摘要
为实现对口蹄疫病毒(FMDV)感染的现场快速检测,本研究于GenBank下载全球具有代表性的107条血清O型、A型和Asia-1型FMDV基因组序列,截取并比对FMDV高度保守的3D(RNA聚合酶)基因序列,设计17套环介导等温扩增引物,并对反应体系和反应条件进行优化,建立FMDV群特异性RT-LAMP技术。该技术在反应前用石蜡密封PicoGreen染料,实现了免开盖可视化判定检测结果;该技术对PRV、PRRSV、AKAV、BTV-1、BTV-16、EHDV、CHUV等偶蹄动物病毒未见非特异性扩增;最低可检测3.4×10;ng/μL的病毒RNA,灵敏度是RT-qPCR技术的10倍,是一步法RT-PCR的100倍;尝试应用RT-LAMP对94份RNA临床样品进行检测比对,该检测方法与RT-PCR方法和RT-qPCR方法对临床样品检测的符合率均为82.8%。本研究建立的FMDV群特异性RT-LAMP快速核酸检测技术,具有准确性高、快速简便的优点,可用作家畜FMDV早期感染的现场诊断技术。
The aim of the present study was to develop a rapid method for detection of FMDV infection on sites.One hundred and seven genomic sequences expressing global serotypes O,A and Asia-1 FMDV deposited in GenBank were downloaded and the highly conserved 3D(RNA polymerase)gene was then selected and aligned for designing 17 sets of primers for loop-mediated isothermal amplification,followed by optimizing the reaction system and conditions in order to develop a FMDV group-specific RT-LAMP technique.PicoGreen dye sealed with paraffin was used before amplification reaction,which made the test results visible without opening the lid.No amplification was observed for other viruses of cloven-hooved animals such as PRV,PRRSV,AKAV,BTV-1,BTV-16,EHDV and CHUV.The detection limit of this technique was 3.4×10;ng/μL of viral RNA,which was 10 times more sensitive than that of conventional RT-qPCR,and 100 times more sensitive than one step RT-PCR.Furthermore,RT-LAMP achieved a coincidence up to 82.8%in detection of 94 RNA samples along with RT-qPCR and RT-PCR.In conclusion,the FMDV group-specific RT-LAMP developed in this study was of high accuracy and rapid detection,providing a suitable technique for field diagnosis of early infection.
作者
谢佳芮
寇美玲
苗海生
XIE Jiarui;KOU Meiling;MIAO Haisheng(Yunnan Key Laboratory of Tropical and Subtropical Animal Virology,Key Laboratory of Tropical and Subtropical Animal Virology,Ministry of Agriculture and Rural Affairs,Yunnan 650224,China)
出处
《中国动物传染病学报》
CAS
北大核心
2022年第3期119-126,共8页
Chinese Journal of Animal Infectious Diseases
基金
云南省技术创新人才培养对象(2018HB076)
国家重点研发计划(2017YFC1200502)。
关键词
口蹄疫病毒
环介导等温核酸扩增
群特异性引物
FMDV
loop-mediated isothermal nucleic acid amplification
group-specific primers
