虾肝肠胞虫芯片式数字PCR定量检测方法的建立及应用OA

Enterocytozoon hepatopenaei(EHP)infection has challenging global shrimp industry with a quick increasing prevalence over the past decade.In order to establish an absolute quantitative method for EHP,a set of specific primers and fluorescent probes were designed based on the conserved region of EHP SWP gene registered in GenBank,and a chip digital PCR(cdPCR)for EHP was established through optimizing reaction conditions.The results showed that the method had excellent linearity(R2=0.998 1);its detection limit was 4.4 copies/μL,indicating a high sensitivity;it failed to crossly react with other common shrimp pathogens,indicating a strong specificity;the variable coefficients of both the intra-and inter-group repeated tests were less than 8%,indicating a good repeatability.It was found that,for the detection of 28 clinical samples,the results by the established cdPCR method were 100%consistent with those by nested PCR proposed by the fishery industry standard of Enterocytozoon hepatopenaei disease Diagnosis Protocol(SC/T 7232-2020).In conclusion,the established method had good linear relationship,high sensitivity,strong specificity,good reproducibility,good accuracy and could achieve absolute quantification,indicating a good application prospect.