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  • 徐博文,李楠*.一种新的同时定量检测乙型肝炎病毒前基因组RNA和DNA的检测方法[J].第二军医大学学报,2020,41(5):564-569    [点击复制]
  • XU Bo-wen,LI Nan*.A new method for simultaneous quantitative determination of hepatitis B virus pregenomic RNA and DNA[J].Acad J Sec Mil Med Univ,2020,41(5):564-569   [点击复制]
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一种新的同时定量检测乙型肝炎病毒前基因组RNA和DNA的检测方法
徐博文,李楠*
0
(海军军医大学(第二军医大学)基础医学院免疫学研究所暨医学免疫学国家重点实验室, 上海 200433
*通信作者)
摘要:
目的 设计并建立一种新的能够同时测定核酸提取物中乙型肝炎病毒(HBV)前基因组RNA(pgRNA)和DNA的检测方法。方法 建立检测HBV pgRNA和DNA的探针法双重荧光定量PCR体系,通过DNA凝胶电泳、定量PCR等实验考察该检测体系的特异性和灵敏度,验证以该方法测定HepG2.2.15细胞及其培养上清中HBV pgRNA和DNA的可行性及准确度。结果 建立的探针法双重荧光定量PCR体系具有较好的特异性和灵敏度,测定不同稀释倍数的细胞培养上清时,在稀释倍数和测定结果间具有较好的相关性。但该方法更适用于测定细胞培养上清中的HBV pgRNA和DNA,而不适用于测定细胞样本。结论 本实验建立的方法能够避免核酸提取物中由于HBV DNA污染造成HBV RNA定量不准的问题,并且在一管PCR反应中同时实现了HBV pgRNA和DNA的双重检测,极大提高了检测效率,具有潜在的临床应用价值。
关键词:  乙型肝炎病毒  慢性乙型病毒性肝炎  定量聚合酶链反应  双重聚合酶链反应  前基因组RNA  DNA  共价闭合环状DNA
DOI:10.16781/j.0258-879x.2020.05.0564
投稿时间:2020-03-15修订日期:2020-04-20
基金项目:国家自然科学基金(81872232,81672798).
A new method for simultaneous quantitative determination of hepatitis B virus pregenomic RNA and DNA
XU Bo-wen,LI Nan*
(Institute of Immunology & National Key Laboratory of Medical Immunology, College of Basic Medical Sciences, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To design and establish a new method for simultaneous determination of hepatitis B virus (HBV) pregenomic RNA (pgRNA) and DNA from nucleic acid extracts. Methods We established a duplex fluorescence quantitative PCR system to determine HBV pgRNA and DNA. DNA gel electrophoresis and quantitative PCR were used to test the specificity and sensitivity. We tested the feasibility and accuracy by determining the HBV pgRNA and DNA in HepG2.2.15 cells and the culture supernatants. Results The established duplex fluorescence quantitative PCR system has a good specificity and sensitivity. When it was used to determine cell culture supernatants with different dilution ratios, the dilution ratios and results were well correlated. However, this method was more suitable for the determination of HBV pgRNA and DNA in cell culture supernatants, rather than cell samples. Conclusion Our method can avoid inaccuracy of HBV RNA determination caused by HBV DNA contaminant in nucleic acid extracts, and realize simultaneous detection of HBV pgRNA and DNA in one PCR reaction, which greatly improves the determination efficiency and has potential clinical application value.
Key words:  hepatitis B virus  chronic hepatitis B  quantitative polymerase chain reaction  duplex polymerase chain reaction  pregenomic RNA  DNA  covalently closed circular DNA