Polymerase Chain Reaction Amplification of Human Papillomavirus DNA from Archival, Papanicolaou-Stained Cervical Smears

Detection of High-Risk Human Papillomavirus Nucleic Acid in Archival Cervical Smears

“…4 The exact method of fixation used by individual smear takers is uncertain, and differences in sampling could produce varying amounts of cellular material in the original smear. Puranan et al 6 assessed the quality of smears according to the area of the slide covered by stained material. Since red blood cells and mucus can contribute significantly to the macroscopic appearance of a smear, we decided to estimate quality using the volume of scraped pellet after washing.…”

Detection and Genotyping of Human Papillomavirus DNA Using Polymerase Chain Reaction Short PCR Fragment 10–Line Probe Assay in Abnormal Papanicolaou-Stained Cervicovaginal Smears

“…9 The resulting HPV DNA-positive samples for the PCR-SPF10 were tested for genotyping of the associated HPV DNA using the HPV INNOLiPA (Innogenetics). In this test, the product of PCR-SPF10 was mixed with an alkaline solution containing EDTA and placed in contact with the nitrocellulose strip that contains immobilized parallel lines of oligonucleotides for identifying the 25 specific types of HPV (types 6,11,16,18,31,33,34,35,39,40,42,43,44,45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 70 and 74) simultaneously in a single test. The strips were incubated for 1 hour at 49°C.…”

“…Simple protocols in which the proteinase K buffer is used for cellular digestion seem to be the most adequate, given that loss or destruction of genomic material is minimal. [15][16][17][18] However, DNA extracted in the traditional way with phenol-chloroform, although it is not the most commonly recommended method, 16 also permits procuring optimum DNA samples. 7,19,20 Successful amplification of genome segments of HPV using PCR depends, to a great extent, on the sensitivity of the primers used determined by the fragment size.…”

“…8,11,21 The viral load can also influence exact detection of HPV DNA, being greater if it is found in an elevated number of copies. 16,18,22 Cervical scrapes provide a limited quantity of epithelial cells. Moreover, if some of these cells contain low viral copies, they can produce errors, depending on the samples, that have repercussions on the sensitivity of the test, above all in the case of infection by ≥ 2 HPV genotypes present in different viral loads.…”

A simplified and reliable HPV testing of archival Papanicolaou-stained cervical smears: application to cervical smears from cancer patients starting with cytologically normal smears

“…a proteinase K/Tris-HCl method as described by Puranen et al, (1996) 2. a proteinase K/salting-out method as described by Poljak et al (1995) 3. a GTC/silica method as described by de Roda Husman et al (1995), with the exception that larger diatoms were used instead of smaller silica particles because this appeared to improve the DNA yield in reconstruction experiments after one extraction round (data not shown) 4. the High Pure PCR Template assay (provisionally named HPPTP assay), which uses GTC in combination with silica columns, according to the manufacturers' instructions (Boehringer-Mannheim).…”