单管多重荧光定量RT-PCR方法鉴别新甲型H1N1及甲型和乙型流行性感冒病毒  

Discrimination of novel influenza A （H1N1） and influenza A and influenza B viruses using a single-tube multiplex RT-real time PCR

作　　者：张弘[1] 何永强 张严峻[3] 王真[1] 李榛[3] 

机构地区：[1]浙江中医药大学附属第一医院医务部,杭州310006 [2]浙江出入境检验检疫局技术中心 [3]浙江省疾病预防控制中心病毒检测所

出　　处：《中华预防医学杂志》2012年第3期273-276,共4页Chinese Journal of Preventive Medicine

基　　金：国家质量监督检验检疫总局科研项目（2007IK017）;浙江省科技厅重大科技项目（2010C12005-2）

摘　　要：目的建立和评价鉴别新甲型H1N1、甲型和乙型流行性感冒（简称流感）病毒的单管多重荧光定量RT-PCR方法。方法2010年10月至2011年4月，采集流感样患者咽拭子样本213份。分别选用新甲型H1N1流感病毒HA基因、甲型流感病毒M基因和乙型流感病毒NP基因中的152、128和107bp片段作为荧光定量PCR检测的靶片段，设计特异性引物和不同发光基团标记的TaqMan探针，采用体外转录构建标准品，绘制标准曲线，评价检测方法的重复性、特异度和灵敏度，并对213份流感样患者咽拭子样本进行检测及测序验证。结果3种病毒对应的标准曲线分别为：新甲型H1N1流感病毒：Y=-3．461gX＋46．985；甲型流感病毒：Y=-3．491gX＋37．709；乙型流感病毒：Y=-3．51lgX＋38．889，Y为Ct值，lgX为病毒RNA拷贝数的对数值。质粒标准曲线的方差系数为0．998，灵敏度达到每个反应10^2拷贝／μl，特异度强。213份样本中检出新甲型H1N1流感病毒核酸39份（18．3％），甲型流感病毒核酸阳性63份（29．6％），乙型流感病毒核酸阳性23份（10．8％），阳性样品经过测序验证。结论为鉴别新甲型H1N1、甲型和乙型流感病毒建立的单管多重荧光定量RT-PCR方法快速、特异且灵敏。Objective To establish and evaluate a single-tube muhiplex RT-real time PCR assay for detecting novel influenza A H1N1, influenza A and influenza B viruses （called ＂IV＂ for short） simultaneously. Methods A total of 213 clinical specimens of influenza-like patient＇s throat swab were collected during October 2010 and April 2011. 152 bp fragment in HA gene of novel influenza A H1N1 virus, 128 bp fragment in M gene of influenza A virus and 107 bp fragment in NP gene of influenza B virus were chosen as the target genes for multiplex RT-real time PCR, a specific primers and probes labeled with different fluoresceins were designed. The standard plasmid was constructed using in vitro transcription assay, and the standard curve was established. The reproducibility, specificity and sensitivity of the assay were evaluated. Furthermore,RNA extracted from 213 clinical specimens of throat swab was detected and verified by sequencing. Results The corresponding standard curves of novel influenza A H1N1 virus, influenza A virus and influenza B virus were Y = - 3. 46lgX ＋ 46. 985, Y = - 3. 49lgX ＋ 37. 709, Y = - 3. 51lgX ＋ 38. 889, respectively; Y was eycle threshold （ Ct ） , and lgX was logarithm value of virus replication number. The standard curve coeffieient was 0. 998. The detection limit of this assay was 102 copies/μl in one reaction. The specificity was strong. 39 （ 18. 3% ） ,63 （29.6%） and 23 （ 10. 8% ） of 213 clinical specimens detected were positive for novel influenza A H1 N1 virus RNA, influenza A virus RNA and influenza B virus RNA respectively. The positive samples were verified by sequencing. Conclusion The single-tube multiplex RT-real time PCR assay developed in this study for deteeting and identifying novel influenza A H1 N1 ,influenza A and influenza B viruses simultaneously was rapid, specifie and sensitive.

关 键 词：逆转录聚合酶链反应 流感病毒A型 HIN1亚型 流感病毒A型 流感病毒B型 

分 类 号：R4[医药卫生—临床医学] R51