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建立一种可同时检测诺如病毒GⅠ和GⅡ型的双重荧光定量一步法RT-PCR方法。应用针对GⅠ型和GⅡ型诺如病毒的特异性引物以及Taqman探针,优化反应体系及条件,建立双重荧光定量一步法RT-PCR方法。对该方法的灵敏度、特异性、重复性进行评估,并对粪便标本进行检测,以传统RT-PCR方法作为参考评价此方法。结果表明,该方法特异性强,与肠道腺病毒、轮状病毒、星状病毒均无交叉反应,对GⅠ和GⅡ型诺如病毒核酸的最低检出限分别可达103拷贝/μL,具有很好的重复性。对100份粪便标本进行检测,诺如病毒的阳性率为31%,其中GⅠ型为3%,GⅡ型为28%,而传统RT-PCR法的检出率为22%。本文建立的双重荧光定量一步RT-PCR法能同时对GI和GII型诺如病毒进行快速检测,其灵敏度高、特异性好,可应用于胃肠炎病人中检测诺如病毒。
Abstract：The object of this study is to develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GⅠ and GⅡ norovirus.The specific primers,Taqman probes,optimized reaction solution and condition were used to develop the duplex fluorescent quantitative one-step RT-PCR assay.The sensitivity,specificity and reproducibility of the assay were evaluated.The assay was evaluated by testing the 100 specimen samples and compared with the reference assay conventional RT-PCR.The assay possessed high specificity for norovirus detection without any evident cross-reaction with enteric adenovirus,rotavirus or astrovirus.The detection limit of the real-time RT-PCR assay,for GⅠ and GⅡ norovirus was up to 103 copy/μL respectively.Compared with the conventional RT-PCR assay,the assay in this study had higher sensitivity with higher detection rate of norovirus in stool specimens.The duplex fluorescent quantitative one-step RT-PCR assay provides rapid,sensitive and reliable detection of GⅠ and GⅡ norovirus,and could be used as a laboratory diagnosis of norovirus in acute gastroenteritis patients.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.002392
中图分类号:R440
引用信息:
[1]周冬梅,靳淼,李慧莹,等.双重荧光定量一步RT-PCR法同时检测GⅠ、GⅡ型诺如病毒[J].病毒学报,2013,29(03):310-315.DOI:10.13242/j.cnki.bingduxuebao.002392.
基金信息:
国家自然科学基金项目(81000727);; 国家科技重大专项(2012ZX10004215)
2013-05-21
2013-05-21
2013-05-21