鹅星状病毒和鹅坦布苏病毒双重nano-PCR检测方法的建立及初步应用

Establishment and primary application of a duplex nano-PCR method for detection of goose astrovirus and goose tembusu virus

为快速鉴别诊断鹅星状病毒(GoAstV)和鹅坦布苏病毒(TMUV),本研究根据GenBank上公布的GoAstV和TMUV基因保守区设计了2对特异性引物,结合金纳米材料,通过对反应条件的优化,建立了一种可同时检测这两种病毒的双重nano-PCR诊断方法。结果显示,该方法对鹅细小病毒(GPV)、鹅圆环病毒(GoCV)、新城疫病毒(NDV)和禽腺病毒(FAdV)这4种鹅常见病毒的扩增结果均为阴性。该方法对GoAstV和TMUV的最低检测浓度分别为1.21×10^(1) copies/μL和1.21×10^(3) copies/μL,敏感性较普通PCR分别高100倍和10倍。对42份临床样品的检测结果显示,共检出GoAstV 13份,TMUV 1份。上述结果表明,本研究建立的双重nano-PCR方法快速、特异、敏感,可用于鹅星状病毒和鹅坦布苏病毒的临床检测和流行病学调查。

In order to rapidly identify goose astrovirus(Go Ast V) and goose tembusu virus(TMUV)simultaneously,a duplex Nano-PCR method was established in this study by designing two pairs of specific primers according to the conserved regions of Go Ast V and TMUV genes published in Gen Bank,and optimizing the reaction conditions.Specific results showed that the amplification results were negative for goose parvovirus(GPV),goose circovirus(Go CV),Newcastle disease virus(NDV) and avian adenovirus(FAd V).Sensitivity results showed that the minimum concentrations of Go Ast V and TMUV were1.21×10^(1)copies/μL and 1.21×10^(3)copies/μL,respectively.and the sensitivity was 100 times and 10times higher than that of conventional PCR,respectively.Clinical sample detection results showed that13 Go Ast V and 1 TMUV were detected in 42 clinical samples.These results indicate that the duplex nano-PCR method established in this study is rapid,specific and sensitive,and can be used for clinical detection and epidemiological investigation of goose astrovirus and goose tembusu virus.