Niu Peihua, Zhang Chen, Lu Roujian, Wang Ji, Lou Yongliang, Tan Wenjie, Ma Xuejun
Institute of Medical Virology, Wenzhou Medical University, Wenzhou 325035, China.
Email:
Zhonghua Yu Fang Yi Xue Za Zhi. 2014 May;48(5):416-9.
To develop an one-tube multiplex RT-PCR assay for simultaneous detection of six human coronaviruses (HCoVs).
Genbank sequences of six human coronaviruses, including HCoV-NL63, HCoV-229E, SARS-CoV, HCoV-OC43, MERS-CoV, and HCoV-HKU1, were included as reference sequences. Primers were designed based on multiple alignment of reference sequences, targeting the conserved regions of each species of HcoV. Virus strains and nucleic acid preserved in our lab were used as template in developing this automatic-electrophoresis-based one-tube multiplex RT-PCR assay. Detection limits and reproducibility were also evaluated with these templates. Samples with infection of other respiratory viruses preserved in our lab were used to evaluate specificity of this assay. Finally, we tested this assay with 140 clinical samples that were validated by real-time PCR in parallel.
This automatic-electrophoresis-based multiplex RT-PCR assay was able to detect six human coronaviruses simultaneously. All positive samples in this study were detected with at least one specific fragment of anticipated length (195, 304, 332, 378, 415, 442 bp) . No fragment was detected in negative controls. Detection limits of 1.0×10(1-1.0)×10(2) copies/µl were achieved in tests of single virus. No cross reaction was observed with other respiratory viruses. This multiplex RT-PCR assay showed the same sensitivity and specificity to that of individual real-time RT-PCR validated with clinical samples. Both methods detected 28 positive samples (20%) .
Six HCoVs can be detected in one tube by this novel multiplex RT-PCR assay with high sensitivity and specificity.
开发一种用于同时检测六种人类冠状病毒（HCoV）的单管多重逆转录聚合酶链反应（RT-PCR）检测方法。
将六种人类冠状病毒的基因库序列，包括HCoV-NL63、HCoV-229E、SARS-CoV、HCoV-OC43、MERS-CoV和HCoV-HKU1，作为参考序列。基于参考序列的多重比对设计引物，靶向每种HCoV的保守区域。在开发这种基于自动电泳的单管多重RT-PCR检测方法时，使用保存在我们实验室的病毒株和核酸作为模板。还使用这些模板评估了检测限和重现性。使用保存在我们实验室的感染其他呼吸道病毒的样本评估该检测方法的特异性。最后，我们用140份经实时PCR平行验证的临床样本对该检测方法进行了测试。
这种基于自动电泳的多重RT-PCR检测方法能够同时检测六种人类冠状病毒。本研究中的所有阳性样本均检测到至少一个预期长度（195、304、332、378、415、442 bp）的特异性片段。阴性对照中未检测到片段。在单病毒测试中实现了1.0×10(1-1.0)×10(2)拷贝/微升的检测限。未观察到与其他呼吸道病毒的交叉反应。这种多重RT-PCR检测方法对临床样本验证的单个实时RT-PCR显示出相同的敏感性和特异性。两种方法均检测到28份阳性样本（20%）。
通过这种新型的多重RT-PCR检测方法可以在一管中检测六种HCoV，具有高敏感性和特异性。
