Venturoli S, Zerbini M, La Placa M, D'Antuono A, Negosanti M, Gentilomi G, Gallinella G, Manaresi E, Musiani M
Department of Clinical and Experimental Medicine, University of Bologna, Italy.
J Clin Pathol. 1998 Feb;51(2):143-8. doi: 10.1136/jcp.51.2.143.
To evaluate different hybridisation techniques to detect and type human papillomavirus (HPV) DNAs amplified by consensus primer polymerase chain reaction (PCR) in biopsy and cytological specimens.
A hybrid capture-immunoassay in microtitre wells was performed to detect HPV sequences amplified by PCR and typed by specific oligoprobes. Consensus primers were used to amplify a sequence within the L1 open reading frame, and direct digoxigenin labelling of amplified products was performed during the amplification reaction. The amplified product was separately hybridised with six biotinylated type specific probes (HPV6, 11, 16, 18, 31, and 33); hybrids were then captured into streptavidin coated microtitre wells and detected by a spectrophotometer as an ELISA using antidigoxigenin Fab fragment labelled with peroxidase and a colorimetric substrate. The results were compared with the dot-blot immunoassay used to detect and type PCR amplified HPV DNA sequences. Consensus primers were used to generate the same unlabelled PCR product; digoxigenin labelled type specific probes for HPV6, 11, 16, 18, 31, and 33 were used and hybrids visualised by colorimetric immunoenzymatic reaction. Thirty nine biopsy specimens and 31 cytological samples were tested by the PCR-ELISA and by standard PCR followed by dot-blot hybridisation.
The PCR-ELISA proved to be more sensitive than standard PCR with dot-blot hybridisation typing. All samples positive for HPV-DNA in standard PCR with dot-blot hybridisation method were confirmed positive by the PCR-ELISA assay; however, seven samples were positive only by PCR-ELISA.
The PCR-ELISA assay, which can be performed in one day, is easily standardised and therefore seems to be a practical, sensitive, and reliable diagnostic tool for the detection and typing of HPV genomes in biopsy and in cytological specimens in the routine diagnostic laboratory.
评估不同的杂交技术，以检测通过共识引物聚合酶链反应（PCR）在活检和细胞学标本中扩增的人乳头瘤病毒（HPV）DNA并进行分型。
进行微孔板杂交捕获免疫测定，以检测通过PCR扩增并由特异性寡核苷酸探针分型的HPV序列。使用共识引物扩增L1开放阅读框内的序列，并在扩增反应期间对扩增产物进行直接地高辛配基标记。扩增产物分别与六种生物素化的型特异性探针（HPV6、11、16、18、31和33）杂交；然后将杂交体捕获到包被链霉抗生物素蛋白的微孔板中，并使用标记有过氧化物酶的抗地高辛配基Fab片段和比色底物，通过分光光度计作为酶联免疫吸附测定（ELISA）进行检测。将结果与用于检测和分型PCR扩增的HPV DNA序列的斑点印迹免疫测定进行比较。使用共识引物生成相同的未标记PCR产物；使用针对HPV6、11、16、18、31和33的地高辛配基标记的型特异性探针，并通过比色免疫酶反应使杂交体可视化。通过PCR-ELISA以及标准PCR随后进行斑点印迹杂交，对39份活检标本和31份细胞学样本进行检测。
PCR-ELISA被证明比标准PCR与斑点印迹杂交分型更敏感。在标准PCR与斑点印迹杂交法中所有HPV-DNA阳性的样本，通过PCR-ELISA测定均被确认为阳性；然而，有7个样本仅通过PCR-ELISA呈阳性。
PCR-ELISA测定可在一天内完成，易于标准化，因此似乎是常规诊断实验室中用于检测活检和细胞学标本中HPV基因组并进行分型的实用、灵敏且可靠的诊断工具。
