肝肠胞虫和致AHPND的副溶血弧菌双重荧光PCR方法的建立及在环境DNA样品中的应用  

Establishment of dual fluorescence PCR method for Enterocytozoon hepatopenaei and AHPND-causing Vibrio parahaemolyticus and its application in environmental DNA samples

作　　者：李家侨 赵优 谢艳辉 杨金金 斯泽恩 郑舒尹 尹伟力 张娜 LI Jiaqiao;ZHAO You;XIE Yanhui;YANG Jinjin;SI Zeen;ZHENG Shuyin;YIN Weili;ZHANG Na(Technology Center of Zhanjiang Customs,Zhanjiang 524000,China;Research Center of General Administration of Customs,Beijing 100010,China;Technology Center of Yantai Customs,Yantai 264000,China;Zhanjiang International Travel Healthcare Center,Zhanjiang 524000,China)

机构地区：[1]湛江海关技术中心,广东湛江524000 [2]海关总署研究中心,北京100010 [3]烟台海关技术中心,山东烟台264000 [4]湛江国际旅行卫生保健中心,广东湛江524000

出　　处：《中国兽医科学》2024年第4期498-503,共6页Chinese Veterinary Science

基　　金：海关总署项目(2023HK037,2019HK038,2021HK007,2021HK163);湛江市科技计划项目(2023B01020);烟台市科技计划项目(2022MSGY058)。

摘　　要：为了快速检测致对虾急性肝胰腺坏死病的副溶血弧菌(VP_(AHPND))和肝肠胞虫(EHP)这2种病原,根据NCBI中副溶血弧菌pirAB^(VP)毒素基因和肝肠胞虫SWP1基因的保守序列设计并合成特异性引物和Taq Man探针,通过优化反应条件,评估方法的特异性、灵敏度、重复性,并且把该方法应用于环境DNA(eDNA)样品检测。结果显示所建立的双重荧光PCR方法特异性强,仅能扩增出V_(PAHPND)和EHP的阳性核酸,与对虾常见病原白斑综合征病毒、传染性皮下和造血器官坏死病毒、十足目虹彩病毒1等均无交叉反应;VP_(AHPND)和EHP的最低检测限分别为14和1.4 copies/μL;组内和组间重复的变异系数均≤1.9%。从56批e DNA样品检测结果发现,VPAHPND阳性7批(12.5%),EHP阳性4批(7.14%),其中VPAHPND-EHP双重感染2批(3.57%)。这表明基于e DNA识别技术,该VPAHPND和EHP双重荧光PCR可以大大提升临床实验室的对虾疫病诊断速度,为海关等监管部门提供可靠高效的快速检测方法。To rapidly dectet acute hepatopannecrosis disease infection with strains of Vibrio parahaemolyticus(VP_(AHPND))and Enterocytozoon hepatopenaei(EHP),specific primers and TaqMan probes were designed and synthesized according to conserved sequences of pirAB^(VP)toxin gene in V.parahaemolyticus and SwPl gene in EHP in NCBI.The specificity,sensitivity and repeatability of the method were evaluated by optimizing reaction conditions.The method was applied to dectet environmental DNA(eDNA)samples.The results showed that the dual fluorescence PCR method was highly specific and could only amplify positive nucleic acids of VP_(AHPND)and EHP.White spot syndrome virus,infectious hypodermal and haematopoietic necrosis virus and decapod iridescent virus 1 had no cross-reaction.The lowest detec-tion limits of VP_(AHPND)and EHP were 14 copies/μL and 1.4 copies/μL respectively.The coefficient of variation for both intra-group and inter-group repeats was≤1.9%.From the 56 batches of eDNA samples,7 batches(12.5%)were V positive and 4 batches(7.14%)were EHP positive,among them 2 batches(3.57%)were VP_(AHPND)-EHP double infected.Based on eDNA recognition technology,VP_(AHPND)and EHP dual fluorescence PCR can greatly improve the diagnostic speed of shrimp diseases in clinical laboratories,providing reliable and efficient rapid detection methods for customs and other regulatory departments.

关 键 词：急性肝胰腺坏死病 副溶血弧菌 肝肠胞虫 双重荧光PCR 环境DNA 监测 

分 类 号：S852.612[农业科学—基础兽医学]