Optimization of the steps of TaqMan real-time fluorescence quantitative PCR for the detection of Enterocytozoon hepatopenaei(EHP)
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.
万方数据
维普
