中国实用儿科杂志

中国实用儿科杂志 ›› 2012, Vol. 27 ›› Issue (08): 615-618.

• 论著 • 上一篇    下一篇


荧光定量聚合酶链反应方法检测呼吸道标本人偏肺病毒临床研究

吴    栩a，汪天林b，陈志敏b，吴亦栋c    

  1. 浙江大学医学院附属儿童医院 a在职研究生，现就职于慈溪市人民医院b呼吸科c基因诊断中心，浙江杭州    310003
  • 出版日期:2012-08-06 发布日期:2012-08-27
  • 基金资助:

    浙江省教育厅基金项目（20061408）

Detection of human metapneumovirus in respiratory specimen by fluorescent quantitative PCR.

WU Xu， WANG Tian-lin， CHEN Zhi-min， WU Yi-dong. Children’s Hospital，Zhejiang University School of Medicine， Hangzhou 310003，China   

  • Online:2012-08-06 Published:2012-08-27

摘要：

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HMPV N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。

Abstract:

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（χ2=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（χ2＝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%（16/122）， in＞3～6y group it was 3.4%（2/58）， and  in 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（χ2＝26.44，P＝0.000）.Conclusion    By chosing 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. 

Key words:  , human metapeumovirus；fluorescent quantitative RT-PCR；direct immunofluorescence assay