伪狂犬病病毒野毒株与gE基因缺失疫苗株TaqMan实时荧光定量PCR鉴别方法的建立

doi:10.16431/j.cnki.1671-7236.2018.07.010

Abstract

Abstract:

In order to establish a TaqMan Real-time PCR for rapid,sensitive and specific distinguishing the wild strain and gE-deleted vaccine strain of pseudorabies virus (PRV),two sets of primers and TaqMan probes were designed for gD and gE genes of PRV,respectively,then a set of 2 novel Real-time PCR were developed for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.The concentration of primers and probes,the annealing temperature in TaqMan Real-time PCR were optimized,the sensitivity,specificity and reproducibility of the assays were determined,and were applied to the detection of clinical samples.The R² value of the TaqMan Real-time PCR standard curve were 0.996 and 0.980,respectively,both showed good linear response.The detection limit of the assays were 39.4 and 12.1 copies/μL,respectively.The assays were highly specific for PRV,without cross-reaction with other common swine viral pathogens,such as porcine circovirus type 2 (PCV2),classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV).Intra-batch and inter-batch reproducibility tests showed that the coefficient of variation (CV) were 1.43% to 1.86%,1.10% to 2.07% (gD gene) and 0.98% to 1.41%,1.12% to 1.86% (gE gene),respectively.Applying the TaqMan Real-time PCR and the conventional PCR to detect 11 PR suspected clinical samples,they got 36.4% and 27.3% positivity,respectively.All these results indicated that the TaqMan Real-time PCR were both sensitive,specific and reproducible,and could be used as a useful tool for quantitative detection and differentiation of wild-type strain from gE-deleted vaccine strain of PRV.

Key words: pseudorabies virus (PRV); wild strain; gE-deleted vaccine strain; TaqMan Real-time PCR; differentiation

CLC Number:

S852.65

LAN Desong, GU Guibo, HOU Zhengzhong. Development of a TaqMan Real-time PCR for Differentiation of Wild-type Strain from gE-deleted Vaccine Strain of Pseudorabies Virus[J]. , 2018, 45(7): 1804-1812.

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Development of a TaqMan Real-time PCR for Differentiation of Wild-type Strain from gE-deleted Vaccine Strain of Pseudorabies Virus

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