摘要
目的探讨优化的逆转录聚合酶链反应(RT-PCR)检测试剂在流感病毒检测中的应用。方法选取乙型流感病毒Yamagata系细胞毒株1株对其倍比稀释,取高、中、低三个稀释浓度提取流感病毒核酸作为模板,采用具有高扩增效率的SpeedStar HS DNA聚合酶和Hi ScriptⅡReverse Transcriptase逆转录酶进行PCR反应体系优化,并用优化试剂对三个浓度的流感病毒模板进行批间和批内重复性试验,与常规试剂TaKara One Step PrimeScript RT-PCR Kit反应试剂盒进行对比研究。结果优化试剂的PCR反应体系整个扩增反应时间从96 min缩短至55 min;通过Ct值比较,中、低浓度条件下的扩增效率与常规TAKARA试剂比较差异有统计学意义(P<0.05);组间比较,差异有统计学意义(P<0.05);该试剂的组内试验和组间试验的变异系数均小于2%,显示其具有良好的可重复性。优化试剂比TAKARA公司One Step PrimeScript RT-PCR Kit成品试剂盒节约1/4的成本,有一定的经济效益。结论优化的RT-PCR反应试剂,缩短了PCR反应时间,具有良好的稳定性和重复性,同时节约了试剂检测成本,有一定的经济效益和社会推广价值。
Objective To discuss the application of optimized RT-PCR detection reagent in influenza virus detection. Methods One strain of influenza B virus Yamagata was selected and multiply diluted. Three concentrations （high, mid- dle, and low） were selected and viral nucleic acid was extracted-as templates. The PCR system was optimized by SpeedStar HS DNA polymeras and HiScript II Reverse Transcriptase with high amplification efficiency. Intra-batch and inter-batch repetitive tests were conducted on the three templates by optimized reagent. The results were compared with those of routine reagent TaKara One Step PrimeScript RT-PCR Kit. Results With optimized reagent, the reaction time of PCR was shortened from 96 min to 55 min. There were significant differences in Ct values between the amplifi- cation efficiency of middle and low concentration and that of routine TAKARA kit （P〈0.05）. There were significant dif- ferences in Ct values among groups（P〈0.05）. The variable coefficients were lower than 2% in both intra-batch and inter- batch tests, showing good repeatability. The cost of optimized reagent was about 1/4 lower than that of One Step Prime- Script RT-PCR Kit, showing a certain economic benefit. Conclusion Optimized RT-PCR detection reagent can shorten the response time of PCR, with good stability and repeatability, and can lower the cost, showing a certain economic benefit and promotional value.
出处
《中国现代医生》
2017年第21期99-102,共4页
China Modern Doctor
基金
浙江省嘉兴市科技计划项目(2014AY21036-3)
