Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay
Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay
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DOI：
10.1128/jcm.44.4.1376-1381.2006
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发表时间：
2006-04-01
影响因子：
9.4
通讯作者：
Miyazaki, K
中科院分区：
文献类型：
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作者：
Fukuda, S;Takao, S;Miyazaki, K
在这项研究中，我们开发了一种一步法、单管基因组特异性逆转录环介导的等温扩增（RT-LAMP）检测方法，用于检测靶向从RNA依赖性RNA聚合酶基因C末端到衣壳N末端/壳结构域区域的诺如病毒（NoV）基因组。这是关于用于检测NoV基因组的RT-LAMP测定的开发的第一份报告。由于NoV基因型的多样性，我们分别使用了9个和13个含有基因组I（GI）和11（GII）的混合碱基的特别设计的引物。RT-LAMP检测方法具有快速、简便、特异、选择性强等优点，在62 ℃恒温条件下，可在90 min内，一般在60 min内得到结果。对于GI和GII，NoV基因组的检测限在10（2）和10（3）拷贝/管之间，并根据基因型进行区分，并且没有发现NoV GI和GII与其他胃肠炎病毒（例如，沙波病毒、人星状病毒、腺病毒40型和41型以及A组和C组轮状病毒）之间的交叉反应。RT-LAMP测定法对RT-PCR的灵敏度和特异性对于GI分别为100%和94%，对于GII分别为100%和100%。这些发现确立了RT-LAMP测定法对于在食源性和人际传播的胃肠炎爆发中从粪便标本中快速检测NoV基因组是潜在有用的。
In this study, we developed a one-step, single-tube genogroup-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of norovirus (NoV) genomes targeting from the C terminus of the RNA-dependent RNA polymerase gene to the capsid N-terminal/shell domain region. This is the first report on the development of an RT-LAMP assay for the detection of NoV genomes. Because of the diversity of NoV genotypes, we used 9 and 13 specially designed primers containing mixed bases for genogroup I (GI) and 11 (GII), respectively. The RT-LAMP assay had the advantages of rapidity, simplicity, specificity, and selectively and could obtain results within 90 min, generally even within 60 min, under isothermal conditions at 62 degrees C. The detection limits for NoV genomes were between 10(2) and 10(3) copies/tube for GI and GII with differentiation by genotype, and no cross-reactions among NoV GI and GII and other gastroenteritis viruses, such as sapovirus, human astrovirus, adenovirus type 40 and 41, and group A and C rotavirus, were found. In the evaluation tests with fecal specimens obtained from gastroenteritis outbreaks, the sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 94% for GI and 100 and 100% for GII, respectively. These findings establish that the RT-LAMP assay is potentially useful for the rapid detection of NoV genomes from fecal specimens in outbreaks of food-borne and person-to-person-transmitted gastroenteritis.