呼吸道合胞病毒A与B亚型多重荧光PCR检测方法的建立及应用  

Establishment and application of a novel multiplex real-time PCR for detection of respiratory syncytial virus subgroup A and B

作　　者：刘文宽[1] 周荣[1] 陈德晖[1] 檀卫平[2] 邱淑燕[1] 许多[1] 李潇[1] 

机构地区：[1]广州医科大学附属第一医院呼吸疾病国家重点实验室,广州510230 [2]中山大学孙逸仙纪念医院儿科,广州510120

出　　处：《临床检验杂志》2016年第10期751-754,共4页Chinese Journal of Clinical Laboratory Science

基　　金：广州市科技计划重大专项(201508020252;201504010032)

摘　　要：目的建立一种新的呼吸道合胞病毒(RSV)分型检测方法,用于快速检测和监测。方法针对RSV G基因设计引物、探针,并引入人β-actin基因(ACTB)作为内参质控,建立RSV-A、RSV-B多重荧光PCR检测方法。使用多种常用呼吸道病原体及体外转录RNA产物考核其特异性和灵敏度。并对2015年1月至12月广州两家医院的儿童急性呼吸道感染患者进行检测。结果所建立的多重检测方法未见非特异性反应。RSV-A、RSV-B和ACTB分别可检测低至4、8和12 copies/μL的RNA模板,且分别在10~1×10^(10)copies/μL、100~1×10^(10)copies/μL和100~1×10^(10)copies/μL时具备良好的线性关系,线性相关系数r2均大于0.99。儿童急性呼吸道感染患者监测中,RSV-A为期间优势亚型。结论本研究所建立的RSV-A、RSV-B多重荧光PCR检测方法,具有较好的特异性、灵敏度、可操作性,可用于相关研究。Objective To establish a novel method for detection of respiratory syncytial virus （RSV） subgroups to diagnose and proceed epidemiologic survey. Methods The primers and probes of the novel multiplex real-time PCR method for testing RSV-A and RSV-B were designed based on the differences of G genes between RSV-A and RSV-B. Human β-actin gene（ACTB） was used as inner-control. A panel of respiratory pathogens, RNA transcripts of RSV-A and RSV-B in vitro and ACTB were used for testing the specificity and sensitivity. The clinical samples of pediatric patients with acute respiratory tract infection from two hospitals in Guangzhou between January and December 2015 were analyzed. Results No nonspecific reactions were observed. The minimal amounts of detectable RNA of RSV-A and RSV-B calculated from the fluorescent signals were 4 and 8 copies/μL respectively, and ACTB RNA was 12 copies/μL. The fine linear relationships were found from 10 to 1×10^10copies/μL of RSV-A, 100 to 1×10^10copies/μL of RSV-B, and 100 to 1×10^10copies/μL of ACTB RNA. The liner factor r2 was greater than 0.99. RSV-A was the dominant subgroup in monito- ring for pediatric patients with acute respiratory tract infection. Conclusion The established multiplex real-time PCR showed high spe- cificity, sensitivity and operability in the detection of RSV-A and RSV-B, and could be used for further study.

关 键 词：呼吸道合胞病毒 呼吸道合胞病毒A亚型 呼吸道合胞病毒B亚型 荧光定量PCR 多重检测 

分 类 号：R446.5[医药卫生—诊断学]