A method and a detection reagent for detecting the gD gene of a virus vaccine strain and the gE gene of a wild virus strain. Background technique [0002] Porcine pseudorabies is an acute infectious disease caused by porcine pseudorabies virus (PRV), with fever, itching and encephalomyelitis as the main clinical symptoms, which can cause abortion, stillbirth and fetal mummification in pregnant sows To newborn piglets, it causes neurological symptoms, ataxia, paralysis, exhaustion and death, and the mortality rate is 100%; adult pigs are often recessively infected, and adult pigs that are resistant to recessive infection are the main ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/705C12Q2600/16C12Q2531/113C12Q2537/143C12Q2561/101
Inventor 闫若潜吴志明曹伟伟赵雪丽刘梅芬靳冬方先珍王淑娟张盼盼闫志玲刘先敏王英华宋宏晓赵胜杰于增源赵协
Owner HENAN CENT FOR ANIMAL DISEASE CONTROL & PREVENTION
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