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目的建立人冠状病毒NL63(human coronavirus NL63)的实时荧光定量PCR(real-time fluorescentquantitative PCR,FQ-PCR)方法,了解呼吸道疾病患儿感染人冠状病毒NL63(HCoV-NL63)的情况。方法收集2008年10月至2009年4月因急性呼吸道感染(ARTI)而就诊于福建医科大学省立临床医学院患儿的咽拭子、鼻咽抽吸物、痰标本共151份,设计多聚酶蛋白1a基因的引物和Taqman探针,扩增1a基因片段,并将其克隆到PMD18-T载体上,构建质粒标准品,建立FQ-PCR检测方法,进行敏感性、特异性试验。扩增核衣壳蛋白N基因,对其序列进行初步分析。结果所建立的FQ-PCR方法特异性好,线性范围为101～1010copies/μL,变异系数小于5%。151份临床标本中共检测到2份HCoV-NL63阳性,阳性率为1.3%(2/151)。结论采用FQ-PCR方法可以检测急性呼吸道疾病患儿感染HCoV-NL63的情况。
Objective To establish a real-time fluorescent quantitative PCR (FQ-PCR) method of human coronavirus NL63 to understand the infection of human coronavirus NL63 (HCoV-NL63) in children with respiratory diseases. Methods A total of 151 samples of throat swabs, nasopharyngeal aspirates and sputum were collected from children with acute respiratory infection (ARTI) who were admitted to Provincial School of Clinical Medicine, Fujian Medical University from October 2008 to April 2009. Polymerase chain reaction Protein 1a gene and Taqman probe, amplification of 1a gene fragment, and cloned into PMD18-T vector to construct plasmid standards, the establishment of FQ-PCR detection methods for sensitivity and specificity of the test. Amplify the nucleocapsid protein N gene, preliminary analysis of its sequence. Results The established FQ-PCR method was of good specificity with a linear range of 101-1010 copies / μL with a coefficient of variation of less than 5%. Two out of 151 clinical samples were positive for HCoV-NL63, the positive rate was 1.3% (2/151). Conclusion The detection of HCoV-NL63 in children with acute respiratory diseases can be detected by FQ-PCR.