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。
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.
作者
徐博文
李楠
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)
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2020年第5期564-569,共6页
Academic Journal of Second Military Medical University
基金
国家自然科学基金(81872232,81672798).
