双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。

The Study of a Novel Nucleic Acid Detection Technology by Double-stranded Probe Real-time PCR

Objective: Using a “double-stranded probe” real-time fluorescent PCR technology to improve the sensitivity of HBV nucleic acid detection, complete the genotype detection of metabolic enzyme CYP2C19 ^*2 in a tube. Methods: The double-stranded probe and the TaqMan probe was used to simultaneously detect different concentrations of HBV in serum samples by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Then, according to the Ct value of nucleic acid detection by instrument to statistical analysis of results; the double-stranded probe was used to detect samples of different genotypes of metabolic enzyme CYP2C19^*2 in a tube, and the detection of nucleic acid Ct value and genotype analysis were performed by Shanghai Hongshi SLAN 96 real-time fluorescent PCR instrument. Results: In the detection of HBV serum samples at different concentrations, the fluorescence background of the double-stranded probe was low and the detection sensitivity was higher than the TaqMan probe. And significant differences were noted between the two probes (P<0.05);The metabolic enzyme CYP2C19^*2 genotypes of 36 samples were detected using the double-stranded probe, the results were consistent with those of Sanger sequencing. Conclusion: The double-stranded probe real-time fluorescent PCR detection technology can complete the highly sensitive nucleic acid detection of the target gene and also the genotype analysis.