荧光重组酶介导等温核酸扩增技术在虾肝肠胞虫病害现场检测中的应用及条件优化

Application and detection condition optimization of a fluorescent recombinase-aided amplification method for field detection of Enterocytozoon hepatopenaei in prawn

摘要:

目的 针对制约对虾养殖业发展的重要病原之一的虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)建立一种新的荧光重组酶聚合酶扩增(recombinase aided amplification, RAA)快速检测技术。方法 以EHP的高保守性基因18S rRNA为检测靶基因，设计多对RAA引物，通过基础型RAA和荧光型RAA筛选出核酸扩增效率最高的引物组合。在此基础上，对荧光型RAA的扩增体系和扩增条件进行优化。优化结果显示，本方法在37 ℃、20 min即可完成检测。随后，对方法的灵敏度、特异性、精密度、稳定性及实际样本应用情况进行评估。结果 本方法特异性高，仅对EHP敏感，对其他与EHP混合或继发感染的病原无反应。灵敏度高，最低检出限为5 copies/μL。精密度高，重复性结果的变异系数小于5%。预混引物探针的原位冻干检测试剂稳定性良好，在室温(25 ℃)和夏日高温(37 ℃)条件下分别可稳定运输、使用和保藏10 d和5 d，在-20 ℃条件下保质期超过1年。本方法对90份对虾临床样品的检测结果与水产行业标准SC/T 7232—2020推荐的荧光定量PCR方法一致，检测符合率为97.78% (88/90)，灵敏度为100.00% (30/30)，特异度为96.67% (58/60)。结论 本研究建立的EHP荧光RAA方法具有快速、操作简便、高灵敏度、高特异性、高精密度、良好稳定性等特点，可为EHP的现场快速检测提供技术参考，在水产疫病精准快速检测中具有广阔的市场应用前景。

Abstract:

Objective To develop a new and rapid fluorescent recombinase-aided amplification (RAA) method for the detection of Enterocytozoon hepatopenaei (EHP), one of the major pathogens restricting the development of the prawn farming industry.Methods Multiple pairs of RAA primers were designed based on the highly conserved 18S rRNA gene of EHP, and the primers with the highest amplification efficiency were screened by basic RAA and fluorescent RAA. The reaction system and conditions of fluorescent RAA were then optimized. The optimization results showed that the developed method achieved detection at 37 ℃ within 20 min. Finally, the sensitivity, specificity, precision, stability, and actual sample application of the developed fluorescent RAA method were evaluated.Results The fluorescent RAA method had high specificity and was only sensitive to EHP, and it had no response to other pathogens causing mixed infection or secondary infection. Moreover, it had high sensitivity and high precision, with the limit of detection being 5 copies/μL and the coefficient of variation less than 5% for repeat test results. The lyophilized premix of primers and probe demonstrated good stability. It can be stably transported, used, and stored for 10 d at room temperature (25 °C) and 5 d at the high temperature (37 °C) in summer, with the shelf life longer than one year at -20 ℃. Additionally, the detection results of ninety clinical samples of prawn by the developed method were consistent with those obtained by the fluorescence quantitative PCR method recommended by fishery industry standard Code of Diagnosis for Enterocytozoon hepatopenaei Disease (SC/T 7232—2020), with the result coincidence rate of 97.78% (88/90). In addition, the fluorescent RAA method showed the sensitivity of 100.00% (30/30) and the specificity of 96.67% (58/60).Conclusion The established fluorescent RAA method has the characteristics of rapid detection, simple operation, high sensitivity, high specificity, high precision, and good stability, providing a technical reference for the field rapid detection of EHP. It has a wide market application prospect in the accurate and rapid detection of aquatic diseases.