福建农业学报2024，Vol.39Issue(9)：1044-1050,7.DOI:10.19303/j.issn.1008-0384.2024.09.005
新型番鸭细小病毒荧光RPA恒温检测方法的建立
A Constant Temperature Fluorescent RPA Assay for Detecting Novel Muscovy Duck Parvovirus
摘要
Abstract
[Objective]A constant temperature fluorescent RPA assay for detecting novel Muscovy duck parvovirus(N-MDPV)was developed.[Method]EXO fluorescent probes were specifically bound to the targeted conserved VP3 fragment of N-MDPV.RPA amplification primers were designed to amplify the segment by using recombinant enzyme polymerase.An assay for detecting N-MDPV was established with reaction time and temperature optimized and specificity,sensitivity,and accuracy tested on the collected nucleic acid of disease material in comparison with traditional PCR and virus isolation identification methods.[Result]The optimized assay reaction temperature and time were 39℃for 30 min to achieve a lowest sensitivity for nucleic acid detection at 10 fg·μL-1.The nucleic acid of N-MDPV was specifically amplified without any cross reaction with those of duck adenovirus type 3,fowl adenovirus type 4,duck circovirus,duck plague virus,duck hepatitis virus,duck tembusu virus,and novel duck reovirus.Along with the conventional PCR and the virus isolation and identification methods,the newly developed assay detected the nucleic acid on 38 duck tissue specimens with a positive rate of 36.8%(14/38)in comparison to those at 36.8%(14/38)for the PCR and 31.6%(12/38)for the isolation and identification method.In addition,100%coincidence rates of the assay and the two other methods on positive samples as well as of the assay and the PCR on the positive clinical samples were secured.[Conclusion]The new RPA method to rapidly and visually detect N-MDPV demonstrated to be highly specific,sensitive,and accurate.It was deemed appropriate for clinical applications.关键词
新型番鸭细小病毒/RPA恒温检测/重组酶聚合酶扩增技术Key words
novel Muscovy duck parvovirus/constant temperature fluorescent RPA assay/recombinase polymerase amplification technology分类
农业科技引用本文复制引用
陈炜,黄瑜,梁齐章,张佳雪,焦文龙,林永强,刘荣昌,傅秋玲,傅光华,朱婷..新型番鸭细小病毒荧光RPA恒温检测方法的建立[J].福建农业学报,2024,39(9):1044-1050,7.基金项目
国家现代农业产业(水禽)技术体系建设专项(CARS-42) （水禽）
福建省百千万工程领军人才项目(FJTMTP-2014) （FJTMTP-2014）
福建省农业科学院自由探索科技创新项目(ZYTS202423) （ZYTS202423）
福建省种业创新与产业化工程项目(Zycxny2021014) （Zycxny2021014）
