摘要
为快速高效地检测新型鹅细小病毒(Novel goose parvovirus,NGPV),试验根据NGPV VP3基因序列设计特异性引物及探针,以NGPV KC3S3株提取的DNA为模板,在构建质粒标准品的基础上,建立TaqMan探针荧光定量PCR检测方法。结果显示:该方法的标准曲线为y=-3.0816x+35.389,相关系数(R^(2))为0.9981,质粒标准品浓度为1.0×10^(2)~1.0×10^(8) copies/µL时具有良好的线性关系;该方法对禽流感病毒(Avian influenza virus,AIV)、星状病毒(Astrovirus,AstV)、小鹅瘟病毒(Goose parvovirus,GPV)、番鸭细小病毒(Muscovy duck parvovirus,MDPV)、新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)、禽腺病毒(Fowl adenovirus,FAV)等鸭常见病毒性病原均无特异性扩增;所设计引物及探针的基因序列与GPV及MDPV的相同区域有4~11个碱基发生突变,可以避免GPV及MDPV对检测结果的影响;该方法批内和批间检测变异系数(CV)均小于1.5%,对66份疑似感染NGPV鸭的肝脏样品检测阳性率为59.1%,高于常规PCR检测结果(31.8%)。结果表明,研究所建立的TaqMan探针荧光定量PCR检测方法具有良好的特异性、敏感性和重复性,可以有效区分GPV与MDPV,在NGPV感染的临床诊断和早期防控方面具有良好的应用前景。
To detect novel goose parvovirus(NGPV)rapidly and efficiently,a TaqMan-based real-time quantitative PCR method was developed in this experiment.Specific primers and probes were designed based on the VP3 gene of NGPV,and DNA extracted from NGPV KC3S3 strain was used as the template and to construct the standard plasmid.The results showed that the method exhibited a good linear relationship,with a standard curve equation of y=-3.0816x+35.389,a correlation coefficient(R^(2))of 0.9981 and the standard plasmid content of 1.0×10^(2) to 1.0×10^(8) copies/µL.No specific amplification to major viruses of ducks such as avian influenza virus(AIV),astrovirus(AstV),goose parvovirus(GPV),muscovy duck parvovirus(MDPV),novel duck reovirus(NDRV),fowl adenovirus(FAV)by this mehtod.The designed primers and probe had 4 to 11 nucleotide mutations in the same region betweem GPV and MDPV,allowing accurate detection without interference from GPV and MDPV.The coefficient of variation(CV)was less than 1.5%for intal and interassay evaluation of the mehtod.The positive ratio of NGPV in duck liver samples detected using the established real-time quantitative PCR was 59.1%,which was higher than that using the conventional PCR(31.8%).The results suggested that the real-time quantitative PCR based on TaqMan probe established in this study had good specificity,sensitivity and repeatability,and could effectively distinguish GPV from MDPV,which had a potential to be used for clinical diagnosis and early prevention and control of NGPV infection.
作者
梁国洋
毛明田
郭华
李芳
张帅
唐熠
刁有祥
LIANG Guoyang;MAO Mingtian;GUO Hua;LI Fang;ZHANG Shuai;TANG Yi;DIAO Youxiang(College of Animal Science and Veterinary Medicine,Shandong Agricultural University,Tai'an,Shandong 271018;Yebio Bioengineering Co.,Ltd of Qingdao,Qingdao,Shandong 266003)
出处
《中国家禽》
2024年第12期69-75,共7页
China Poultry
基金
国家现代农业产业技术体系项目(CARS-42-19)。
