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Multiple PCR primer set and kit for simultaneous detection of porcine pseudorabies virus and porcine parvovirus

A porcine pseudorabies and parvovirus technology, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, can solve the problems of high detection cost, long diagnosis time, and low sensitivity, and achieve simple detection procedures, Low-cost, high-sensitivity effects of detection

Active Publication Date: 2016-06-08
哈药集团生物疫苗有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Virus isolation and detection have problems such as cumbersome operation methods, long diagnosis time, and uncertain results due to sample contamination; serological diagnostic methods are subject to many limitations in practical application due to the continuous emergence of mutant strains of the virus (Izumida et al., 1988; JooandChu, 1999; Klugeetal., 1999; Mengeling, 1999; PrietoandCastro, 2005; Takashimaetal., 1988); In addition, both detection methods have the disadvantage of low sensitivity
[0004] Commercialized PCR kits are mostly single-item PCR, and one kit can only detect one virus in one test. For the same pig disease material mixed with three viruses, three kinds of kits are needed to confirm the pathogen. Three PCR experiments are not only time-consuming and labor-intensive, but also costly

Method used

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  • Multiple PCR primer set and kit for simultaneous detection of porcine pseudorabies virus and porcine parvovirus
  • Multiple PCR primer set and kit for simultaneous detection of porcine pseudorabies virus and porcine parvovirus
  • Multiple PCR primer set and kit for simultaneous detection of porcine pseudorabies virus and porcine parvovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The establishment of embodiment 1 multiplex PCR method

[0041] 1. Experimental method

[0042] 1.1PCR primer design

[0043] Based on the partially known sequences of PRV (Genbank No. AF257079) and PPV (Genbank No. AY502114) published by GenBank, several pairs of primers were designed, as shown in Table 1 and Table 2. Use DNAstar to analyze the primer-dimers of the two designed viral primers to avoid the formation of stable primer-dimers between the primers, and analyze the homology or complementarity of the amplified sequences of each pair of primers to avoid them. There is a high degree of homology or complementarity between them.

[0044] Table 1 Primer design for multiplex PCR

[0045]

[0046] Table 2 Primer design for multiplex PCR

[0047]

[0048] 1.2 Establishment of PCR reaction conditions

[0049] 1.2.1 Extraction of viral nucleic acid

[0050] 1.2.1.1 Extraction of PPV and PRV DNA (CTAB-NaCl method)

[0051] 1) Take 0.5mL sample and place in a ...

experiment example 1

[0074] Specificity experiment of experimental example 1 primer set

[0075] 1. Experimental method

[0076] Detect respectively the primer pair of the multiplex PCR method of embodiment 1 (the primer pair 1 that is made up of the nucleotide sequence shown in SEQIDNo.1, SEQIDNo.2 and is made up of the nucleotide sequence shown in SEQIDNo.3, SEQIDNo.4 The specificity of the primer pair 2).

[0077] 1.1 Specificity of PPV primer pairs

[0078] Respectively with PPV and PRV genomic DNA, respectively with the cDNA of PRRSV, TGEV, PEDV, CSFV and BVDV as template, with PPV primer pair (primer pair 1 consisting of the nucleotide sequence shown in SEQIDNo.1, SEQIDNo.2) As primers, amplify according to the amplification system and reaction conditions in Example 1, and detect the amplification results by electrophoresis.

[0079] 1.2 Specificity of PRV primer pairs

[0080] Respectively with PPV and PRV genomic DNA, respectively with the cDNA of PRRSV, TGEV, PEDV, CSFV and BVDV as te...

experiment example 2

[0085] Sensitivity test of experimental example 2 multiplex PCR

[0086] The OD was measured by a spectrophotometer, and the concentration of the PPV genomic DNA obtained in Example 1 was 15000 μg / μL, and the concentration of the PRV genomic DNA was 15000 μg / μL. The concentration was adjusted to 3 μg / μL before the experiment.

[0087] PPV and PRV samples were diluted 10 times (from 3×10 2 ng to 3×10 -3 ng), using PPV and PRV mixed genomic DNA as a template (volume ratio 1:1), or respectively using PPV and PRV genomic DNA as a template, amplify according to the multiplex PCR reaction system and amplification system of Example 1, electrophoresis Check the amplification result.

[0088] see results Figure 4 , it can be seen that when the content of PPV and PRV mixed genomic DNA is greater than 3×10 -1 ng can obtain effective amplification.

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Abstract

The invention discloses a multiple PCR primer group and a detection kit for simultaneously detecting porcine pseudorabies virus and porcine parvovirus, and belongs to the field of detection of porcine pseudorabies virus and the porcine parvovirus. According to the multiple PCR primer group, a plurality of pairs of primers are firstly designed according to partial known sequences of the porcine pseudorabies virus and the porcine parvovirus exposed by GenBank and then a primer group high in specificity and sensitivity is screened out of the primers. The primer group is composed of a first pair of primers as shown in SEQ ID No.1 and SEQ ID No. 2 and a second pair of primers as shown in SEQ ID No.3 and SEQ ID No.4. The invention further discloses a multiple PCR detection kit for simultaneously detecting the porcine pseudorabies virus and the porcine parvovirus. The multiple PCR detection kit is capable of synchronously detecting the two viruses namely porcine pseudorabies virus and porcine parvovirus in one step, and is high in specificity and sensitivity, simple and convenient as well as efficient in detection procedures, and low in detection cost.

Description

technical field [0001] The present invention relates to the multiplex PCR primer set of detection virus, relate in particular to the multiplex PCR primer set of detection porcine pseudorabies virus (PseudorabiesVirus, PRV) and porcine parvovirus (PorcineParvovirus, PPV) 2 kinds of DNA viruses, the present invention also relates to using said primer The multiple PCR detection kit prepared by the group belongs to the multiple PCR detection field of porcine pseudorabies virus and porcine parvovirus. Background technique [0002] In recent years, multi-pathogen mixed infection has become a common phenomenon in pig production. The disease in pig herds is often not caused by a single pathogen, but caused by the joint action of two or more pathogens, which delays diagnosis and control, leads to high morbidity and mortality in pig herds, and causes huge economic losses (AllanandEllis, 2000; Dorre et al., 2007; Ellis et al., 2000; Rodriguez-Arrioja et al., 1999; Segales et al., 2002...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/701C12Q2600/16
Inventor 丁国杰李彦伟杨末崔艳丽刘鑫莹闫冰张影李来旭魏宏宇张凤强陈欣李淑红
Owner 哈药集团生物疫苗有限公司