对虾桃拉病毒(TSV)RT-PCR快速诊断技术的研究

Rapid Diagnosis of Taura Syndrome Virus (TSV) by RT-PCR

对虾桃拉病毒(Taura Syndrome Virus,TSV)最早于1992 年在凡纳对虾(Penaeus vannamei)体内发现,此病毒是对虾养殖业危害较严重的病毒之一.将该病毒基因组的一段序列克隆至转录载体 pSP64(PolyA)上,经体外转录、磁珠法分离、纯化获得了人工的TSV RNA模板;用定量的TSV RNA阳性对照模板进行RT PCR条件优化,灵敏度检测以及样品制备方法等试验,结果表明:RT PCR检测的灵敏度可达到0.1 fg,相当于 100 个病毒粒子;所制备的样品对病毒 RNA的逆转录及扩增无抑制,适合于对虾桃拉病毒快速检测.

Taura Syndrome Virus (TSV),as one of RNA viruses first discovered in Penaeus.vannamei in 1992,has become one of the most serious viruses affected shrimp culture.This viral agent has spread throughout the shrimp growing regions of Central and South to become established in North America in the short span of 5 a.Now Penaeus.vannamei has been cosmically cultured in our country,so a sensitive and fast diagnostic tool should be provided for screening shrimp for TSV infections in early time. In this study,a DNA fragment of TSV genome was cloned into the transcriptional vector pSP_(64) (PolyA).After in vitro transcription,isolation and purification by magnetic beads method,an artificial TSV-RNA template was obtained.The reverse transcriptase polymerase chain reaction (RT-PCR ) procedure,sensitivity and sample preparation were assayed with the TSV-RNA template.The results showed that the sensitivity of RT-PCR was 0.1 fg (equivalent to 100 copies of virus particles).The prepared samples did not affect the transcription and amplification of virus RNA.By RT-PCR with this primer set,17% in different species including adult and juvenile population of Penaeus.vannamei in suspected infection found TSV.Find and eliminate the affected shrimp with this method might be significant in preventing the spread of this viral agent.