柞蚕核型多角体病毒PCR检测方法的建立

Development of a PCR-based Method for Detection of Antheraea pernyi Nucleopolyhedrovirus

为了建立柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedrovirus,ApNPV)的早期检测技术,采用NCBI提供的BLAST软件在线检索ApNPV特异基因ORF142的序列后设计引物AP1,对ApNPV基因组DNA进行PCR扩增,得到约130 bp的片段,最低检测量为0.5 fg/mL病毒DNA。分别以感染ApNPV的柞蚕幼虫总DNA和ApNPV多角体悬液为模板,相同扩增条件下可扩增出大小一致的一条特异性片段,最低检测量分别为1 fg/mL幼虫总DNA和20～25 PIBs/mL ApNPV多角体。以柞蚕4龄幼虫为材料,人工模拟感染ApNPV后取幼虫血淋巴为检测物可以直接检出ApNPV,当添毒量为2.3×108PIBs/mL时,添毒36 h后即可检出,且病毒感染后的检出时间与添毒浓度呈正相关。采用该方法的检测过程只需3 h,快速而方便。

In order to develop an early detection technology against Antheraea pernyi nucleopolyhedrovirus （ApNPV）, we conducted online BLAST search in NCBI databases and obtained the sequence of ApNPV specific gene 0RF142 which was further used to design gene specific AP1 primers. PCR amplification to ApNPV genomic DNA yielded a fragment of around 130 bp and the minimum detectable amount was 0.5 fg/mL of viral DNA. When the total DNA of Antheraea pemyi larvae infected by ApNPV and the polyhedron suspension of ApNPV were used as the template respectively, the specific DNA fragment could be obtained under the same amplification condition. The minimum detectable amount was 1 fg/mL of larval total DNA and 20 -25 PIBs/mL of polyhedron of ApNPV. Moreover, when the haemolymph from the 4th instar Antheraea pernyi larvae from artificially imitated infection by ApNPV was used as the template, ApNPV could be detected directly. When the virus was administered at a dosage of 2.3 xl08 PIBs/mL, ApNPV could be detec-ted at 36 h after virus treatment. I ne time ofsuccessful detection was positively correlated with the dosage of virus admini-stered. The whole detection procedure took only 3 h, being very efficient and convenient.