H1亚型禽流感病毒RT-RPA-LFD快速检测方法的建立及应用
Development of Rapid Detection Method for H1 Subtype Avian Influenza Virus RT-RPA-LFD
曾婷婷 1李孟 1谢芝勋 1李丹 1谢丽基 1罗思思 1张民秀 1王盛 1谢志勤 1范晴 1阮志华 1粟永春2
作者信息
- 1. 广西壮族自治区兽医研究所 广西兽医生物技术重点实验室/农业农村部中国(广西)-东盟跨境动物疫病防控重点实验室,广西 南宁 530001
- 2. 广西金陵农牧集团有限公司/广西博士后创新实践基地,广西 南宁 530049
- 折叠
摘要
以H1亚型AIV的HA基因为研究对象,采用重组聚合酶核酸等温扩增技术(RPA),通过RT-PCR方法优选出1套引物,进而优化体系的反应温度、最佳探针及引物浓度,使用优化后的最佳反应体系进行敏感性及特异性试验,并探索该方法能达到的最短反应时间,利用侧向流动免疫(LFD)试纸条观测反应后的结果,最终建立检测H1亚型AIV的 RT-RPA-LFD快速检测方法.结果表明:建立的 RT-RPA-LFD快速检测方法在探针工作浓度为0.14 mmol/L,37℃条件下反应20 min,可最低检测到1.5×102 copies/反应的H1亚型AIV RNA,其他常见家禽病原体未见阳性反应.该方法与RT-PCR及鸡胚分离鉴定测序结果完全一致,可满足H1亚型AIV临床快速鉴别诊断的需求,为基层兽医现场快速检测提供技术支撑.
Abstract
Taking the HA gene of H1 subtype AIV as the research object,recombinant polymerase nucleic acid isothermal amplification technology(RPA)was used to select a set of primers by RT-PCR method,and the temperature of the reaction system and the probe and primer concentration were optimized.The sensitivity and specificity of the method were tested using the optimized reaction system,and the optimized system was used to explore the shortest reaction time.Based on the results of lateral flow dipstick(LFD),the RT-RPA-LFD rapid detection method for H1 subtype AIV was established.The results showed that the rapid RT-RPA-LFD assay for detection of H1 subtype AIV RNA could detect 1.5×102 copies/response of H1 subtype AIV RNA at the probe working concentration of 0.14 mmol/L and 37℃for 20 min,and no positive reaction was found for other common avian pathogens,which was consistent with the results of RT-PCR and chicken embryo isolation and sequencing.The rapid detection method can meet the need of rapid clinical differential diagnosis of H1 subtype AIV,and provide technical support for the rapid detection of grass-roots veterinary field.
关键词
H1亚型AIV/等温扩增技术/快速检测/RT-RPA-LFDKey words
H1 subtype AIV/Isothermal amplification technology/Rapid detection/RT-RPA-LFD引用本文复制引用
基金项目
广西壮族自治区重点研发计划(桂科AB21076004)
广西自然科学基金(2022GXNSFAA035445)
广西自然科学基金(2021GXNSFBA196031)
广西科技基地和人才专项(桂科AD23049005)
广西八桂学者项目(2019-A50)
出版年
2024
国家科技期刊平台
NSTL
万方数据