三种贝类原虫多重PCR检测方法的建立

Development of a multiplex PCR assay for detection of three species of protozoa in shellfish

根据基因库中单孢子虫、派琴虫和马尔太虫的基因序列,分别设计了3对特异性引物,通过对多重PCR扩增条件的优化,建立了可同时检测鉴别这3种原虫的多重PCR方法。用该技术对同一样品中的单孢子虫、派琴虫和马尔太虫模板进行扩增,结果均同时得到3条大小与试验设计相符的244 bp(单孢子虫)、596 bp(派琴虫)和478 bp(马尔太虫)的特异性扩增带,对其他贝类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10 pg的单孢子虫、派琴虫和马尔太虫DNA,提示该技术适用于这3种原虫的临床快速检测和鉴别诊断。

Three pairs of specific primers were designed according to the conserved regions on the sequences of Haplosporidium sp. , Perkinsus sp. and Marteilia refringens in GenBank. A multiplex polymerase chain reaction（multiplex PCR） was developed to simultaneously detect the three pathogens, Haplosporidium sp. ,Perkinsus sp. and M. refringens in shellfish. The specific band of 244 bp,596 bp and 478 bp were amplified from the samples with Haplosporidium sp. Perkinsus sp. , and M. refringens, respectively,but no specific band was amplified from other shellfish pathogens by this multiplex PCR. As little as 10 pg DNA of Haplosporidium sp. , Perkinsus sp. and M. refringens was detected. The results showed that this multiplex PCR could be used as a sensitive tool to detect Haplosporidium sp. , Perkinsus sp. and M. refringens in clinical samples.