摘要
目的 建立偏肺病毒逆转录实时荧光定量聚合酶链反应（RT-PCR）,并检测临床样本,了解偏肺病毒感染患儿的临床流行病学特点.方法 针对保守基因F合成引物和探针,制备偏肺病毒转录RNA标准品并建立标准曲线,检测线性范围、最低检测限、重复性、灵敏度和特异性.对采集自2010年12月至2011年11月兰州地区急性呼吸道疾病患儿的222份下呼吸道临床标本进行偏肺病毒筛查,同时对阳性标本进行常见呼吸道病毒的共感染筛查,并对偏肺病毒进行相关流行病学分析.结果 方法线性检测范围是10-108拷贝/μl,最低检测限是10拷贝/μl,曲线相关系数是1,扩增效率是91.62％,Ct值组内CV值小于2％.以传统PCR产物的测序结果为参照,逆转录实时荧光定量PCR的敏感性和特异性分别是100％和96.17％.偏肺病毒检出22例（9.46％）,其中男童13例（5.86％）,女童8例（3.60％）.春、夏、秋、冬的检出率分别是15.71％、0％、5.08％、11.11％.偏肺病毒载量与混合感染、疾病种类、临床症状差异均无统计学意义.结论 该方法是检测偏肺病毒灵敏度、特异性和重复性均较好的方法;偏肺病毒在兰州地区儿童呼吸道感染中占有重要地位,长期系统的监测具有重要意义.
Objective To establish and evaluate a Metapneumovirus real-time quantitative reverse transcription polymerase chain reaction （RT-PCR）,detect clinical specimens and explore the clinical prevalence characteristics of Metapneumovirus-infected children.Methods The primers and probes which targeted the conserved genes F were designed,RNA standards were prepared to establish a standard curve,the sensitivity,specificity and reproducibility had been tested.222 lower respiratory clinical specimens were collected from children in Lanzhou area with ARI.Metapneumovirus and co-infection viruses were detected simultaneously;further Metapneumovirus related epidemiology was studied.Results Metapneumovirus linear detection range was 10-10s copies/μl,the lowest detection limit was 10 copies/μl,the correlation coefficient was 1,the amplification efficiency was 91.62%,the CV of Ct value was less than 2%.Take conventional PCR product sequence results as reference,real-time quantitative RT-PCR sensitivity and specificity were 100% and 96.17%.Metapneumovirus detection rate were 9.46%,13 cases for boys （5.86%）,8 case for girls （3.60%）.The detection rate of spring,summer,autumn and winter were 15.71%,0%,5.08%,11.11%.There were no significant differences between the Metapneumovirus viral load and mixed infection or the types of disease,clinical symptoms.Conclusions Metapneumovirus realtime quantitative RT-PCR has been confirmed as a sensitive and specific method.Metapneumovirus was an important agent of children respiratory tract infection in Lanzhou region.We should take the long-term systematic surveillance seriously.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2016年第2期216-219,共4页
Chinese Journal of Experimental and Clinical Virology
关键词
逆转录实时荧光定量PCR
偏肺病毒
Real-time quantitative reverse transcription PCR
Metapneumovirus
