TaqMan实时荧光定量PCR法检测对虾肝肠胞虫(EHP)步骤优化

Optimization of the steps of TaqMan real-time fluorescence quantitative PCR for the detection of Enterocytozoon hepatopenaei(EHP)

优化虾肝肠胞虫(EHP)的TaqMan实时荧光定量PCR检测流程,目的是更准确、快速地检测出虾苗,饵料,亲虾体内携带的EHP。本研究采用酚/氯仿抽提法与水生动物病原体核酸提取试剂盒两种DNA提取方法,对等量的病原组织样本进行了对比提取。此外,还设置不同浓度的裂解液以探究其对病原组织裂解效果的影响,并设定不同裂解时间以优化裂解步骤。最后,通过调整qPCR反应的循环数,确定最佳的检测条件。结果显示,水生动物病原体试剂盒提取的DNA检出率高于酚/氯仿抽提法提取,裂解液浓度90%时检测出的EHP含量最高;裂解时间30 min时检测出的EHP含量较高;循环数在40次时足以检测出EHP。综上,优化后的检测流程为用水生动物病原体核酸提取试剂盒提取病原组织DNA,裂解液浓度90%,裂解时间30 min及qPCR检测条件的循环数控制在40次。

Optimize the TaqMan real-time fluorescent quantitative PCR detection process of Enterocytozoon hepatopenaei(EHP)to detect EHP in larvae,bait,and parent shrimps more accurately and quickly.Two DNA extraction methods,phenol/chloroform extraction and aquatic animal pathogen nucleic acid extraction kit,were used to compare and extract the same amount of pathogenic tissue samples.In addition,different concentrations of lysates were set up to investigate the effect of lysates on the lysate of pathogenic tissues,and different lysate times were set up to optimize the lysate steps.Finally,the optimal detection conditions were determined by adjusting the number of qPCR cycles.The results showed that the detection rate of DNA extracted by the aquatic pathogen kit was higher than that of the phenol/chloroform extraction method,and the highest content of EHP was detected when the concentration of lysis solution was 90%;The highest content of EHP was detected when the lysis time was 30 min;The number of cycles of 40 was sufficient to detect EHP.To sum up,the optimized procedure was as follows:use aquatic animal pathogen nucleic acid extraction kit to extract DNA from pathogenic tissues,the concentration of lysate was set at 90%,the lysis time was set at 30 min,and the number of cycles of qPCR was controlled at 40 times for the reaction.