中国兽医科学2024，Vol.54Issue(4)：498-503,6.DOI:10.16656/j.issn.1673-4696.2024.0067
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
摘要
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分类
农业科技引用本文复制引用
李家侨,赵优,谢艳辉,杨金金,斯泽恩,郑舒尹,尹伟力,张娜..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[J].中国兽医科学,2024,54(4):498-503,6.基金项目
海关总署项目(2023HK037,2019HK038,2021HK007,2021HK163) （2023HK037,2019HK038,2021HK007,2021HK163）
湛江市科技计划项目(2023B01020) （2023B01020）
烟台市科技计划项目(2022MSGY058) （2022MSGY058）
