基于PCR及焦磷酸测序技术的单孢子虫鉴定技术研究与应用

Establishment and application of Haplosporidium nelsoni identification based on PCR amplification and pyrosequencing

为适应口岸单孢子虫快速、准确、高通量检测的需求,建立一种基于PCR及焦磷酸测序技术平台的单孢子虫鉴定方法。以OIE推荐的PCR扩增方法获得单孢子虫特异基因,根据此基因的保守序列利用焦测序软件PyroMarkQ96ID设计专用引物进行PCR扩增及焦磷酸测序,测得序列经比对分析确定为单孢子虫序列。同时采用PCR焦磷酸测序方法和OIE推荐的PCR方法对牡蛎样品进行检测。结果表明,所建立的检测方法可从基因序列水平上准确鉴定牡蛎样品中的单孢子虫,且检测结果与OIE方法的检测结果一致。

For rapid, accurate and high throughput detection of Haplosporidium nelsoni, pyrosequencing analysis coupled with PCR amplification of the target sequence was developed. H. neZsoni DNA sequence was obtained by the OIE reference PCR method. Pyrosequencing special primers were designed targeting the conserved region of the sequence. The DNA of Haplosporidium-positive oyster samples was chosen to amplify the target sequence using pyro- sequencing primers, and the sequence was analyzed by PyroMarkTM ID System. BLAST online showed that the sequence was specific for H. nelsoni. Oyster samples were detected by both the PCR-pyrosequencing method and the OIE reference PCR method. The results showed that the PCR-pyrosequencing detection method could identify H. nelsoni and the result was consistent with the OIE reference PCR examination. The method meets the requirements of H. neZsoni quarantine and provides a new approach for the examination of other animal diseases.