农业生物技术学报2024，Vol.32Issue(4)：961-973,13.DOI:10.3969/j.issn.1674-7968.2024.04.020
重组酶聚合酶扩增技术检测南美白对虾急性肝胰腺坏死病
Detection of Acute Hepatopancreatic Necrosis Disease in Pacific White Shrimp(Penaeus vannamei)by Recombinase Polymerase Amplification
摘要
Abstract
Acute hepatopancreatic necrosis disease(AHPND)is a newly emerged bacterial disease in shrimps(Penaeidae),causing significant economic losses in the global shrimp farming industry.AHPND is primarily caused by Vibrio spp.carrying virulent pirA and pirB.In this study,specific primers and probes were designed and screened for pirA and pirB,and a recombinase polymerase amplification(RPA)method was developed.This RPA reaction ran in 39℃for 20 min.The results revealed successful screening of primer/probe combinations for both pirA and pirB genes.Through a series of tests using the RPA method,these primer/probe combinations demonstrated specific detection of the target virulence genes,without amplification of genomic DNA from 13 microorganisms,such as Vibrio alginolyticus,V.parahaemolyticus,and Enterocytozoon hepatomegaly.The detection limit of the RPA method was 0.01 and 0.1 fg/μL of recombinant plasmid pirAB-pMD19,when the primers and probes targeting pirA or pirB were used.The primer concentrations were optimized at 0.4 µmol/L each,while the optimal concentration for the probe was 0.16 µmol/L.The RPA method was used to detect suspicious tissues of Penaeus vannamei using optimized sets of primers and probe.The results showed that the RPA method could accurately detect pirA and pirB from shrimp tissues,which was consistent with the PCR method recommended by the World Organization for Animal Health(WOAH).In summary,this PRA methods targeting pirA and pirB respectively could rapid and accurately detect ANPND-causing bacteria with easy operation and short time-consumption,making it a promising supplementary tool for routine monitoring and rapid detection of AHPND.关键词
重组酶聚合酶技术(RPA)/急性肝胰腺坏死病(AHPND)/pirA/pirB/检测Key words
Recombinase polymerase amplification(RPA)/Acute hepatopancreatic necrosis disease(AHPND)/pirA/pirB/Detection分类
农业科技引用本文复制引用
金东升,漏懿荣,王瑶华,段丽君,李其昂,陈琛,闫茂仓,陈炯,周前进..重组酶聚合酶扩增技术检测南美白对虾急性肝胰腺坏死病[J].农业生物技术学报,2024,32(4):961-973,13.基金项目
浙江省公益技术研究计划/农村农业(LGN22C010001) （LGN22C010001）
浙江省重点研发项目(2021C02059) （2021C02059）
国家自然科学基金(42276110) （42276110）
宁波市科技特派员项目(2022S210) （2022S210）
浙江省"三农九方"农业科技协作计划项目(2023SNJF062) （2023SNJF062）
浙江省近岸水域生物资源开发与保护重点实验室开放基金(J2022001) （J2022001）
宁波大学One health交叉学科研究项目(HZ202201) （HZ202201）
温州市基础性科研项目(S2020020) （S2020020）
