Establishment and Preliminary Application of One-step Reverse Transcriptase Droplet Digital PCR Assay for Bovine Viral Diarrhea Virus  

作　　者：Bing Liyuan Ye Jingfei Liu Jiwei Wang Shuai Zheng Dingcheng Meng Tingting Shang Yumo Ciren Qiongda Sun Liang Guo Li 

机构地区：[1]Jilin Agricultural Science and Technology University,Changchun 132109,China [2]Jilin Provincial Animal Disease Prevention and Control Center,Changchun 130000,China [3]Jilin Academy of Agricultural Sciences,Changchun 130000,China

出　　处：《Animal Husbandry and Feed Science》2023年第1期30-35,共6页动物与饲料科学（英文）

基　　金：Supported by Science and Technology Development Plan Fund Project of Jilin Province(20210202101NC);YDZJ202203C G Z H 050.

摘　　要：[Objective]The paper was to establish a one-step reverse transcriptase droplet digital PCR(RT-ddPCR)assay for bovine viral diarrhea virus(BVDV).[Method]Based on one-step real-time quantitative PCR(RT-qPCR)assay,BVDV-specific primers and probes were designed in this study.The reverse transcriptase,annealing temperature,primer and probe concentrations and reaction conditions of RT-ddPCR assay were optimized.Meantime,the specificity,sensitivity and repeatability of RT-ddPCR assay were evaluated.[Result]The optimal reverse transcription system for the established RT-ddPCR assay was as follows:commercial one-step reverse transcriptase droplet digital PCR kit with matching reagents,a final primer concentration of 900 nmol/L,a final probe concentration of 250 nmol/L and an optimal annealing temperature of 57℃.The results were negative when the method was used to detect other common epidemic viruses;the minimum detection limit was 3.2 copies/μL with good repeatability,and the coefficient of variation was less than 5%.RT-ddPCR and RT-qPCR assays were used to test 24 bovine swab samples and the test results showed that the established RT-ddPCR assay was superior to RT-qPCR assay.[Conclusion]The RT-ddPCR assay established in this study has strong specificity,high sensitivity and good repeatability,and is suitable for nucleic acid detection of clinical samples.This study provided a technical support for early detection and quantitative diagnosis of BVDV infection.

关 键 词：Bovine viral diarrhea virus One-step procedure Droplet digital PCR Quantitative detection 

分 类 号：S855[农业科学—临床兽医学]