贝类单孢子虫和折光马尔太虫二重PCR检测方法的建立

Development of a Duplex PCR Assay for Detection of Haplosporidium and Marteilia refringens in Shellfish

根据基因库中单孢子虫和折光马尔太虫的基因序列,分别设计了2对特异性引物,通过对二重PCR扩增条件的优化,研究建立了可同时检测鉴别这2种原虫的二重PCR。对同一样品中的单孢子虫和折光马尔太虫模板DNA进行扩增,得到2条大小与试验设计相符的244 bp(单孢子虫)和478 bp(折光马尔太虫)的特异性扩增带,而对派琴虫、嗜水气单胞菌、荧光假单胞菌、副溶血弧菌、溶藻弧菌、河弧菌和拟态弧菌等病原体的检测,结果均为阴性。敏感性试验表明,该技术最低能检测到10 pg的单孢子虫和折光马尔太虫DNA。

A duplex polymerase chain reaction was optimized to simultaneously detect two pathogens, Haplosporidium sp. and Marteilia refringens in shellfish. Two pairs of specific primers were designed according to the conserved regions on the sequences of Haplosporidium sp. and Marteilia refringensin Gen- Bank. All samples containing Haplosporidium sp. and Marteilia refringenscould be amplified into two specific bands, 244 bp for Haplosporidium sp. and 478 bp for Marteilia refringensby this duplex PCR, but no specific bands of the same sizes were amplified from other shellfish pathogens, such as Perkinsus sp. , Aeromonas hydrophila, Pseudomonas fluorescens, Vibrio parahaemolyticu, Vibrio alginolyticu, Vibrio fluvialis and Vibrio mimicus. As little as 10 pg of Haplosporidium sp. and Marteilia refringens DNA could be detected.