摘要
旨在建立一种高灵敏度的检测马疱疹病毒4型的3D数字PCR(3D-dPCR)方法,为马鼻肺炎的早期诊断提供技术支持。通过优化3D-dPCR反应体系中引物和探针的浓度和退火温度,评估其灵敏性、特异性、重复性。结果显示,建立的3D-dPCR方法中引物和探针的最佳浓度分别为0.4、0.13μmol/L,最佳退火温度为60℃。该方法的R^2为0.9964,具有良好的线性关系,灵敏度高,最低检测限为5.4 copies/μL;与马疱疹病毒1型、马泰勒虫、马动脉炎病毒的核酸无交叉反应;RSD小于5%。结果表明,本研究中建立的3D-dPCR方法灵敏度高、特异性强,可作为马疱疹病毒4型定量检测的方法。
To establish a high-sensitivity 3D digital PCR(3D-d PCR)method for detection of equine herpesvirus type 4(EHV-4)and provide technical support for early diagnosis study of equine rhinopneumonitis,by optimizing the working concentration and annealing temperature of the primers and probes,the limit of detection,specificity and repeatability of this method were evaluated.The results indicated that the optimal concentration of primers and probes was 0.4μmol/L,0.13μmol/L respectively,and the optimal annealing temperature was 60℃.The R^2 value of 3D-d PCR standard curve was 0.9964,with a good linear response.Furthermore,the limit of detection was 5.4 copies/μL.There was no cross reaction with the nucleic acid of equine herpesvirus type 1,Theileria equi and equine arteritis virus.The RSD was less than 5%.The results showed that the established 3D-d PCR method had high sensitivity and specificity,thus could be used for quantitative detection of EHV-4.
作者
陈凯云
王科珂
雷程红
徐新峰
胡都斯·艾尔肯
徐军
白梅花
史茜
肖媛媛
赵相鹏
CHEN Kai-yun;WANG Ke-ke;LEI Cheng-hong;XU Xin-feng;Hudusi·AIERKEN;XU Jun;BAI Mei-hua;SHI Qian;XIAO Yuan-yuan;ZHAO Xiang-peng(Beijing Institute of Inspection and Quarantine Science and Technology,Beijing 101113,China;Urumqi Customs Technology Center,Urumqi 830063,China;College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2020年第7期833-839,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2017YFF0210305)
2019海关总署科研计划项目(2019HK049)。
