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目的建立实时荧光定量聚合酶链反应(RT-PCR)检测呼吸道标本人偏肺病毒(HMPV)方法;探讨HMPV感染现状及其临床特征。方法自行设计引物,建立检测HMPV荧光定量PCR的方法并与直接免疫荧光检测法(DFA)比较,对2009年12月至2010年3月浙江大学附属儿童医院623例下呼吸道感染患儿的临床标本进行检测与分析。结果 (1)建立的RT-PCR以HMPV为模板获得阳性结果,对其他常见呼吸道病毒均阴性;(2)623例样本中DFA检出HMPV感染10例,检出率1.61%,RT-PCR检出HMPV感染28例,检出率4.49%;(χ2=16.05,P<0.01);(3)DFA阳性者10份RT-PCR均阳性,595份两者均阴性,18份标本DFA阴性RT-PCR呈阳性,显示很好的相关性(r=0.59,P<0.01);(4)哮喘患儿中HMPV检出率为15.4%(4/26),显著高于肺炎组(20/515,3.9%)及气管支气管炎组(0/36,0%),差异有统计意义(χ2=11.22,P=0.011);(5)HMPV检出率与年龄有关,0～1岁阳性率2.2%(9/404),>1～3岁组13.1%(16/122),>3～6岁组3.4%(2/58),>6岁组2.6%(1/39),其中>1～3岁年龄组患儿HMPV检出率明显高于其他组(χ2=26.44,P=0.000)。结论选择HMPVN基因保守区域设计了一对引物和一条TaqMan探针,建立了检测HMPV荧光定量RT-PCR方法;荧光定量RT-PCR检测HMPV敏感性明显高于直接免疫荧光法;哮喘患儿中HMPV检出率明显高于肺炎患儿;HMPV感染以>1～3岁幼儿发生率最高。
Objective To establish a real-time fluorescent quantitative polymerase chain reaction (RT-PCR) detection of respiratory syncytial human metapneumovirus (HMPV) method; investigate the status and clinical features of HMPV infection. Methods The primers were designed to establish a method for detecting HMPV fluorescence quantitative PCR and compared with direct immunofluorescence detection (DFA). The clinical data of 623 children with lower respiratory tract infection from Children’s Hospital of Zhejiang University from December 2009 to March 2010 were analyzed. For testing and analysis. Results (1) The positive results of RT-PCR established by HMPV template were negative for other common respiratory viruses. (2) In 623 samples, 10 cases were detected by DFA, the detection rate was 1.61%, RT-PCR (3) DFA positive were 10 positive by RT-PCR, 595 were negative by both, 18 samples were negative by DFA-negative RT-PCR (P <0.01) PCR showed positive correlation (r = 0.59, P <0.01). (4) The detection rate of HMPV in children with asthma was 15.4% (4/26), which was significantly higher than that in pneumonia group (20/515, (Χ2 = 11.22, P = 0.011); (5) The detection rate of HMPV was related to age, and the positive rate of 0 to 1 year old was 2.2% (9/404), 13.1% (16/122)> 1-3 years old, 3.4% (2/58)> 3-6 years old and 2.6% (1/39)> 6 years old, The detection rate of HMPV in 3-year-old children was significantly higher than that in other groups (χ2 = 26.44, P = 0.000). Conclusion A pair of primers and a TaqMan probe were designed in the conserved region of HMPVN gene and a quantitative RT-PCR method for detecting HMPV fluorescence was established. The sensitivity of HMPV by fluorescence quantitative RT-PCR was significantly higher than that of direct immunofluorescence. HMPV The detection rate was significantly higher in children with pneumonia; HMPV infection was the highest incidence of children aged> 1-3 years.