Lo E S, Lo Y M, Tse C H, Fleming K A
University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.
J Clin Pathol. 1992 Aug;45(8):689-92. doi: 10.1136/jcp.45.8.689.
Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV).
PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA.
Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus.
This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.
开发一种用于检测乙型肝炎病毒（HBV）前核心、终止密码子突变体的特异性聚合酶链反应（PCR）检测方法。
利用已发表的序列数据合成PCR引物，其3'端对前核心、终止密码子突变体或野生型HBV的核苷酸1896具有特异性。通过PCR合成两种病毒类型的阳性对照模板，在适当位置掺入每种病毒类型的特异性序列。这些模板用于优化该程序的特异性。然后对来自香港或牛津的急性或暴发性HBV肝炎的福尔马林固定、石蜡包埋的人体组织进行突变型或野生型病毒检测。使用两步法从该组织中扩增HBV DNA，首先是初始扩增阶段，然后是对最佳稀释的靶DNA进行第二次诊断阶段。
实现了对突变型或野生型HBV的特异性检测。决定特异性的一个重要因素是退火温度，70℃被证明具有高度特异性。为了克服临床样本中靶标拷贝数的固有差异并提供内源性阳性对照，生成并标准化用于特异性PCR的靶标HBV量很重要。来自香港的2例暴发性肝炎和4例急性肝炎病例，以及来自牛津的1例暴发性肝炎病例，仅含有野生型HBV，没有突变病毒的证据。
该方法可应用于福尔马林固定石蜡包埋组织。它快速、无放射性，对HBV核苷酸1896处的终止密码子突变具有特异性。对来自牛津和香港的少数暴发性肝炎病例的初步调查仅显示野生型病毒。该结果与日本和以色列发表的结果不同。
