| Autor: |
Snijders PJ; Section Molecular Pathology, Department of Pathology, VU University Medical Center, The Netherlands., van den Brule AJ, Jacobs MV, Pol RP, Meijer CJ |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular medicine [Methods Mol Med] 2005; Vol. 119, pp. 101-14. |
| DOI: |
10.1385/1-59259-982-6:101 |
| Abstrakt: |
Polymerase chain reaction (PCR)-based assays that use consensus primers to detect DNA of a broad spectrum of human papillomavirus (HPV) types in a single assay belong to the most frequently used methods to detect HPV in clinical specimens. Here, we describe in detail one of these assays, the so-called GP5+/6+ PCR method, which can be used to detect and type HPV DNA in crude extracts of cervical scrapes and biopsy specimens. Following PCR with GP5+ and GP6+ primers, the latter of which is biotinylated at its 5' end, the presence of DNA of any of the high-risk genotypes can easily be determined by an enzyme immunoassay (EIA). In this assay, PCR products are captured in streptavidin-coated wells of a microtiter plate, denatured by alkaline treatment, and hybridized to cocktails of digoxigenin-labeled oligonucleotides specific for high-risk or low-risk HPV types. The resulting hybrids can then be detected by alkaline phosphatase conjugated anti-digoxigenin polyclonal antibodies and substrate followed by optical density reading. Subsequently, EIA-positive PCR products can be typed by a reverse line blot genotyping procedure that, using a miniblotter device, enables typing of up to 39 samples with specific oligonucleotide probes for 37 different HPV (sub)types in a single assay. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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