基于扩增后分析的单管双重荧光PCR方法的建立  

Development of duplex fluorescent PCR assay based on post amplification analysis

作　　者：郑夔[1] 袁帅[1] 孙芳芳[1] 李小波[1] 师永霞[1] 戴俊[1] 黄吉城[1] 康晓平[2] 秦成峰[2] 

机构地区：[1]广东出入境检验检疫局检验检疫技术中心,广东广州510700 [2]军事医学科学院微生物流行病研究所

出　　处：《中国国境卫生检疫杂志》2017年第2期77-82,共6页Chinese Journal of Frontier Health and Quarantine

基　　金：国家质检总局科技计划项目(2015IK066)

摘　　要：目的建立单通道双重荧光PCR方法检测寨卡病毒和基孔肯雅病毒,为探索超多重荧光PCR方法奠定基础。方法根据TOCE原理,首先在寨卡病毒的Taq Man探针荧光PCR技术基础上,设计两条含有不同标签序列的杂交探针,通过寨卡病毒毒株选择合适的标签序列,再根据合适的标签序列设计合成两条理论上解链温度不同的检测探针,同样用寨卡病毒毒株评估两条检测探针;接着按相同原则设计合成基孔肯雅病毒的杂交探针和检测探针,进行单管内单通道双重荧光PCR试验。结果标签序列的选择结果表明,只有当标签序列的解链温度低于荧光PCR的退火延伸温度时,阳性样本才能获得相应的扩增曲线;根据合适的标签序列设计合成的两条理论上解链温度不同的检测探针,实际检测时可以在同一检测通道中通过融解曲线分析准确区分。单管双重荧光PCR可准确检测出基孔肯雅病毒、寨卡病毒的毒株和阳性样本。结论建立了一种针对基孔肯雅病毒和寨卡病毒的单通道双重荧光PCR检测方法,为后续多种虫媒病毒超多重荧光PCR检测方法的开发提供借鉴。Objective To develop a duplex fluorescent PCR assay for detection of Zika virus and Chikungunya virus simultaneously,and to play foundation for the establishment of multiplex fluorescent PCR assay. Methods Based on the principle of tagging oligonucleotide cleavage and extension（TOCE）,two hybridization probes with different tag se- quences were designed,according to the TaqMan probe technology for Zika virus fluorescent PCR. Then an appropri- ate tag sequence was selected through the tests of known Zika virus sample ,and two tag sequence relative detection probes with distinct theoretical melting temperature were designed and evaluated by using known Zika virus sample. With the same procedure ,hybridization probes and detection probes to Chikungunya virus were designed and synthe- sized with that a single tube single channel duplex fluorescent PCR test was proceeded. Results The results of tag sequence selection showed that amplification curve with positive samples could be seen only when the melting tem- perature of the tag sequence was lower than the annealing/extention temperature of fluorescent PCR. Both of the de- tection probes with distinct theoretical melting temperature were designed and synthesised according to the appropri- atetag sequence,could be successfully distinguished in a single channel by melting curve analysis.This method could accurately detect Chikungunya virus and Zika virus in single tube. Conclusion A single tube duplex fluorescent PCR method for the detection of Chikungunya virus and Zika virus had been established,which provide reference for the subsequent development of high-throughput multiplex fluorescent PCR assay for the detection of arboviruses.

关 键 词：多重 荧光PCR 基孔肯雅病毒 寨卡病毒 虫媒病毒 

分 类 号：R184.35[医药卫生—流行病学]