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 establish a single-channel double-fluorescent PCR method for the detection of Zika virus and Chikungunya virus and lay a foundation for exploring the method of supermultiple fluorescence PCR. Methods According to the principle of TOCE, two hybridization probes with different tag sequences were designed based on the Taqman probe fluorescent PCR technique of Zika virus. The suitable tag sequences were selected by Zika virus strains, Sequence design and synthesis of two theoretically different melting temperature detection probe, also used Zika virus strain assessment of two detection probes; followed by the same principle design and synthesis of chikungunya hybridization probe and detection probe, Single-tube single-channel double-fluorescent PCR test. Results The results of the selection of the tag sequence showed that the positive sample could obtain the corresponding amplification curve only when the melting temperature of the tag sequence was lower than the annealing extension temperature of the fluorescent PCR. According to the suitable tag sequences, two theoretical melting Different temperature detection probe, the actual test can be in the same test channel by melting curve analysis accurate distinction. Single-tube double-fluorescence PCR can accurately detect Chikungunya virus, Zika virus strains and positive samples. Conclusion A single-channel double-fluorescent PCR assay for Chikungunya virus and Zika virus was established, which can be used as a reference for the subsequent development of a variety of multi-fluorescence PCR detection methods of Arbovirus.