7种猪场常见高致死病原GeXP检测方法的建立

doi:10.16431/j.cnki.1671-7236.2019.05.004

Abstract

Abstract:

A new multiplex PCR method was established to simultaneously detecting classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV),pseudorabies virus (PRV),porcine parvovirus (PPV),porcine circovirus type 2 (PCV2), Fusobacterium necrophorum (Fn) and Haemophilus parasuis (Hps) seven kinds of common highly lethal epidemic pathogen in hoggery.Seven specific primers refer to each pathogen were designed,and one pair Cy-5 conjugated universal primers was designed.In this study,the universal primers were respectively fused in the 5'end of each specific primer to form seven pairs of specific chimeric primers.In optimized reaction condition,each chimeric primer was mixed with universal primer respectively and amplified cDNA/DNA of seven pathogens to verify the specificity of single PCR.This study further relied on GenomeLab Genetic Analysis System (GeXP),we mixed all the seven specific chimeric primers and universal primers,amplified cDNA/DNA of a single pathogen to verify multiplex PCR specificity.The genomes of other common pig disease pathogenes were taken as positive internal.Seven pairs of specific chimeric primers and universal primers were used to amplify cDNA/DNA of seven pathogens,then we mixed templates with positive samples to determine their anti-interference.The sensitivity of GeXP multiplex PCR assay was determined by gradient recombinant plasmid dilution.The results showed that GeXP multiplex and single detection both could detect the signal of specific target fragments,no other obvious interference signals were detected.GeXP multiplex anti-interference showed that mixed 3 pathogen genomes of other common pig diseases,the assay could also detect the seven pathogens simultaneously.GeXP multiplex sensitivity analysis showed that specific results of seven genes were detected,and the detection limit was 10³ copies/μL.The GeXP multiplex assay was established in this study for simultaneous detection of 7 kinds of common and highly lethal epidemic pathogens in pig farms,and it had the characteristics of high throughput,specificity and sensitivity,providing a new detection method for rapid diagnosis of cross-infection and mixed infection of swine epidemic diseases.

Key words: highly lethal disease; epidemic pathogen; GeXP; specific chimeric primers; multiplex PCR

CLC Number:

S858.28

WANG Qiushi, LI Jiangling, ZHAO Sujun, LIU Rui. Establishment of GeXP Detection Method for 7 Common Hyper Lethal Pathogens in Pig Farms[J]. , 2019, 46(5): 1283-1289.

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Establishment of GeXP Detection Method for 7 Common Hyper Lethal Pathogens in Pig Farms

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