摘要
为建立一种能够快速检测非洲猪瘟病毒(ASFV)的普通PCR方法,本研究分别以ASFV B646L和F778R基因为靶序列设计了两对特异性引物,建立ASFV B646L和F778R双基因普通PCR方法,并对其特异性、灵敏性及重复性进行检测。结果显示:该PCR体系(25.0μL体系)的最优组合为Pfu酶0.2μL、10×Reaction Buffer 2.5μL、2.5 mmol/L d NTP Mixture 2.5μL、B646L上下游引物各1.5μL、F778R上下游引物各0.5μL、DNA模板1.0μL;退火温度为57℃、退火时间为30 s、72℃延伸2.5 min,30个循环。该方法仅针对ASFV B646L和F778R基因进行特异性扩增,无交叉反应。同时对B646L和F778R阳性质粒的最低检测下限分别为7.2×10^(7)copies/μL、1.5×10^(6)copies/μL,试验重复性良好。本研究对于提高ASFV临床诊断的准确性、特异性和高效性具有重要价值,为其快速检测提供了可靠的技术支撑。
In order to detect African swine fever virus(ASFV)quickly by ordinary PCR,In this study,we established a com-mon PCR method for ASFV B646L and F778R dual genes by two pairs of specific primers which were designed based on the target sequences of ASFV B646L and F778R genes respectively.And its specificity,sensitivity and reproducibility were tested.It showed that the optimal combination of PCR system(25.0μL system)was Pfu enzyme 0.2μL,10×Reaction Buffer 2.5μL,2.5 mmol/L dNTP Mixture 2.5μL,B646L upstream primer and B646L downstream primer were 1.5μL,F778R upstream primer,F778R downstream primer each 0.5μL,DNA template 1.0μL,annealing temperature 57℃,annealing time 30 s.The method was only used for specific amplification of ASFV B646L and F778R genes,without cross-reaction.The lower limits of detection of positive plasmid B646L and F778R were 7.2×10^(7)copies/μL and 1.5×10^(6)copies/μL.The test repeatability is good.This study has important value for improving the accuracy,specificity and efficiency of clinical diagnosis of ASFV,and provides reliable technical support for rapid detection of ASFV.
作者
王慧
钟菊花
杨俊
杜丽飞
王红兵
刘俊琦
WANG Hui;ZHONG Juhua;YANG Jun;DU Lifei;WANG Hongbing;LIU Junqi(Hunan Institute of Animal Husbandry and Veterinary Medicine,Changsha 410131,China;College of Veterinary Medicine,Hunan Agricultural University,Changsha 410128,China)
出处
《激光生物学报》
CAS
2023年第6期554-560,共7页
Acta Laser Biology Sinica
基金
湖南现代农业产业技术体系项目(湘[2022]222号)。
