贝类奥尔森派琴虫实时荧光定量PCR检测方法的建立及初步应用

Development and application of a real-time PCR assay for detection of Perkinsus olseni

目的建立一种快速、灵敏、特异的用于检测贝类奥尔森派琴虫的实时荧光定量PCR方法。方法根据基因库中奥尔森派琴虫的基因保守序列,设计合成1对引物和1条TaqMan探针,建立荧光定量PCR方法,对采自广西沿海的49份贻贝标本进行检测,并与常规PCR比较。结果建立的荧光定量PCR方法灵敏度可达20个拷贝,比常规PCR灵敏度高100倍。49份贻贝标本的阳性率为16.3%,检测的奥尔森派琴虫基因组DNA含量为2.38×106～9.21×102拷贝/μl。结论建立的荧光定量PCR方法可以用于贝类奥尔森派琴虫感染的快速检测。

Objective To establish a rapid, sensitive, and specific method of real-time PCR to detect Perkinsus olseni. Methods A pair of primers and a TaqMan probe were designed and synthesized according to the conserved gene sequences of P. olseni, and then reaction parameters were optimized to develop a real-time PCR assay. The real-time PCR assay thus developed was used in 49 Guangxi Mussel clinical samples and was compared with routine PCR. Results The real-time PCR assay thus developed detected 20 template copies of plasmid DNA, and its sensitivity was 100 times greater than that of the routine PCR. In 49 Mussel clinical samples, the real-time PCR thus developed had a positive rate of P. olseni detection of 16.3%, and the DNA concentration of P. olseni was 2.38×10^6 9.21×10^2 eopies/μl. Conclusion This method of real-time PCR can be used as a tool for the rapid detection of P. olseni.