中东呼吸综合征冠状病毒半巢式PCR检测方法的建立

Development of a semi-nested PCR assay for detection of Middle East respiratory syndrome coronavirus

目的建立特异、灵敏的半巢式PCR方法以期用于检测中东呼吸综合征冠状病毒(MERS-CoV)。方法根据MERS-CoV N蛋白基因设计并合成特异性内侧、外侧引物。通过条件优化,建立MERS-CoV半巢式PCR检测体系,扩增产物测序后与目的序列进行同源比对。以10倍系列稀释重组质粒为标准品,与普通PCR反应进行比较,检测半巢式PCR方法的灵敏度。以其他6种呼吸道病原体及冠状病毒基因为对照,检测半巢式PCR方法的特异性。结果使用建立的半巢式PCR方法扩增的特异片段与GenBank中发表的MERS-CoV基因序列同源性为100%;半巢式PCR检测方法的灵敏度为1.17×101拷贝,比普通一轮PCR扩增的灵敏度(1.17×105拷贝)提高了104倍;用该方法检测MERS-CoV基因为阳性,其他6种呼吸道病原体及冠状病毒基因检测均呈阴性。结论建立的MERS-CoV半巢式PCR方法具有良好的准确性、灵敏性和特异性,为MERS-CoV感染的诊断提供了一种新的、可靠的检测手段。

Objective To establish a sensitive and specific assay for detection of Middle East respiratory syndrome coronavirus （MERS-CoV） using semi-nested PCR. Methods Semi-nested primers were designed in accordance with the MERS-CoV nucleoeapsid （N） protein gene. Semi-nested PCR was carried out using optimized parameters and the seminested PCR product was sequenced. The sensitivity of the assay was tested using positive DNA prepared by 10-fold serial dilution of the recombinant plasmid and the assay was compared to conventional PCR. In addition, specificity was examined by detecting six control respiratory disease pathogens and coronavirus pathogens. Results The sequence of thesemi-nested PCR product was compared to that of MERS-CoV in GenBank, and the sequence similarity was found to be100%. The assay was sensitive enough to detect as few as 1.17 ×10^1 copies/reaction, which was much more sensitivethan conventional PCR （1.17 × 10^5 copies/reaction）. The assay detected the MERS-CoV recombinant plasmid in samplesand did not detect six control respiratory disease pathogens and coronavirus pathogens. Conclusion The semi-nestedPCR detection assay that was successfully established in this study is highly accurate, sensitive, and specific, thus provi- ding a new, reliable method of detecting MERS-CoV infection.