中国动物传染病学报2025，Vol.33Issue(2)：61-69,9.
口蹄疫病毒O型、A型和Asia Ⅰ型三重实时荧光定量RT-PCR检测方法的建立及初步应用
Development and Preliminary Application of a Triplex FQ-PCR for Detection of Foot-and-Mouth Disease Virus Types O,A and Asia Ⅰ
摘要
Abstract
To develop a triplex fluorescence quantitative real-time PCR(FQ-PCR)assay for detection of foot-and-mouth disease virus(FMDV)types O/A/Asia Ⅰ,this study designed universal inversion transcription primers for O/A/Asia Ⅰ based on the comparison of multiple FMDV genes and the optimal conserved region of 2B genes.According to the comparison results of VP1 gene,3 pairs of specific primers and TaqMan MGB probes for types O/A/Asia Ⅰ of FMDV were then designed with the variation regions as the amplification targets.Afterwards,a triplex FQ-PCR assay for detection of FMDV types O/A/Asia Ⅰ based on probe technology was developed followed by the optimization of the reaction system.The triplex FQ-PCR assay was also evaluated for its specificity,sensitivity and reproducibility.Finally,15 clinically suspected samples of FMDV infection were tested and the results were compared with genetic sequencing and RT-PCR methods.The results indicated that the triplex FQ-PCR assay developed here realized one-step identification of FMDV types O/A/Asia Ⅰ samples.The triplex FQ-PCR assay amplified FMDV types O/A/Asia Ⅰ in the inoculated cell cultures but not seven other pathogens(vesicular stomatitis virus etc.)and negative controls.The minimum template concentration for FMDV types O/A/Asia Ⅰ was 10 copies/μL,which showed its high sensitivity.The coefficients of variation of intra-batch and inter-batch tests ranged from 0.48％-1.55％,indicating its high repeatability.The triplex FQ-PCR assay was then used to test 15 clinically suspected FMDV infection samples.The results were completely consistent with the gene sequencing,which was superior to the RT-PCR assay.In conclusion,the triplex FQ-PCR assay was developed with high sensitivity and specificity,which simultaneously identified and detected FMDV O/A/Asia Ⅰ in the same reaction system,thus meeting the needs of clinical differentiation.关键词
口蹄疫病毒/三重实时荧光定量RT-PCR/应用Key words
Foot-and-mouth disease virus/triplex fluorescence quantitative real-time PCR/application分类
农业科技引用本文复制引用
王翠,闫若潜,赵雪丽,王东方,王淑娟,马震原,杨海波,刘影,柴茂,谢彩华..口蹄疫病毒O型、A型和Asia Ⅰ型三重实时荧光定量RT-PCR检测方法的建立及初步应用[J].中国动物传染病学报,2025,33(2):61-69,9.基金项目
2022年度河南省重点研发与推广专项(222102110227,222102110121,222102110289) （222102110227,222102110121,222102110289）
2022年度河南省重大科技专项(221100110600) （221100110600）
2022年河南省现代农业产业技术体系(HARS-22-12-T) （HARS-22-12-T）
