摘要
本研究设计的一种4重荧光定量RT-PCR检测方法,以A型流感病毒各亚型的血凝素基因(HA)为检测靶标,实现了同时检测新甲型H1N1流感病毒、人季节性H1N1流感病毒和人季节性H3N2流感病毒。本法使用人细胞RNA酶P基因作为内参,以判断标本来源和实施质量控制。利用不同来源和亚型的流感病毒验证了该方法的特异性;利用连续稀释的新甲型H1N1流感病毒HA全基因体外转录物进行灵敏度分析。结果表明该方法灵敏度高,可检测低至20个拷贝的RNA;特异性强,每对引物只检测出对应的病毒,无交叉反应;并且成功地验证性检验了34份新甲型H1N1流感病毒阳性临床标本和20份人季节性H1N1和H3N2流感病毒及人乙型(HB)流感病毒阳性临床标本。因此,该多重荧光定量RT-PCR法是一种可同时检测2009年新甲型流感病毒及季节性流感病毒的有效方法。
In this study,we established a rapid and sensitive multiplex Real-time reverse transcription polymerase chain reaction （mrtRT-PCR） to simultaneously detect the novel human influenza A H1N1 virus,human seasonal influenza A H1N1 and H3N2.This assay had three pairs of primer to target the conserved regions of the HA gene for each of the HA types including novel H1N1,seasonal H1N1 and seasonal H3N2,and one pair of primer designed to detect the internal control-RnaseP gene. This assay was performed in one-step in one tube. To validate the specificity of the multiplex Real-time RT-PCR assay,different human influenza virus including human seasonal influenza A H1N1 and H3N2,human influenza B and reference A/California/07/2009（H1N1）sw1 was tested. To evaluate the sensitivity of the assay,serial dilutions of RNA from in vitro transcription of the novel human influenza A H1N1 HA gene was tested. Finally this assay was evaluated with clinical samples from 54 fever patients diagnosed with novel influenza A H1N1 or seasonal H1/H3 or HB infection either by real-time PCR recommended by the WHO or HI assay by the National Influenza Center. Our results showed that the assay could achieve a sensitivity of 20 RNA copies of novel influenza A H1N1 with high specificity and could detect potential mixed co-infection.In conclusion,this multiplex real-time RT-PCR assay combines both rapidity and sensitivity for not only detecting the novel human influenza A H1N1 virus,but also monitoring the human seasonal influenza A H1N1 and H3N2 simultaneously.
出处
《病毒学报》
CAS
CSCD
北大核心
2010年第2期97-102,共6页
Chinese Journal of Virology
基金
传染病防治重大专项(2009ZX10004-101
2008ZX10004-002
2008ZX10004-001
2008ZX10004-004)
关键词
新甲型H1N1
实时定量PCR
多重检测
novel influenza A H1N1 virus
Real-time RT-PCR
multiplex detection
