中国兽医科学2024，Vol.54Issue(1)：63-69,7.DOI:10.16656/j.issn.1673-4696.2024.0004
急性肝胰腺坏死病副溶血弧菌实时荧光RPA快速检测方法的建立
Establishment of a real-time RPA method for rapid detection of Vibrio parahaemolyticus causing acute hepatopancreas necrosis disease
摘要
Abstract
To establish a simple and rapid method for the detection of AHPND-causing Vibrio para-haemolyticus(VpAHPND),multiple pairs of primers and exo-probe were designed according to the conserved region of pirB gene,a real-time recombinase polymerase amplification(RPA)method was developed through screening the primers,exo-probes and optimizing experimental conditions.The method can be completed within 15 min at 42 ℃,VpAHPND detected quickly and specifically,and has no cross reaction with other shrimp pathogens and pathogenic bacteria in the breeding environment,the minimum detectable cell con-centration of VpAHPND was 3.8 × 102 CFU/mL,plasmid detection limit was 1.17 × 101 copies/μL,and the re-peatability was good.A total of 50 Litopenaeus vannamei samples of artificial infection were tested by the established RPA method and the test results were consistent with the WOAH recommended real-time qPCR.These results showed that the real-time RPA method developed in this study was a simple,sensi-tive,low instrument dependence and reliable method which could potentially be applied for the rapid detection of VpAHPND in the research laboratory and on site diagnosis.关键词
急性肝胰腺坏死病/副溶血弧菌/重组酶聚合酶等温扩增技术/快速检测Key words
acute hepatopancreas necrosis disease/Vibrio parahaemolyticus/recombinase polymerase amplification/rapid detection分类
农业科技引用本文复制引用
田飞焱,孟霞,裴建明,黄海莉,徐节华,顾泽茂,王淑兵,吴葵..急性肝胰腺坏死病副溶血弧菌实时荧光RPA快速检测方法的建立[J].中国兽医科学,2024,54(1):63-69,7.基金项目
江西省重点研发计划项目(20202BBG73031) （20202BBG73031）
江西省农牧渔业科研指导性课题项目[赣农厅办函(2021)36号-序号29] （2021）
