The association of human papillomaviruses (HPV) with invasive cervical carcinoma and its precursor lesions is well characterized. The clinical utility of a touchdown approach for HPV detection requires further investigation as increased assay analytical sensitivity may not necessarily equate with improved clinical sensitivity or specificity. A touchdown approach may be beneficial as an analytical test for the re-evaluation of (apparently) HPV negative abnormal cervical cytological or histological samples, and for investigating the association of HPV with disease conditions at diverse organ sites. The use of standardized DNA quantities in a PCR rather than standard sample volumes containing arbitrary amounts of DNA is supported. Touchdown modification of the GP5+/GP6+ PCR assay enables the detection of HPV undetected under regular assay conditions. Of 26 breast tumors, 5 (19%) tested HPV positive by the standard assay and 15/26 (58%) using a touchdown protocol. Multiple infections were also detectable using a touchdown approach. Twenty-one different HPV types were recorded in 111/114 (97%) vaginal lesions. Seventeen different HPV types were demonstrated in 47/65 (72%) abnormal cytology samples recorded as HPV negative by standard GP5+/GP6+ conditions. HPV-52 was not amplified by the standard protocol at the dilutions tested. HPV-31 was better amplified under standard annealing conditions (1.5fg in 100 ng background DNA) than by a touchdown approach (15fg detection limit). HPV recombinant plasmids were each detected with high (albeit varying) sensitivity by a touchdown protocol. Under standard GP5+/GP6+ annealing conditions, HPV-16 went undetected when the DNA content of a PCR was 2 μg or 1 μg, and with 500 ng C-33A DNA the sensitivity limit was 1 ng SiHa cell DNA. Touchdown protocols supported HPV-16 detection from 1 ng or 0.5 ng SiHa cell DNA in a background of 2 μg or 1 μg C-33A DNA respectively, and from 0.1 ng of SiHa cell DNA (~28 copies HPV-16) in 500 ng or 100 ng background DNA. HPV detection sensitivity was dependent on the total amount of DNA in a PCR. HPV was detected by gel electrophoresis, and by Southern and dot blot hybridization. Six different PCR protocols were assessed. Thirdly, clinical sample DNA extracts (cervical smears, formalin-fixed vaginal lesions and breast tumors) were tested for HPV. Secondly, the detection of small quantities (15ag-1.5pg) of HPV recombinant plasmids (types 16, 31, 33, 45, 51, 52, and 56) diluted in C-33A DNA was investigated. Methodsįirstly, HPV-16 was amplified from SiHa cell DNA (0.1 ng–100 ng) diluted in a background of C-33A DNA (100 ng-2 μg). In addition, dTTP was substituted with dUTP to permit contamination control measures against carry-over PCR product. This study has examined the effects of incorporating 'hot start' and 'touchdown' steps into the protocol. The GP5+/GP6+ PCR assay is a well-established HPV detection technique.
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