Wu J, Sullivan D E, Gerber M A
Department of Pharmacology, Tulane University School of Medicine, New Orleans, LA.
J Virol Methods. 1994 Oct;49(3):331-41. doi: 10.1016/0166-0934(94)90148-1.
To monitor and compare the effect of drugs on hepatitis B virus (HBV) replication in hepatitis patients, an accurate quantitative method with high sensitivity is needed. Polymerase chain reaction (PCR) is the most sensitive method for the detection of HBV DNA, but because of sample to sample variability in amplification efficiency, the quantification of target nucleic acids by conventional PCR is inaccurate. Therefore, we developed a competitive PCR method with an internal standard which uses the same primers as the target HBV DNA. The standard was generated from the HBV S gene by PCR site-directed mutagenesis and differed from the native S gene by a single base which introduced an internal restriction enzyme site. For practical application, this method was used to quantitate the effect of the carbocyclic analogue 2'-deoxyguanosine (2'-CDG) on HBV DNA replication in 2.2.15 cells, an HBV transfected HepG2 cell line. A decrease of HBV DNA level in the attomole range was demonstrated by the assay in the media of 2.2.15 cells treated with 2'-CDG. The results were verified by conventional HBV DNA slot blot analysis followed by densitometric scanning. Competitive PCR proved to be a sensitive, accurate and reliable method for HBV quantitation.
为监测和比较药物对肝炎患者体内乙型肝炎病毒（HBV）复制的影响，需要一种高灵敏度的准确定量方法。聚合酶链反应（PCR）是检测HBV DNA最灵敏的方法，但由于不同样本间扩增效率存在差异，常规PCR对目标核酸的定量并不准确。因此，我们开发了一种带有内标的竞争性PCR方法，该方法使用与目标HBV DNA相同的引物。该标准品通过PCR定点诱变从HBV S基因产生，与天然S基因仅相差一个碱基，该碱基引入了一个内部限制性酶切位点。在实际应用中，该方法用于定量碳环类似物2'-脱氧鸟苷（2'-CDG）对HBV转染的HepG2细胞系2.2.15细胞中HBV DNA复制的影响。在用2'-CDG处理的2.2.15细胞培养基中，该检测方法证明HBV DNA水平在阿托摩尔范围内有所下降。结果通过常规HBV DNA斑点杂交分析及光密度扫描进行验证。竞争性PCR被证明是一种用于HBV定量的灵敏、准确且可靠的方法。
