锁核酸结合分子信标技术检测乙型肝炎病毒DNA G1896A点突变研究

The detection of HBV DNA G1896A point mutation with locked nucleic acid technology combined with molecular beacon

目的研究和评估乙型肝炎病毒(HBV)DNA G1896A点突变的实时荧光PCR检测方法。方法收集经测序验证HBV DNA G1896A未突变的野生型样本150例和已发生突变的突变型样本58例,在突变区域设计分子信标探针,点突变处进行锁核酸处理,利用荧光PCR方法检测HBV前C区G1896A点突变;再从临床标本中随机抽取18例、8例和19例荧光PCR结果分别显示为G1896A突变的标本、杂合的标本以及野生型的标本的PCR产物进行序列测定。结果突变型质粒和野生型质粒的检测灵敏度均可以达到100copies/ml;突变型探针检测高浓度的野生型质粒时无检测信号,野生型探针检测高浓度突变型质粒时无检测信号;突变型模板在总杂合模板中的比例达到5%时则都可以将突变型检测出来;序列测定的8例G1896A突变标本、6例杂合标本和19例野生型标本的PCR产物结果和荧光PCR检测结果完全吻合。结论实时荧光PCR检测方法可以快速、简便、准确地检测HBVDNAG1896A点突变,是检测点突变的重要方法,具有重要的临床应用价值。

Objective The aim of this study is to investigate and evaluate the real-time fluorescent PCR detection method of hepatitis B virus （HBV） DNA G1896A point mutations. Methods A total of 158 HBV samples which had been analyzed for G1896A mutation were selected, including 150 non-mutation samples and 58 mutant samples. The molecular beacon probe was designed in the mutant region, and the sample was processed with locked nucleic acid in the point mutation region. The pre C region G1896A point mutations of HBV was detected with Fluorescent PCR, then use DNA sequencing to confirm 8 mutant samples, 6 heterozygous samples, and 19 non-mutation samples which had been detected by real-time fluorescent PCR detection method. Results （1）Both the sensitivity of mutant plasmids and of wild-type plasmids achieved 100 copies / ml. （2）There was no detection signal when we used mutant probe to detect the high concentration of wild-type plasmids. It was the same when we used wild-type probe to detect the high concentration of mutant plasmids. （3）The mutant could be detected when the mutational template accounted for 5% of the total heterozygous. （4）All the result of detection with sequencing coincided with that of detection with real-time fluorescent PCR detection method. Conclusion Because real-time fluorescent PCR detection method can rapidly, easily, and exactly detect the HBV DNA G1896A point mutation, thus it is an important method to detect point mutations and has important clinical value.