实时荧光聚合酶链反应方法在2006年广州地区儿童流行性感冒病毒监测中的应用

目的 应用实时荧光聚合酶链反应(PCR)方法检测流感病毒,并对2006年广州地区儿童感染流感病毒进行流行病学分析.方法 以流感病毒的NP基因序列为参考,综合分析GenBank上的NP基因序列,使用引物和探针设计软件Primer Express设计A/B型流感病毒及A亚型流感病毒各1对特异性引物和1条TaqMan荧光探针,用于分别检测A/B型流感病毒及A亚型流感病毒,建立实时荧光PCR方法,进行灵敏度和特异性检测.采用实时荧光PCR方法,对来自2006年门诊或住院呼吸道感染患儿的12 301份咽拭子或鼻腔抽洗液样本进行检测,并与病毒分离方法进行对照.结果 实时荧光PCR方法共检出1687份阳性样本,总阳性率为13.71%,其中A型773份(45.8%),全部为H1N1型;B型914份(54.2%).1-4月份检出流感病毒阳性525例,其中B型流感病毒455例(86.7%),A型(H1N1)流感病毒70例(13.3%);5-8月份检出流感病毒阳性1118例,其中B型流感病毒419例(37.5%),A型(H1N1)流感病毒699例(62.5%).病毒培养分离阳性为1380份,均为实时荧光PCR方法阳性,占实时荧光PCR方法阳性者的81.80%.结论 实时荧光PCR方法应用于流感病毒的检测具有简便快捷、特异、敏感的特点,2006年广州地区儿童流行性感冒病毒的流行1-4月份以B型流感病毒为主,5-8月份以H1N1亚型流感病毒为主.

Application of fluorescent real-time polymerase chain reaction in analyzing the epidemic of influenza among children in Guangzhou area in 2006

Objective To investigate the prevalence of influenza virus infections in children in 2006 using the real-time PCR method.Methods (1) Consulting the most conserved sequence NP gene of influenza virus,after comparing with the NP gene sequences of influenza virus in GenBank,one pair of specific primers and one TaqMan probe were designed for each subtype of influenza virus by the software Primer Express.The sensitivity of influenza was evaluated by testing known positive samples which had been two-fold diluted.The specificity of real-time PCR for influenza virus detection was assessed by cross testing 60 isolates of influenza A,16 isolates of influenza B,and by testing a variety of other respiratory viruses positive samples;(2) 281 nasopharyngeal aspirate samples were detected by real-time PCR and virus isolation;(3)the 12 301 specimens from the patients of Guangzhou Children's Hospital were tested by using the real-time PCR method.Furthermore,the real-time PCR reagent was evaluated by comparing with the result of virus isolation.Results ( 1 ) The sensitivity of real-time PCR developed in this study for influenza A detection was 1:222 and for influenza B was 1 :220 in two-fold serially diluted way.(2) No positive results were found in cross testing of other viruses positive specimens.(3) Influenza virus was detected from 1687 cases (13.71%) out of the 12 301 cases,including 773 cases (45.8%) positive for subtype A and 914 eases (54.2% ) positive for subtype B;455 out of 525 (86.7% ) of influenza B positive specimens and 70 out of 525 (13.3% ) of influenza A (H1N1) positive specimens were from patients seen during January to April;419 out of 1118 (37.5%) specimens positive for influenza B and 699 out of 1118 (62.5%)specimens positive for influenza A(H1N1) were from patients seen from May to August.Influenza virus could be identified from 1380 samples by the methods of virus isolation,accounting for 81.80% of the 1687 positive samples detected by real-time PCR.All the influenza virus subtype A was H1N1.Conclusion The real-time PCR method developed in this study was sensitive and specific for detecting influenza A and B in clinical specimens.During 2006,influenza A and influenza B co-circulated.The predominant vires was influenza B from January to April,peaking i'n ApriL Influenza A (HIN1) prevailed from May to August,with the peak in June.