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目的建立能同时检测登革热4种血清型的一步法多重荧光定量PCR检测方法,用于登革热病毒分型的早期实验室诊断。方法针对病毒Ⅰ~Ⅳ型使用优化的引物探针,调整反应温度和条件,稀释阳性样本,进行特异性、灵敏度、重复性检测,评估该反应体系的性能。结果 16份样本中,6份经抗体确证的登革热阳性样本使用自建方法检测全为阳性,且1型3份,2、3、4型各1份,均与预期结果一致。流感、乙脑、诺如阳性样本以及健康人血清检测未见特异性扩增曲线,特异性高。登革热病毒2、3、4型最低检测限为10~2 copies/ml。1型最低检测限为10~3 copies/ml。不同型别各浓度梯度检测变异系数小于10%。结论本实验设计的登革热单管多重荧光定量PCR检测体系特异性、灵敏度、重复性都较好。
Objective To establish a one-step multiplexed fluorescent quantitative PCR method for simultaneous detection of four serotypes of dengue fever for the early laboratory diagnosis of dengue virus typing. Methods The optimized primers were designed for virus Ⅰ ~ Ⅳ. The reaction temperature and conditions were adjusted, and the positive samples were diluted. The specificity, sensitivity and reproducibility were tested to evaluate the performance of the reaction system. Results Of the 16 samples, 6 positive samples of dengue fever confirmed by antibody test were all positive using self-built method, and 3 copies of type 1, 1 piece of type 2, 3, and 4 each were consistent with the expected results. Influenza, JE, Norovirus positive samples as well as healthy human serum detection no specific amplification curve, high specificity. The detection limit of dengue virus 2,3,4 type was 10 ~ 2 copies / ml. The lowest detection limit of type 1 is 10 ~ 3 copies / ml. Different types of concentration gradient detection coefficient of variation of less than 10%. Conclusion The dengue single tube multiplex PCR assay designed in this study has better specificity, sensitivity and repeatability.