VanDevanter D R, Warrener P, Bennett L, Schultz E R, Coulter S, Garber R L, Rose T M
PathoGenesis Corporation, Seattle, Washington 98119, USA.
J Clin Microbiol. 1996 Jul;34(7):1666-71. doi: 10.1128/JCM.34.7.1666-1671.1996.
A consensus primer PCR method which amplifies a region of herpesviral DNA-directed DNA polymerase (EC 2.7.7.7) and which uses degenerate primers in a nested format was developed. Primers were designed to target sequences coding for highly conserved amino acid motifs covering a region of approximately 800 bp. The assay was applied to 22 species of herpesviruses (8 human and 14 animal viruses), with PCR products obtained for 21 of 22 viruses. In the process, 14 previously unreported amino acid-coding sequences from herpesviral DNA polymerases were obtained, including regions of human herpesviruses 7 and 8. The 50 to 60 amino acid-coding sequences recovered in the present study were determined to be unique to each viral species studied, with very little sequence variation between strains of a single species when studied. Template dilution studies in the presence of human carrier DNA demonstrated that six human herpesviruses (herpesviruses 1, 2, 3, 4, 5, and 6B) could be detected at levels at or below 100 genome equivalents per 100 ng of carrier DNA. These data suggest that consensus primer PCR targeted to herpesviral DNA polymerase may prove to be useful in the detection and identification of known herpesviruses in clinical samples and the initial characterization of new herpesviral genomes.
开发了一种共识引物PCR方法，该方法可扩增疱疹病毒DNA导向的DNA聚合酶（EC 2.7.7.7）的一个区域，并以巢式形式使用简并引物。引物设计用于靶向编码覆盖约800 bp区域的高度保守氨基酸基序的序列。该检测方法应用于22种疱疹病毒（8种人类病毒和14种动物病毒），22种病毒中有21种获得了PCR产物。在此过程中，获得了14个疱疹病毒DNA聚合酶以前未报道的氨基酸编码序列，包括人类疱疹病毒7型和8型的区域。本研究中回收的50至60个氨基酸编码序列被确定为所研究的每种病毒物种所特有，在研究单个物种的毒株时，序列变异很小。在存在人类载体DNA的情况下进行的模板稀释研究表明，六种人类疱疹病毒（疱疹病毒1、2、3、4、5和6B）可以在每100 ng载体DNA中100个基因组当量或更低的水平被检测到。这些数据表明，靶向疱疹病毒DNA聚合酶的共识引物PCR可能被证明可用于临床样本中已知疱疹病毒的检测和鉴定以及新疱疹病毒基因组的初步表征。
