鹅星状病毒基因Ⅰ型和Ⅱ型双重荧光定量RT-PCR检测方法的建立与应用

Establishment and Application of Double Real-time PCR Detection Simultaneously for GAstVⅠand GAstVⅡ

鹅星状病毒(goose astroviruses,GAstV)作为一种雏鹅新发病原,对养鹅业造成巨大经济损失,在尚无有效治疗手段情况下,加强疫病及时检测和防控十分重要。为建立快速、特异且灵敏的鉴别GAstVⅠ型和GAstVⅡ型的病毒检测方法,本研究根据GAstVⅠ和GAstVⅡ的保守区域RdRp基因序列,设计合成2对特异性引物和2种不同荧光基团标记的TaqMan探针,建立了一种双重荧光定量PCR检测方法,以达到在同一反应体系中同时定量检测GAstVⅠ和GAstVⅡ的目的。结果显示,GAstVⅠ和GAstVⅡ的最小检出量分别为43.3、6.49 copies·μL^(-1);重复试验的变异系数不超过0.5%;该方法对DPV、GPV、GTMUV、GRV、AIV(H9N2)等病毒核酸无交叉反应。综上表明,本研究建立的可鉴别GAstVⅠ和GAstVⅡ的双重荧光定量PCR方法灵敏度高、特异性强、重复性好。可用于临床GAstVⅠ和GAstVⅡ的快速鉴别诊断,也可用于两种基因型GAstV的定量分析。

Goose astrovirus(GAstV),as an emerging pathogen causing visceral gout and high mortality in goslings and result in huge economic losses.Thus,it is very important to develop credible detection methods to prevent this diseases wide spread in the absence of effective treatment measure.In order to establish a rapid,specific and sensitive method for detection of genotype GAstVⅠand GAstVⅡviruses,a duplex TaqMan real-time quantitative reverse transcription PCR(RT-qPCR)assay was established.Two pairs of specific primers and two kinds of TaqMan probes labeled with different fluorescent groups according to the RdRp gene sequences were designed and synthesized.The results showed that the minimum detectable amounts of GAstVⅠand GAstVⅡwere 43.3 and 6.49 DNA copies·μL^(-1),respectively;the coefficient of variation of repeated tests was less than 0.5%;and the method had no cross reaction to viral nucleic acids such as DPV,GPV,GTMUV,GRV,AIV(H9N2),etc.In summary,the results above indicate that this duplex real-time PCR method can distinguish GAstVⅠand GAstVⅡwith high sensitivity and specificity.It can be used for the clinical rapid differential diagnosis of GAstVⅠand GAstVⅡ,and quantitative analysis of two genotypes of GAstV.