六重荧光RT-PCR快速检测A、B型流感等呼吸道病毒方法的建立  

Establishment of a method for rapid detection of respiratory viruses such as influenza A and B by sextuple fluorescent real-time RT-PCR

作　　者：连昌虎 唐屹君[2] 刘涛[3] LIAN Chang-hu TANG Yi-jun LIU Tao(Longgang District Pingdi Division for Disease Prevention and Hygiene, Shenzhen , Guangdong 518117 Shenzhen Center for Disease Control and Prevention , Shenzhen , Guangdong 518055 Shenzhen Chronic Disease Prevention and Control Center, Shenzhen , Guangdong 518000, China)

机构地区：[1]深圳市龙岗区坪地预防保健所,广东深圳518117 [2]深圳市疾病预防控制中心,广东深圳518055 [3]深圳市慢性病防治中心,广东深圳518000

出　　处：《热带医学杂志》2017年第7期872-876,共5页Journal of Tropical Medicine

摘　　要：目的利用六重荧光RT-PCR法建立起一种快速检测A、B型流感病毒/人偏肺病毒/NL63型、HKU1型和OC43型冠状病毒的方法。方法根据A、B型流感病毒/人偏肺病毒/NL63型、HKU1型和OC43型冠状病毒的保守序列,设计相应的引物和Taqman探针,利用一步法RT-PCR试剂盒优化反应体系后,采用十倍稀释体外转录RNA检验建立体系的灵敏度和重复性,并建立相对定量的标准曲线;利用具有多种相似临床症状的呼吸道病毒检验建立体系的特异性。结果 A、B型流感病毒/人偏肺病毒/NL63型、HKU1型和OC43型冠状病毒的检测灵敏度为100 copies/μL,标准曲线的相关系数都在99%以上,重复性良好,特异性实验未发现有非特异性扩增。结论本研究建立的六重荧光RT-PCR技术可以快速、准确地检测A、B型流感病毒/人偏肺病毒/NL63型、HKU1型和OC43型冠状病毒等六种呼吸道病毒。Objective To develop a method to rapidly detect Influenza Virus A ,Influenza Virus B,metapneumovirus, HCOV-NL63,HCOV-HKU1 and HCOV-OC43 by sextuple fluorescence real-time RT-PCR. Methods According to the conservative sequences of Influenza Virus A,Influenza Virus B,metapneumovirus,HCOV-NL63,HCOV-HKU1 and HCOV-OC43,primers and Taq-man probes were designed. One step RT-PCR kit was used to set up sextuple fluorescence real-time RT-PCR system to detect Influenza Virus A and Influenza Virus B,metapneumovirus,HCOV-NL63,HCOV-HKU1 and HCOV-OC43. The 10-fold serial dilution of in-vitro transcribed RNA was used to determine the sensitivity and reproducibility of the sextuple fluorescence real-time PCR assay. Other similar respiratory virus was used to evaluate the specificity of this assay. Results The sensitivity of the system to detect Influenza Virus A ,Influenza Virus B,metapneumovirus,HCOV-NL63,HCOV-HKU1 and HCOV-OC43 were identified accurately 100 copies/μL,and the regression coefficient of the quantitative curve exceeded 99%without nonspecific amplification. Conclusion The detection system based on real-time RT-PCR could be utilized to rapidly and sensitively detect Influenza Virus A and B,metapneumovirus,HCOV-NL63,HKU1 and OC43.

关 键 词：流感病毒A 流感病毒B 偏肺病毒 冠状病毒NL63 冠状病毒HKU1 冠状病毒OC43 六重荧光RT-PCR 

分 类 号：R373.1[医药卫生—病原生物学]