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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。

Detection of human metapneumovirus in respiratory specimen by fluorescent quantitative PCR

Objective To establish fluorescent quantitative RT-PCR of detecting human metapneumovirus ( HMPV ), and to explore the HMPV infectious status and clinical features. Methods Searching conservative HMPV N gene segment for PCR amplification by DNAstar,compare and analyze whole genome sequence of series of HMPV ; primers were designed and the method of RT-PCR to detect HMPV were established. HMPV in 623 clinical specimens from hospitalized children from December 2009 to March 2010 were detected by fluorescent quantitative RT-PCR and direct immunofluorescence assay simultaneously. Results Taking HMPV as template to carry out fluorescent quantitative PCR,we got positive result,but other common respiratory virus and Mycoplasma pneumoniae showed negative results. By direct immunofluorescence assay,10 cases were HMPV positive ( 1.61% ), and by fluorescent quantitative PCR method 28 cases were positive ( 4.49% ) . Statistical significance between the two methods was found ( χ 2 =16.05 ,P < 0.01 ) . Of 10 cases with positive direct immunofluorescence assay,PCR all were positive,and 595 cases were negative by the two methods . A good correlation was found (r= 0.59 ,P <0.01 ) . The other 18 PCR-positive cases were negative by direct immunofluorescence method.The positive rate of HMPV in asthma was 15.4% ( 4/26 ), much higher than 3.9% of the pneumonia group ( 20/515 ) and the tracheobronchitis group 0% ( 0/36 ), showing statistical significance ( χ 2 = 11.22,P= 0.011 ) .The positive rate correlated with age,which in 0 ～ 1y group was 2.2% ( 9/404 ), in > 1 ～ 3y group it was 13.1%(),>～(), larger-than-6 y group it was 2.6% ( 1/39 ) . The positive rate of HMPV in > 1 ～ 3 y group was much higher than other groups ( χ 2 = 26.44 ,P= 0.000 ) . Conclusion Bychosing conservative area of HMPV N gene a pair of primers and single band of TaqMan probe are established,and the method of fluorescent quantitative RT-PCR is bulit up to detect HMPV. The sensitivity of fluorescent quantitative RT-PCR in detecting HMPV is much higher than that of direct immunofluorescence assay. The positive rate of HMPV in asthma is obviously higher than that of pneumonia. The positive rate of HMPV is the highest in children among > 1 ～ 3 years old.