鸭坦布苏病毒荧光定量RT-PCR方法的建立及应用

Establishment and Application of a Fluorescence Quantitative RT-PCR Assay for Duck Tembusu Virus

为建立鸭坦布苏病毒(duck tembusuvims,DTMUV)荧光定量RT-PCR (qRT-PCR)检测方法,对DTMUV的NS5基因设计特异性引物和MGB探针,使用RT-PCR扩增NS5基因并将其连接到pEASY-T1载体上构建重组质粒,提取阳性重组质粒作为qRT-PCR阳性模板绘制标准曲线,用SYBRGreenI法检测MGB探针法qRT-PCR的引物是否有非特异性扩增,并进行特异性、敏感性、重复性试验和临床样品检测。结果显示:MGB探针法qRT-PCR标准曲线的相关系数为0.999 3,其引物没有非特异性扩增;对于其他阳性样本,如鸭肝炎病毒(DHV)、新城疫病毒(NDV)、禽流感病毒(AIV)等抗原无扩增曲线,只能检出DTMUV;qRT-PCR敏感性可达3×10^(2) copies/μL,是普通RT-PCR的10 000倍;批内和批间重复性试验变异系数(CV)为0.08%~0.49%,均小于0.5%。从云南省不同地区采集20份临床出现产蛋下降症状的病鸭组织,应用试验建立的qRT-PCR方法检出12份阳性样品,而普通RT-PCR检出10份,两者的阳性率分别为60% (12/20)和50% (10/20)。结果表明,本试验成功建立了基于DTMUV NS5基因的MGB探针qRT-PCR检测方法,其标准曲线线性关系显著,特异性强,与其他禽源性病原无交叉反应性,敏感性和重复性良好,敏感性和阳性检出率较普通RT-PCR高,更适合于DTMUV感染的临床诊断和流行病学调查。

In order to develop a fluorescent quantitative reverse transcriptase-polymerase chain reaction(qRT-PCR)assay for duck tembusu virus(DTMUV),special primers and MGB probes were designed based on DTMUV NS5 gene that was then amplified by RT-PCR to connect to pEASY-T1 vector to construct recombinant plasmids,then positive recombinant plasmids were extracted as the positive template of qRT-PCR to drew a standard curve,the primers of MGB probe-based qRT-PCR were detected by SYBR Green I method to determine whether there was any non-specific amplification,followed by the tests of specificity, sensitivity and repeatability as well as detection of clinical samples.The results showed that the determination coefficient of the standard curve was 0.999 3,and no any non-specific amplification was found;for other positive samples,no amplification curve was found in the antigens of duck hepatitis virus(DHV),Newcastle disease virus(NDV) and avian influenza virus(AIV),and only DTMUV could be detected;the detection limit of qRT-PCR could be up to 3 × 10^(2) copies/μL,which was 10 000 times higher than that of conventional RT-PCR;the coefficient of variations(CV) of intra-assay and inter-assay repeatability tests were in the range of 0.08% to 0.49%,both of which were less than 0.5%.20 tissues were collected from sick ducks with clinical symptoms of decreased egg production in different regions in Yunnan Province,12 positive samples were detected by the established qRT-PCR, but only 10 by conventional RT-PCR,the positive rates by the two methods were 60%(12/20) and 50%(10/20),respectively.In conclusion,the qRT-PCR of MGB probes based on DTMUV NS5 gene was successfully established with strong specificity,its standard curve was with obvious linear relationship,and no cross-reaction with other avian pathogens was found,its sensitivity and repeatability were good,especially its sensitivity and the positive detection rate was higher than that of conventional RT-PCR.Therefore, the method was more suitable for clinical diagnosis and epidemiological investigation of DTMUV infection.