鸭痘病毒TaqMan荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2015.10.007

Abstract

Abstract: To establishment a TaqMan Real-time PCR method for detection of duck poxvirus (DPV),we cloned the P4b gene of DPV.The specific primers and probe were designed according to the nucleotide sequence of avipoxvirus available in GenBank.Recombinant plasmid pMD-DPV-P4b was employed as positive standard template for Real-time PCR.By optimization of reaction conditions,a TaqMan Real-time PCR method for detection of DPV was established.The results of specificity test proved this method had no cross-react with other waterfowl vial agents and poxviruses including avian influenza virus,duck flavivirus,duck hepatitis virus,Newcastle disease virus,duck entertitis virus,goose parvovirus,goatpox virus and fowlpox virus.The detection limit of the assay was 1.29×10² copies/μL of viral DNA,which was 100 times higher than that of the routine PCR.Reproducibility test showed that the CVs of intra assay and inter assay were both less than 2%.Above results supported that the assay was suitable for the detection of DPV very well.

Key words: duck poxvirus; avipoxvirus; Real-time PCR; TaqMan probe

CLC Number:

S852.65

CAO Hui-hui, SUN Wen-chao, ZHENG Min, WEI Xian-kai, SU Jiao-xiu, LIANG Sheng, ZHENG Lie-feng, LI Jun, LIU Qi. Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus[J]. , 2015, 42(10): 2560-2566.

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Establishment of TaqMan Real-time PCR Method for Detection of Duck Poxvirus

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