猪细环病毒2型荧光定量PCR检测方法的优化

Optimization of the detection method of fluorescent quantitative PCR assay for Torque teno sus virus type 2

【目的】优化1种猪细环病毒2型(TTSu V2)的荧光定量PCR检测方法。【方法】对原有引物进行调整设计,以原有重组质粒为模板来重新进行检测TTSu V2 DNA的SYBR Green I real-time PCR扩增。【结果】新的TTSu V2 SYBR Green I荧光定量PCR的扩增效率为99.3%,标准曲线的决定系数为0.998,可准确检测的最低核酸模板浓度为50 copies/μL。与原方法相比较,新方法在熔解曲线、特异性、重复性方面都非常接近,但在扩增曲线方面得到明显改善,其可准确定量检测的灵敏度提高了至少10倍以上,临床检测数据也更加准确可靠。【结论】建立了一种新的准确灵敏度更高的定量检测TTSu V2的荧光定量PCR方法。

【Objective】The aim of this study was to optimize the detection methods of fluorescent quantitative PCR（ FQ-PCR） assay for Torque teno sus virus type 2（ TTSu V2）. 【Method】The primers specific to TTSu V2 in the original method was adjusted,then,the original plasmid was used as templates to detect the SYBR Green I real-time PCR amplification of TTSu V2 DNA. 【Result】The amplification efficiency of the new TTSu V2 SYBR Green I FQ-PCR was 99. 3%. The coefficient of determination for the new standard curve was 0. 998. The new assay had an accurate detectable limit of 50 copies / μL of nucleotide acid template. The melting curve,specificity and reproducibility of the new method were all very similar to the original method. However,the amplification curve was obviously improved,the accurate detectable sensitivity was increased at least 10 times and the clinical detection data was more accurate and reliable in the new assay. 【Conclusion】A new FQ-PCR assay for quantitative detection of TTSu V2 with higher sensitivity was developed.