Shamanin V, Delius H, de Villiers E M
Division Tumourvirus-Characterisation, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
J Gen Virol. 1994 May;75 ( Pt 5):1149-56. doi: 10.1099/0022-1317-75-5-1149.
A PCR assay was developed to detect known and as yet unidentified papillomaviruses (PVs). For this purpose we analysed the conserved amino acid sequences in the L1 and E1 open reading frames of 45 human and nine animal PVs. Candidate regions for the design of a primer were identified as those having the least number of amino acid and nucleotide sequence variants among the different PVs. These regions in the L1 ORF have been described previously. We modified the sequences of the backward and the forward primers, as well as the sequence of the oligonucleotide used as the degenerate probe, in order to cover a broader spectrum of PVs. The sensitivity of the assay for the human and animal PVs tested after hybridization with a 32P-labelled degenerate oligonucleotide probe was one genome copy per cell for integrated PV DNA and 10 genome copies per cell for plasmid PV DNA. The only exceptions were human papillomavirus (HPV) type 4, HPV60 and HPV65, for which a lower sensitivity was obtained. This group could be detected only by using additional primers. The assay was used to analyse 85 lung cancer biopsies representing different histological types. Using this system no PV DNA sequences were detected in the biopsies when compared with human placental DNA (a negative control) and PV DNA-positive standards.
开发了一种聚合酶链反应（PCR）检测方法来检测已知和尚未鉴定的乳头瘤病毒（PV）。为此，我们分析了45种人类乳头瘤病毒和9种动物乳头瘤病毒的L1和E1开放阅读框中的保守氨基酸序列。将引物设计的候选区域确定为不同PV中氨基酸和核苷酸序列变体数量最少的区域。L1开放阅读框中的这些区域先前已有描述。我们修改了反向引物和正向引物的序列，以及用作简并探针的寡核苷酸序列，以覆盖更广泛的PV谱。在用32P标记的简并寡核苷酸探针杂交后，该检测方法对所测试的人类和动物PV的灵敏度为：整合型PV DNA每细胞一个基因组拷贝，质粒PV DNA每细胞10个基因组拷贝。唯一的例外是4型人乳头瘤病毒（HPV）、HPV60和HPV65，对它们的检测灵敏度较低。只有使用额外的引物才能检测到这一组病毒。该检测方法用于分析85份代表不同组织学类型的肺癌活检样本。与人类胎盘DNA（阴性对照）和PV DNA阳性标准相比，在活检样本中未检测到PV DNA序列。
