多重RT-PCR体系检测4种虾病毒的方法

A multiplex RT-PCR assay for simultaneous detection of four viruses of shrimp

根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV特异性扩增片段435 bp,IHHNV特异性扩增片段301 bp和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。

Viral infection is one of the major causes for the huge economic losses in shrimp farming.A multiplex reverse transcription polymerase chain reaction（mRT-PCR）was developed for simultaneous detection of 4 major shrimp viruses including infectious hypodermal and hematopoietic necrosis virus（IHHNV）,Taura syndrome virus（TSV）,white spot syndrome virus（WSSV）and yellow-head virus（YHV）in this research.Four sets of specifically designed oligonucleotide primers were used in the assay and each of them could amplify viral nucleic acids by PCR products with different sizes,such as：508 bp for TSV,435 bp for WSSV,301 bp for IHHNV and 614 bp for YHV.Specificity of multiplex RT-PCR nucleic acids of individual virus amplified in PCR reaction containing 4 primer sets were performed and they were highly specific and no specific bands were amplified from other penaeid shrimp pathogenic bacteria.Multiplex RT-PCR for detection of two,three or four types of different viruses in a single reaction system,the corresponding DNA samples of IHHNV（I）and WSSV（W）and cDNA of TSV（T）and YHV（Y）were randomly mixed and amplified using four primer sets.The sensitivity of the multiplex RT-PCR was 0.1 pg for IHHNV,1 pg for TSV,0.02 pg for WSSV and 0.2 pg for YHV.In the field application,49 samples were examined by multiplex RT-PCR.The results were consistent with those results of the single PCR detection.It indicated that this multi-PCR method is superior in terms of sensitivity,specificity rapidity and simplicity,and is potentially a valuable diagnostic tool for shrimp viral infections.