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
田飞焱 1孟霞 2裴建明 2黄海莉 2徐节华 2顾泽茂 3王淑兵 4吴葵5
作者信息
- 1. 江西省农业技术推广中心,江西南昌 330046;华中农业大学水产学院,湖北武汉 430070
- 2. 江西省农业技术推广中心,江西南昌 330046
- 3. 华中农业大学水产学院,湖北武汉 430070
- 4. 南昌市新建区动物疫病预防控制中心,江西南昌 330100
- 5. 南昌市疾病预防控制中心,江西南昌 330038
- 折叠
摘要
为建立一种简单、快速的急性肝胰腺坏死病副溶血弧菌(VpAHPND)检测方法,本研究根据pirB基因保守序列设计了多组引物和exo探针组合,经过筛选优化,建立了 VpAHPND实时荧光RPA检测方法.结果显示,该方法在42 ℃恒温反应15 min即可快速、特异性地检测出急性肝胰腺坏死病副溶血弧菌(VpAHPND),与其他虾类病原和养殖环境中的致病菌不发生交叉反应,最低菌体细胞检出浓度为3.8×102CFU/mL,质粒检测限为1.17×101copies/μL,且具有良好的重复性.利用本研究建立的实时荧光RPA方法与WOAH推荐的实时荧光qPCR方法对50份人工感染的南美白对虾样品进行检测,二者检测结果一致.结果表明,本研究建立的检测方法操作简单,反应灵敏,仪器依赖性低,结果可靠,适用于实验室和现场对VpAHPND快速检测.
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引用本文复制引用
基金项目
江西省重点研发计划项目(20202BBG73031)
江西省农牧渔业科研指导性课题项目(赣农厅办函202136号-序号29)
出版年
2024
国家科技期刊平台
NSTL
维普
万方数据