虾肝肠胞虫(EHP)现场快速高灵敏度检测试剂盒的性能评价研究

Validation of a Highly Sensitive Kit for the Rapid Detection of Enterocytozoon hepatopenaei in Field

近年来,虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)流行使我国养殖对虾遭受严重经济损失,现场快速检测是EHP防控的重要技术保证。本研究对本实验室研发的EHP现场快速检测试剂盒的分析特异性(ASp)、分析灵敏度(ASe)、诊断特异性(DSp)、诊断灵敏度(DSe)、重复性和稳定性6项性能参数开展了系统评估。ASp测试显示,该试剂盒与对虾白斑综合征病毒(WSSV)、偷死野田村病毒(CMNV)、虾血细胞虹彩病毒(SHIV)、致急性肝胰腺坏死病副溶血弧菌(VpAHPND)、传染性皮下及造血组织坏死病毒(IHHNV)等5种对虾常见病原及健康对虾无交叉反应;ASe测试显示,该试剂盒检测下限为101 copies/反应;以EHP TaqMan RT-qPCR方法为标准,比较了试剂盒对298份临床样品的测试结果,试剂盒的DSp为99.2%、DSe为91.7%;试剂盒对EHP阴性样品及强阳性样品的检测重复率为100%,弱阳性样品检测重复率为95.8%;试剂盒在–20℃和–40℃条件下分别可保存7个月和12个月以上。本研究表明,本实验室研制的EHP现场快速检测试剂盒具操作简便、快速、灵敏度高、特异性强、重复性好和稳定性强等优点,可满足对虾养殖现场对EHP的高灵敏度检测。

Enterocytozoon hepatopenaei(EHP)has caused serious losses in shrimp aquaculture in China in recent years.For cultured shrimp,disease prevention cannot be carried out by vaccination because of the lack of specific immune mechanisms.The most effective measure to prevent disease outbreaks is to conduct on-site rapid pathogen detection as far as possible for relatives or seedlings or to carry out on-site detection early in the onset of shrimp,timely detection,and identification of pathogens.There is,therefore,an urgent demand for rapid detection techniques and kits for controlling and preventing EHP.A novel,highly sensitive kit,developed in our laboratory,can achieve rapid detection of EHP in the field by optimizing the three steps of tissue nucleic acid extraction,nucleic acid amplification,and nucleic acid detection,and provide a practical solution for early rapid screening and detection of shrimp EHP disease.To validate the newly developed highly sensitive kit for rapid EHP detection in the field,a systematic evaluation of the six performance parameters of the kit was carried out in this study.The analytical specificity results showed that the kit did not cross-react with the DNA/RNA extracted from healthy shrimp or shrimp infected with five other pathogens,including WSSV,CMNV,SHIV,VpAHPND,and IHHNV.The analytical sensitivity analysis showed that the detection limit was 101 copies/μL with shrimp DNA preparation.The diagnostic sensitivity and diagnostic specificity were determined to be 91.7%and 99.2%,respectively,in tests of 298 clinical samples,in comparison with the TaqMan qPCR protocol of EHP.The repeatability was 100%for strongly positive and negative samples and 95.8%for weakly positive samples.The period of validity of the kit was tested and found to be over 7 months at–20℃storage and over 12 months at–40℃.The study results demonstrated that the kit is a simple,sensitive,specific,and accurate tool for the rapid detection of EHP in practical applications.