摘要
根据GenBank已发表的鸭坦布苏病毒(Duck Tembusu virus,DTMUV)基因组(登录号:JX273153)保守基因序列,设计了1对特异性引物及TaqMan探针,以DTMUV重组质粒为阳性标准品模板,建立了快速、准确检测DTMUV的荧光定量RT-PCR方法。结果显示,该方法特异性好,仅能在DTMUV样本中检出荧光信号,与其他病原不发生交叉反应;敏感性高,最低检测限可达25 copies/μL;不同梯度定量模板的拷贝数的对数值与CT值之间的线性关系表达式为Ct=–3.323×Lg(conc)+43.70,相关系数R2=0.997,扩增效率E=100%,DNA在2.5×101～2.5×107个拷贝数内检测曲线有良好的线性关系。对来源于江苏省和安徽省的29份疑似鸭坦布苏病毒感染的病鸭组织进行荧光定量RT-PCR检测,结果有27份呈现阳性,阳性检出率为93.10%,比常规RT-PCR的敏感性高。研究结果表明,该方法快速、灵敏、特异、重复性好且能实时定量检测,可用于DTMUV的临床检测和分子流行病学调查。
A pair of primers and a TaqMan probe were designed according to the reported genomic sequence of Duck Tembusu virus (DTMUV) (GenBank accession No. JX273153) and an accurate and rapid real-time RT-PCR assay was then developed. The real-time RT-PCR method specifically detected DTMUV and had no cross-reaction with of other duck pathogens. The sensitivity of the assay was 25copies/μL. The linear correlation was observed at DNA concentrations from 2.5×101copies/μL to 2.5×107copies/μL. The equation of the assay was Ct=–3.323×Lg (conc)+43.70. The correlation coefficient was 0.997 and PCR efficiency reached up to 100%. Twenty-nine samples were collected from diseased ducks on different farms in Jiangsu and Anhui provinces and detected using this method. As a result, 27 samples were tested positive (93.10%). The real-time RT-PCR assay developed in the present study was a rapid, sensitive and specific method for reliable and quantitative detection of DTMUV, which can be used for clinical diagnostics and molecular epidemiology.
出处
《中国动物传染病学报》
CAS
2013年第4期46-50,共5页
Chinese Journal of Animal Infectious Diseases
