| 张燕,蒙卓成,邢欢,崔小花,杨柳,郑璇,戴尊孝.参照多重PCR片段分析法进行实时荧光PCR法对ABCB1三等位基因测定结果读取方法的确定[J].四川精神卫生杂志,2022,35(2):137-143.Zhang Yan,Meng Zhuocheng,Xing Huan,Cui Xiaohua,Yang Liu,Zheng Xuan,Dai Zunxiao,Interpretation of results obtained using real-time fluorescence PCR based on multiplex PCR fragment analysis for ABCB1 tri-allele[J].SICHUAN MENTAL HEALTH,2022,35(2):137-143 |
| 参照多重PCR片段分析法进行实时荧光PCR法对ABCB1三等位基因测定结果读取方法的确定 |
| Interpretation of results obtained using real-time fluorescence PCR based on multiplex PCR fragment analysis for ABCB1 tri-allele |
| 投稿时间：2021-09-08 |
| DOI：10.11886/scjsws20210908004 |
| 中文关键词: 实时荧光PCR法 多重PCR片段分析 三等位基因 ABCB1 |
| 英文关键词:Real-time fluorescence PCR Multiplex PCR fragment analysis Tri-allele ABCB1 |
| 基金项目:西安市科技计划项目（项目名称：西安市药学（精神卫生）重点实验室，项目编号：201805051ZD2CG35） |
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| 中文摘要: |
| 目的 为解决实时荧光PCR法对三等位基因测定结果无法正常读取的问题，通过参照多重PCR片段分析法，确定实时荧光PCR法读取方法，实现临床对ABCB1三等位基因准确、简便且价格低廉的检测。方法 收集西安市精神卫生中心2020年3月-2021年3月DNA样本2 794例，抽取5%作为实验样本，分别进行实时荧光PCR法和多重PCR片段分析法测定。根据PCR曲线Ct值的比较以及多重PCR片段分析法碱基峰位强度的比较，对比分析两种方法的判读结果，对其中报告结果不相同的样本进行数据核查并确定PCR读取方法。结果 共抽取139例样本，其中存在120例等位基因及19例三等位基因，实时荧光PCR法和多重PCR片段分析法对等位基因的检测结果完全一致。根据多重PCR片段分析结果，对19例三等位基因制定了实时荧光PCR法的读取方法：扩增曲线图中，当∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣<3，分别读取两组碱基Ct值小的碱基，将其组合形成判读结果；当∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣≥3，读取两组碱基Ct值最小的碱基，其纯合型即为判读结果。根据读取方法，修正实时荧光PCR法的测定结果为：1例G/G，1例A/A，4例T/G，5例T/A，8例T/T，与多重PCR片段分析法结果一致。结论 通过多重PCR片段分析法制定实时荧光PCR法对ABCB1三等位基因的读取方法，两者判读结果一致性良好。 |
| 英文摘要: |
| Objective To resolve the problems related to the abnormal interpretations of real-time fluorescence polymerase chain reaction （PCR） results for tri-allele， to formulate the interpretation methods of real-time fluorescence PCR by referring to multiplex PCR fragment analysis， so as to obtain an accurate， simple and cheap detection method for ABCB1 tri-allele.Methods A total of 2 794 DNA samples were collected from Xi'an Mental Health Center from March 2020 to March 2021， and 5% of which were selected as experiments. Real-time fluorescence PCR method and multiplex PCR fragment analysis method were performed respectively. According to the comparison of Ct values of PCR curves and the comparison of base peak intensity in multiplex PCR fragment analysis， comparison and analysis were conducted on the interpretation results of the two methods， and samples with different interpretation results were verified， thereafter， PCR interpretation method was formulated.Results A total of 139 samples were collected， of which 120 alleles and 19 tri-allele were detected. The results of allele detection by two methods were absolutely consistent. In combination with the results of multiplex PCR fragment analysis， a method for the interpretation of real-time fluorescence PCR for 19 cases of tri-allele was developed as follows： when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ in amplification curve was less than 3， the interpretation results were the combination of the base pairs with small Ct values； when ∣∣Ct.G－Ct.T∣－∣Ct.G－Ct.A∣∣ was greater than or equal to 3， the interpretation results were homozygotes from the base pairs with minimum Ct values. According to the interpretation method， the results of real-time fluorescence PCR were revised， and it showed 1 case of G/G， 1 case of A/A， 4 cases of T/G， 5 cases of T/A and 8 cases of T/T， which were consistent with the results of multiplex PCR fragment analysis.Conclusion Referring to the multiplex PCR fragment analysis method， the interpretation of ABCB1 tri-allele by real-time fluorescence PCR is developed， and the two interpretation methods are in good agreement. |
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