虾肝肠胞虫芯片式数字PCR定量检测方法的建立及应用

Establishment and Application of a cd PCR Assay for Enterocytozoon hepatopenaei

虾肝肠胞虫(Enterocytozoonhepatopenaei,EHP)感染是目前对虾行业面临的全球性挑战,在过去10年中其发病率急剧上升。为建立一种可绝对定量检测EHP的方法,根据Gen Bank中EHP胞壁蛋白SWP基因保守区域,设计一套特异性引物和荧光探针,通过优化反应条件,建立了EHP芯片式数字PCR(chipdigital PCR,cd PCR)定量检测方法。结果显示:该方法线性关系良好(R2=0.9981);敏感性高,检测限可达4.4copies/μL;特异性强,与其他对虾常见病原无交叉反应;重复性良好,批内重复与批间重复试验的变异系数均小于8%。采用建立的cd PCR方法对28份临床虾样品进行检测,结果发现,该方法与水产行业标准《虾肝肠胞虫病诊断规程》(SC/T7232—2020)中套式PCR方法检测结果符合率为100%。结果表明,本研究建立的EHPcd PCR定量检测方法线性好、灵敏度高、特异性强,重复性及准确性良好,且能绝对定量,具有较好的应用前景。

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 Gen Bank,and a chip digital PCR(cd PCR)for EHP was established through optimizing reaction conditions.The results showed that the method had excellent linearity(R2=0.9981);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 cd PCR 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.