CHIKV Real-time PCR检测方法的建立及试剂盒研发

目的建立快速特异检测基孔肯雅病毒的Real-time PCR方法及试剂盒。方法根据发表在Genbank上15株基孔肯雅病毒基因序列全长,应用ClustalW2.0和DNAMAN8软件筛选出位于其E1基因上的种内保守种间特异的目的片段,并据此设计出最优引物。人工合成该基因片段并构建重组质粒,对重组质粒梯度稀释后,再利用SYBR Green I Real-time PCR的方法检测该特异基因的浓度,建立标准品曲线,用于临床上基孔肯雅病毒的早期诊断。结果琼脂糖凝胶电泳结果显示,标准品的PCR扩增产物的电泳条带长度与目标条带一致,测序结果显示与目标条带序列一致,说明引物与阳性质粒性能良好。经优化确定CHIKV荧光定量PCR反应体系中最佳的引物浓度为350 nmol/L,最佳的反应条件为：50 ℃ 2 min;95 ℃预变性2 min;以95 ℃ 15 s,60 ℃ 15 s,72 ℃ 1 min进行40个循环扩增,在72 ℃进行荧光采集。根据荧光定量PCR熔解曲线出现特异性单峰,并对黄病毒属成员登革病毒和寨卡病毒的检测为阴性,表明该检测方法特异性高;检测阈值达302 拷贝 ,显示敏感性好,重复试验的变异系数均小于0.1%,说明该实验设计稳定性良好。结论基于该方法的试剂盒具有特异性好、灵敏度高、重复性好、能够快速定量等优点,有利于临床上基孔肯雅病毒的早期诊断,具有良好的应用前景。

Establishment of CHIKV real-time PCR detection method and diagnostic kit

ObjectiveTo establish a real-time PCR method and diagnostic kit for rapid detection of chikungunya virus (CHIKV). MethodsBased on the full length of 15 strains of CHIKV gene sequences published in Genbank, ClustalW2.0 and DNAMAN8 software were used to screen the CHIKV fragment in E1 gene, which was conserved in species and specific among species. The optimal PCR primers were also designed for this fragment. The fragment was synthesized and cloned on a plasmid. The standard curve was constructed by detecting the amount of the fragment on the recombinant plasmids which were sequentially diluted, with SYBR Green I real-time PCR kit. The quantitative criterion (standard curve) was established for the detection of chikungunya virus. ResultsThe results of agarose gel electrophoresis showed that the length of the PCR amplification products was consistent with that of the target bands, and the sequence was consistent with the target gene’s sequence, indicating that the primers and the positive plasmid were viable. The optimal reaction conditions were as follows: 50 ℃ for 2 min, 95 ℃ for 2 min, 95 ℃ for 15 s, 60 ℃ for 15 s, 72 °C for 1 min, for 40 cycles of repeated amplification, followed by the fluorescence capture at 72 °C. According to the fluorescence quantitative PCR melting curve, a specific single peak appeared, which indicated the specificity of the detection system. The specificity was also confirmed by the negative results of detecting dengue virus and Zika virus with this system. The sensitivity of this detection system was prominent with a detection threshold of 302 copies, and the coefficient of variation (CV) of the repeated test was less than 0.1%. ConclusionThe kit based on this method has the advantages of good specificity, sensitivity, rapidity and stability, which are required for the early diagnosis of CHIKV infection in clinical practice, and therefore, it has a good application prospect.