Department of Medical Microbiology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands.
J Virol Methods. 2011 Nov;177(2):174-83. doi: 10.1016/j.jviromet.2011.08.005. Epub 2011 Aug 11.
The ssRNA+ family Coronaviridae includes two subfamilies prototyped by coronaviruses and toroviruses that cause respiratory and enteric infections. To facilitate the identification of new distantly related members of the family Coronaviridae, we have developed a molecular assay with broad specificity. The consensus-degenerated hybrid oligonucleotide primer (CODEHOP) strategy was modified to design primers targeting the most conserved motifs in the RNA-dependent RNA polymerase locus. They were evaluated initially on RNA templates from virus-infected cells using a two-step RT-PCR protocol that was further advanced to a one-step assay. The sensitivity of the assay ranged from 10(2) to 10(6) and from 10(5) to 10(9) RNA copy numbers for individual corona-/torovirus templates when tested, respectively, with and without an excess of RNA from human cells. This primer set compared to that designed according to the original CODEHOP rules showed 10-10(3) folds greater sensitivity for 5 of the 6 evaluated corona-/torovirus templates. It detected 57% (32 of 56) of the respiratory specimens positive for 4 human coronaviruses, as well as stool specimens positive for a bovine torovirus. The high sensitivity and broad virus range of this assay makes it suitable for screening biological specimens in search for new viruses of the family Coronaviridae.
ssRNA+科冠状病毒科包括冠状病毒和环曲病毒两个亚科，它们引起呼吸道和肠道感染。为了方便鉴定冠状病毒科中与远缘相关的新成员，我们开发了一种具有广泛特异性的分子检测方法。我们对依赖 RNA 的 RNA 聚合酶基因座中最保守的基序进行设计，修改了一致性退化杂交寡核苷酸引物（CODEHOP）策略，设计出针对这些基序的引物。我们最初用病毒感染细胞的 RNA 模板，采用两步 RT-PCR 方案对这些引物进行了评估，然后进一步优化为一步法检测。当用和不用人细胞 RNA 过量进行检测时，该检测方法的灵敏度分别为 10(2)到 10(6)和 10(5)到 10(9)个 RNA 拷贝数。与根据原始 CODEHOP 规则设计的引物相比，6 种评估的冠状病毒/环曲病毒模板中有 5 种的灵敏度提高了 10-10(3)倍。该引物组可检测到 57%（32/56）对 4 种人类冠状病毒呈阳性的呼吸道标本，以及对牛环曲病毒呈阳性的粪便标本。该检测方法具有高灵敏度和广泛的病毒检测范围，非常适合筛选用于寻找冠状病毒科新病毒的生物标本。
