多重RT-PCR同时检测鉴别三种对虾病毒的研究与应用

Studies on Development of Multiplex RT-PCR for Simutaneous Detection of Three Kinds of Viruses in Penaeid Shrimp

根据基因库中对虾桃拉综合征病毒(TSV)、白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(I-HHNV)的基因序列,分别设计了三对特异性引物,通过对多重RT-PCR扩增条件的优化,研究建立了可同时检测鉴别TSV、WSSV和IHHNV的多重RT-PCR。该技术对同一样品中的TSV RNA、WSSV DNA和IHHNV DNA模板进行扩增,结果均同时得到3条大小与实验设计相符的231bp(TSV)、593bp(WSSV)和356bp(IHHNV)的特异性多重RT-PCR扩增带,对其它对虾病原核酸的扩增结果为阴性。敏感性试验结果表明,该技术最低能检测到10pg TSV RNA、100pg WSSV DNA和100pg IHHNV DNA。临床检测试验结果表明,该技术对TSV、WSSV和I-HHNV的检出率明显高于传统的临床症状观察和组织病理学检查,提示该技术适用于这三种病毒的临床快速检测和鉴别诊断。

A multiplex reverse transcription polymerase chain reaction （multi-RT-PCR） was optimized to simultaneously detect three pathogens, Taura syndrome virus （TSV）, white spot syndrome virus （WSSV） and infectious hypodermal and hematopoietic necrosis virus （IHHNV） in Penaeid shrimps. Three pairs of specific primers were designed according to the conserved regions on the sequences of TSV （NC003005）, WSSV （AF369029） and IHHNV （NC002190） in GenBank. It was shown that all samples containg TSV, WSSV and IHHNV could be amplified into three specific bands, 231 bp for TSV, 593 bp for WSSV and 356 bp for IHHNV by this multiplex RT-PCR but no specific bands of the same sizes were amplified from other Penaeid shrimp pathogenic virus or bacteria. As little as 10pg of TSV RNA, 100pg of WSSV and IHHNV DNA could be detected. We used this multiplex RT-PCR to detect samples from some farms in south China. Results showed that TSV, WSSV and IHHNV were popularly existed in cultivated Penaeid shrimps in south China and two, or even three of these viruses were detected in some samples. And it also demonstrated that this multiplex RT-PCR could be used as a sensitive tool to detect TSV, WSSV and IHHNV in clinical samples simultaneously.