HPV-DNA detection was performed from the a basic GP5+/GP6+ PCR dependent assay, once the described by de- Roda Husman ainsi que al (1995a)
Briefly, the GP–PCR reaction was carried out using 50 ?l of PCR solution containing 10 m M Tris HCL pH 8.3, 50 m M KCl, 200 ? M of each deoxynucleotide, 3.5 m M of MgCl2, 1 U of DNA polymerase (AmpliTaq; Perkin-Elmer, USA) and 25 pmol of each of the GP5+ and biotinylated GP6+ primers (Eurogentec, Belgium): 40 cycles of amplification were carried out using a Perkin-Elmer 9600,USA thermocycler. Each cycle included a denaturation step at 95°C for 1 min, one annealing step at 40°C for 1 min, and a chain elongation step at 72°C for 1.5 min. The first step was preceded by a denaturation step of 4 min and the last step was followed by an elongation step of 10 min.
About three dilutions of one’s telephone line SiHa which has had step 1–ten duplicates away from HPV16 (100 pg, 1 ng and you may 10 ng) were used since positive control. While the negative PCR regulation, distilled water and you can operating blanks were used all of the 10 samples. HPV positivity is reviewed of the Southern blot hybridization regarding GP5+/GP6+ PCR products having a cocktail probe regarding particular [?- thirty-two P]dCTP labelled DNA fragments away from cloned DNA out of HPV6, eleven, 16, 18, 31 and 33 under lower stringent standards (van den Brule ainsi que al, 1990; de Roda Husman et al, 1995a,b). (suite…)
