摘要
目的使用具有高扩增效率的Speed Star HS DNA聚合酶建立快速、可靠的RT-PCR反应体系,应用于流感病毒核酸检测工作,以提高工作效率、缩短检测周期。方法建立优化的基于Speed Star HS DNA聚合酶的高速RT-PCR体系,并与应用较广的Takara one step Prime Script RT-PCR试剂盒比较。2种方法通过对A型和B型流感的基因标准品的一系列10倍稀释液分别检测,比较两者的分析灵敏度、反应时间、扩增效率等。通过对150份流感阳性样品进行检测,比较两者的诊断灵敏度。结果 2个PCR反应体系的灵敏度相当,最低检出限均为10 copy/μl阳性标准品,均有较好的扩增效率,达到90%以上。对150份阳性样本检测的结果表明2种方法有较好的一致性。结论基于Speed Star HS DNA聚合酶的高速RT-PCR反应体系较之Takara one step Prime Script RT-PCR试剂盒灵敏度相当,都有很好的扩增效率,且反应抑制性小,整个PCR时间更短,可应用于流感病毒检测工作,且能明显提高实验室检测的工作效率。
Objective Using Speed Star Hot Start（ HS） DNA polymerase,a fast and reliable RT-PCR reaction system was established and applied to detect the nucleic acid detection of influenza virus in order to improve the working efficiency and shorten the detection cycle. Methods To establish an optimized high speed RT-PCR system based on HS DNA Speed Star polymerase,and to compare it with the widely used one step Prime Script RT-PCR Takara kit. Two methods were used to detect the A type and B type influenza in a series of 10 times diluted solution,respectively. The sensitivity,reaction time and amplification efficiency of the two methods were compared. The test results of 150 positive samples showed that the two methods had good agreement. Results The sensitivity of the two PCR reaction systems was equivalent,and the minimum detection limit was10 copy / μl of positive standard,which had a good amplification efficiency,reaching more than 90%. The test results of 150 positive samples showed that the two methods were in good agreement. Conclusion The sensitivity of the high speed Speed Star reaction system of HS DNA RT-PCR polymerase is equivalent with that of Takara one step Prime Script RT-PCR kits. Both of them have good amplification efficiency,the little response inhibition and shorter whole PCR time,which can be used in the detection of influenza viruses and can significantly improve the efficiency of laboratory testing.
出处
《中国卫生检验杂志》
CAS
2016年第13期1852-1854,共3页
Chinese Journal of Health Laboratory Technology
基金
嘉兴市科技计划项目(2014AY21036-3)
