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Establishment and Application of a SYBR Green I Real-time Quantitative PCR Assay for the Detection of lat gene of Pseudorabies Virus in Bama Miniature Pigs

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Agricultural Biotechnology 2015年第3期4卷 62-66页

作者： Sufang LU Guangjun GUO Ling MO Feng WEI Bingmei DONG Wentong ZHANG Zhiqiang SHEN Shandong Binzhou Animal Science & Veterinary Medicine Academy/Shandong Research & Development Center of Veterinary Propolis Vaccines Shandong Lvdu Bio-Sciences & Technology Co. Ltd.

[ Objective] This study aimed to establish a rapid, sensitive and specific SYBR Green I real-time quantitative PCR assay for the detection of latent pseudorabies virus （PRV） infection. [ Method ] SYBR Green I real-time quantitative PCR conditions and system for early detection of latent pseudorabies virus in- fection were optimized and compared with conventional PCR to investigate the sensitivity and specificity. Subsequently, the established assay was applied to detect different clinical samples. [ Result] The sensitivity of SYBR Green I real-time quantitative PCR assay （52 copies/μl） was 1 000 times higher than that of conven- tional PCR （5.2×1^04 copies/μl） and the detection time was shortened by 1/2. The established assay could be used to detect PRV but could not be used to detect PCV2, PPV, CSFV or PRRSV. Various tissues were collected from Bama miniature pigs with latent PRV infection under sterile conditions for real-time PCR detection. Results showed that viral copy number in the brain, nasal swab, inguinal lymph node, liver, lung and spleen was above 20, while PRV was not detected in the kidney and heart tissues. [ Conclusion] The established SYBR Green I real-time quantitative PCR assay for PRV/.AT detection was specific, sensitive and rapid, which could be used for pathogen monitoring, epidemiological investigation and quantitative study of PRV.

关键词： SYBR Green I Pseudorabies virus gE deleted virus lat gene latent infection

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