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
李家侨 1赵优 2谢艳辉 1杨金金 1斯泽恩 1郑舒尹 1尹伟力 3张娜4
作者信息
- 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
- 折叠
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(VPAHPND)和肝肠胞虫(EHP)这2种病原,根据NCBI中副溶血弧菌pirABVP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TaqMan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VPAHPND 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;VPAHPND 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eDNA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eDNA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.
Abstract
To rapidly dectet acute hepatopannecrosis disease infection with strains of Vibrio parahaemolyticus(VPAHPND)and Enterocytozoon hepatopenaei(EHP),specific primers and TaqMan probes were designed and synthesized according to conserved sequences of pirABVP toxin gene in V.parahaemolyticus and SWP1 gene in EHP in NCBI.The specificity,sensitivity and repeatability of the method were evaluat-ed by optimizing reaction conditions.The method was applied to dectet environmental DNA(eDNA)sam-ples.The results showed that the dual fluorescence PCR method was highly specific and could only ampli-fy positive nucleic acids of VPAHPND 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 VPAHPND and EHP were 14 copies/μL and 1.4 copies/μL respectively.The coefficient of vari-ation for both intra-group and inter-group repeats was≤1.9％.From the 56 batches of eDNA samples,7 batches(12.5％)were VPAHPND positive and 4 batches(7.14％)were EHP positive,among them 2 batches(3.57％)were VPAHPND-EHP double infected.Based on eDNA recognition technology,VPAHPND and EHP dual fluorescence PCR can greatly improve the diagnostic speed of shrimp diseases in clinical laboratories,providing re-liable 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Key words
acute hepatopannecrosis disease/Vibrio parahaemolyticus/Enterocytozoon hepatope-naei/dual fluorescence PCR/environmental DNA/monitoring引用本文复制引用
基金项目
海关总署项目(2023HK037)
海关总署项目(2019HK038)
海关总署项目(2021HK007)
海关总署项目(2021HK163)
湛江市科技计划(2023B01020)
烟台市科技计划(2022MSGY058)
出版年
2024
国家科技期刊平台
NSTL
万方数据