罗氏沼虾诺达病毒的核酸检测及其部分序列分析

根据安替列群岛分离的罗氏沼虾诺达病毒株基因组序列(MrNV-ant),制备特异性核酸探针,设计特异性引物,用点杂交和RT-PCR的方法检测在中国境内分离的罗氏沼虾诺达病毒(MrNV-chin).点杂交的方法可以检测出少于26ng的患肌肉白浊症的组织样品中的病毒,或少于25ng的病毒RNA样品;RT-PCR可以检测出少于25pg的RNA样品.扩增的MrNV-chin RNA1序列长858个核苷酸,与MrNV-ant的核苷酸一致率为957%,两者翻译后的氨基酸序列的一致率为99.7%.扩增的MrNV-chin RNA 2序列长1121个核苷酸,与MrNV-ant的核苷酸一致率为92%,两者翻译后的氨基酸序列的一致率为93.2%.因此,MrNV-ant和MrNV-chin应属于同一种病毒的不同分离株.用两株罗氏沼虾诺达病毒的RNA聚合酶序列与其它6株诺达病毒RNA聚合酶序列比较后构建的进化树中,罗氏沼虾诺达病毒与Alphanodavirus的亲缘关系近于与Betanodavirus的亲缘,组成了一个新的分支.

Nucleic Acid Detection and Partial Sequence Analysis of Macrobrachium rosenbergii Nodavirus

According to the genomic sequence of Macrobrachium rosenbergii nodavirus(isolated from Antillian postlarvae,MrNV ant),the spectific probes and primers were prepared for detection of Macrobrachium rosenbergii nodavirus isolated from China (MrNV chin) by using Dot blot and RT PCR methods.By Dot blot,virus less than 26ng of diseased postlarvae tissues and 2.5ng of viral RNA could be detected,while RT PCR method could detect 25pg of viral RNA.The product of RT PCR was subsequently cloned,sequenced and analysed.The amplified sequence from MrNV chin RNA1 and RNA2 was 858bp and 1121bp,respectively.Compared MrNV chin with MrNV ant, these two isolates shared 95.7% and 92% identities in RNA1 and RNA2 nucleotide sequences,respectively;99.7% and 93.2% identities in their amino acid sequences,respectively.In phylogenetic tree constructed with RNA dependent RNA polymerases from other 6 nodaviruses, these two MrNV isolates constitute a new branch but are closer to Alphanodavirus than to Betanodavirus.