摘要
背景:在实时荧光定量聚合酶链反应检测过程中,由于设计的热循环参数不同,检测结果常会出现假阳性或假阴性。目的:变性温度、变性时间以及聚合酶链反应运行的温度特性对实时荧光定量聚合酶链反应检测乙型肝炎病毒的影响。设计、时间及地点:对比观察实验,于2007-07-06/08在上海市安图医院PCR实验室完成。材料:LightCycler荧光聚合酶链反应检测仪由美国罗氏公司提供,乙型肝炎病毒核酸扩增聚合酶连反应荧光定量检测试剂盒由深圳匹基公司提供。方法:聚合酶链反应运行程序条件:变性温度分别为88～97℃,变性时间分别为5s和40s,退火和延伸60℃,40s,循环数40。主要观察指标:乙肝病毒遗传物质的数量。结果:聚合酶链反应运行温度上冲和下冲比较严重;变性时间对DNA浓度较低的样本影响很大,DNA很难被检测出;变性温度超过95℃,变性时间超过40s时,检测到的DNA的量明显降低;变性时间小于20s时,容易出现假阴性。结论:变性时间太短、变性温度太高且变性时间太长容易造成乙型肝炎病毒检测出假阴性。
BACKGROUND： False positive/negative results will be induced by different thermal cycling setting in the detection process of polymerase chain reaction （PCR） assay.
OBJECTIVE： To observe the effect of denaturing temperature, time and thermal characteristics of PCR processors on the validity of HBV-DNA obtained by real-time fluorescent PCR.
DESIGN, TIME AND SETTING： The observation was performed at the PCR Laboratory of Shanghai Antu Hospital from July the sixth to July eighth 2007.
MATERIALS： The LightCycler PCR detector was supplied by ROSSI Company （America）, and the HBV quantitative detection reagent was supplied by Shenzhen PG Company （China）.
METHODS： The procedure conditions of PRC： 40 cycles of denaturation temperature from 88 ℃ to 97 ℃ for 5 s and 40 s respectively, and the temperatures of anneal and extension was 60 ℃ for 40 s.
MAIN OUTCOME MEASURES： Amounts of HBV-DNA.
RESULTS： Temperature overshoot and undershoot were severe at the beginning of denaturation and annealing respectively. Denaturation time impacted samples of low DNA obviously. When denaturation temperature was higher than 95 ℃ and denaturation time is longer than 40 s, DNA tested were reduced remarkably. False negative would be induced if denaturation time is lower than 20 s.
CONCLUSION： False negative will be induced when denaturation time is too short or over high denaturation temperature and over long denaturation time.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第44期8677-8680,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金资助项目(50676063)
上海市重点学科(T0503
P0502)~~
