Establishment and Preliminary Application of Duplex Fluorescent RPA for Detection of Pathogens in Shrimp Products
张娜 1谢艳辉 1仇保封 2郑舒尹 1斯泽恩 1李家侨1
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作者信息
1. 湛江海关技术中心,广东湛江 524000
2. 南通海关技术中心,江苏南通 226004
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摘要
为实现对虾产品中两种病原体的简便、快速、高效检测,针对对虾传染性皮下及造血器官坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(EHP)进行双重荧光RPA(recombinase polymerase amplification,重组酶聚合酶扩增)检测方法的研究.根据IHHNV和EHP的保守序列设计引物、探针,建立双重荧光RPA方法,同时对方法的特异性、敏感性和重复性进行验证.结果表明,所建立的方法只对IHHNV和EHP有明显的扩增;用IHHNV和EHP阳性质粒进行倍比稀释检测,结果表明双重荧光RPA方法对IHHNV和EHP质粒检测灵敏度可以达到1×102copies/μL;用10个重复质粒样本进行检测,结果证明方法重复性较好.使用该方法对进口的冷冻对虾样本检测,检测出1例EHP阳性和1例IHHNV阳性,同时样本用荧光PCR方法进行比对检测,两种方法检测结果一致.本研究中所建立的方法对于两种疫病的区分检测具有重要的临床意义,可应用于养殖场、对虾产品生产企业和口岸实验室疫病的快速筛查.
Abstract
To achieve a simple,rapid and efficient detection of two pathogens in shrimp products,a duplex fluorescent recombinase polymerase amplification(RPA)detection method was developed for infectious hypodermal and hematopoietic necrosis virus(IHHNV)and Enterocytozoon hepatopenaei(EHP).Primers and probes were designed based on the conserved sequences of IHHNV and EHP,and the duplex fluorescent RPA method was established.The specificity,sensitivity and repeatability of the method were validated.The results showed that the established method exhibited distinct amplification only for IHHNV and EHP.Detection using diluted plasmids of IHHNV and EHP demonstrated that the sensitivity of the duplex fluorescent RPA method for detecting IHHNV and EHP plasmids could reach 1 ×102 copies/μL.The method showed good repeatability with ten repeated plasmid samples.The method was employed to examine imported frozen shrimp samples,resulting in the discovery of one positive EHP and one positive IHHNV.Comparison of the detection results with fluorescent PCR showed consistency between the two methods.The method established in this research has important clinical significance for the distinguishing and detection of the two diseases,and can be applied to rapid screening methods for disease diagnosis in aquaculture farms,shrimp product production enterprises,and port laboratories.
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