摘要
为了建立一种敏感、特异的牛冠状病毒(BCoV)实时荧光定量RT-PCR检测方法,试验利用GenBank中BCoV参考毒株N基因序列,设计引物及小沟结合物(MGB)探针,建立实时荧光定量RT-PCR方法,对方法的特异性、灵敏度和稳定性进行检验。利用建立的方法对采集自7个省份31个发病牛场的925份临床样本进行检测,并用基于BCoV N基因的普通RT-PCR结合测序的方法对部分阳性样品进行验证。结果表明:建立的实时荧光定量RT-PCR方法的扩增体系为上下游引物(10μmol/L)各1μL,探针(10μmol/L)0.8μL,2×One Step RT-PCR BufferⅢ10μL,Ex Taq HS(5 U/μL)0.4μL,Prime Script RT Enzyme MixⅡ0.4μL,总RNA 2μL,ddH_(2)O 4.4μL;扩增程序为,42℃5 min;95℃10 s;95℃5 s,55℃35 s,40个循环。标准曲线方程为y=40.979-3.512x,可信度(R^(2))为0.999,扩增效率(E)为92.6%,最低检测限为48 copies/μL,重复性变异系数(CV)不超过0.82%。31个发病牛场BCoV阳性场占比为64.52%(20/31),其中腹泻症状阳性场占比为54.55%(12/22),呼吸道症状阳性场占比为65.22%(15/23);阳性样品经基于BCoV N基因的普通RT-PCR方法验证,测序正确。说明建立的BCoV实时荧光定量RT-PCR检测方法灵敏度高、特异性强、稳定性好,能检测出临床样本中的BCoV核酸,可应用于BCoV感染的诊断、监测与流行病学调查。
In order to establish a sensitive and specific real-time fluorescent quantitative RT-PCR detection method for Bovine coronavirus(BCoV),based on the N gene sequences of BCoV reference strains in GenBank,the experiment designed primers and MGB probes to establish real-time fluorescent quantitative RT-PCR method;the specificity,sensitivity and stability of the method were tested.The established method was used to detect 925 clinical samples collected from 31 diseased cattle farms in seven provinces,and some positive samples were verified by ordinary RT-PCR combined with sequencing method based on BCoV N gene.The results showed that the amplification system of the established real-time fluorescent quantitative RT-PCR method was as follows:upstream and downstream primers(10μmol/L)were 1μL each;probes(10μmol/L)were 0.8μL;2×One-Step RT-PCRⅢ10μL,Ex Taq HS(5 U/μL)0.4μL,Prime Script RT Enzyme MixⅡ0.4μL,total RNA 2μL,ddH_(2)O 4.4μL;amplification procedure was 42℃5 min;95℃10 s;95℃5 s,55℃35 s,40 cycles.Standard curve equation was y=40.979-3.512x;the reliability(R^(2))was 0.999;the amplification efficiency(E)was 92.6%;the minimum detection limit was 48 copies/μL,and the repeatability coefficient of variation(CV)did not exceed 0.82%.In 31 sick cattle farms in China,BCoV-positive farms accounted for 64.52%(20/31);among them,the proportion of BCoV positive field in diarrhea was 54.55%(12/22)and the proportion of BCoV positive farms with respiratory symptoms was 65.22%(15/23).The positive samples were verified by the common RT-PCR method based on the BCoV N gene,and the sequencing was correct.The results suggested that the established real-time fluorescent quantitative RT-PCR detection method for BCoV had high sensitivity,strong specificity and good stability,which could detect BCoV nucleic acids in clinical samples,and be applied to the diagnosis,monitoring and epidemiology survey of BCoV infection.
作者
高辉
李晓成
刘莹
袁野
张欣怡
尼博
张慧
段纲
董雅琴
魏荣
GAO Hui;LI Xiaocheng;LIU Ying;YUAN Ye;ZHANG Xinyi;NI Bo;ZHANG Hui;DUAN Gang;DONG Yaqin;WEI Rong(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China;China Animal Health and Epidemiology Center,Qingdao 266032,China;Tianjin Centers for Disease Control and Prevention,Tianjin 300402,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2022年第6期86-91,共6页
Heilongjiang Animal Science And veterinary Medicine
