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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 To establish a rapid and reliable RT-PCR reaction system using Speed Star HS DNA polymerase with high amplification efficiency for the detection of influenza virus nucleic acid in order to improve work efficiency and shorten the test cycle. Methods An optimized high-speed RT-PCR system based on Speed Star HS DNA polymerase was established and compared with the widely used Takara one step Prime Script RT-PCR kit. The two methods were used to detect the sensitivity, reaction time and amplification efficiency of a series of 10-fold dilutions of gene standards of influenza A and B respectively. 150 samples of influenza-positive samples were tested to compare the diagnostic sensitivity of the two. Results The sensitivity of the two PCR reaction systems was comparable, with the lowest detection limits of 10 copies / μl positive standard, all of which had good amplification efficiency, reaching more than 90%. The results of testing 150 positive samples showed good agreement between the two methods. Conclusion The high-speed RT-PCR reaction system based on Speed Star HS DNA polymerase is more sensitive than the Takara one step Prime Script RT-PCR kit, and has good amplification efficiency with less reaction inhibition and shorter PCR time , Can be applied to the detection of influenza virus, and can significantly improve the efficiency of laboratory testing.