常规PCR与巢式PCR法快速检测克氏原螯虾白斑综合征病毒(WSSV)

对传统对虾白斑综合征病毒(WSSV)的PCR检测方法中的病毒模板DNA的提取方法加以改进,建立了一种快速检测克氏原螯虾(Procambarus clarkii)WSSV的PCR方法。本方法将克氏原螯虾肝胰腺组织匀浆液冻融3次进行差速离心后,在病毒沉淀中加入50μL蛋白酶K溶液,室温消化5 min,沸水中煮10 min后,10000 r/min高速离心5 min即可取上清液用于PCR扩增,结果显示:与传统的病毒模板DNA提取方法相比,本方法的PCR结果特异性强,扩增效率高,准确率高,可应用于快速大规模检测克氏原鳌虾中的WSSV。

PCR and Nested PCR Method for Detecting WSSV in Procambarus clarkia

A rapid PCR method was established for detecting WSSV of Procambarus clarkii based on the improvement of the traditional Virus template DNA extraction of PCR method.The hepatopancreas of P.clarkii was homogenated on ice and freezed-thawed 3 times and then differential centrifugated.The pellet was suspended and digested with 50 μL proteinase K solution and digested for 5 min at room temperature.The lysate was boiled for 10 min and then centrifuged at 10000 r/min for 5 min before used for PCR amplification.The results indicated that,compared with the traditional virus template DNA extraction method,the improved method had a specificity result,high amplification efficiency,high rate of accuracy and could be applied for rapidly detecting WSSV in P.clarkii on a large scale.