A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR

Abstract

In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes.

Frequency and Type-distribution of Human Papillomavirus from Paraffin-embedded Blocks of High Grade Cervical Intraepithelial Neoplasia Lesions in Thailand

“…DNA quality was checked by β-globin, house keeping gene, amplification using Polymerase Chain Reaction (PCR) under the following; initial denaturation at 94 o C for 5 min, 40 cycles with the cycling of 94 o C for 1 min, 60 o C for 1 min, 72 o C for 1 min and then final extension at 72 o C for 5 min. The primer sequences were as follows (Jacob et al, 1996): β-globin sense ACA CAA CTG TGT TCA CTA GC and β-globin antisense GAA ACC CAA GAG TCT TCT CT. The amplified products were subjected to electrophoresis and visualized by UV transilluminator.…”

HPV16 semiquantitative viral load and serologic biomarkers in oral and oropharyngeal squamous cell carcinomas

“…16 A previous study demonstrated that, within the range of 1 Â 10 to 1 Â 10 6 genome equivalents, the EIA OD value obtained after 1 hr of substrate incubation shows a linear relation with the amount of input DNA. 17 Consequently, EIA ODs can semiquantitatively assess the relative viral load.…”

Detection and Type-Distribution of Human Papillomavirus in Vulva and Vaginal Abnormal Cytology Lesions and Cancer Tissues from Thai Women

“…After deparaffinization with xylene, twice rehydration in absolute ethanol and discharge the remaining ethanol, all of samples were dried at room temperature to ensure that no residual ethanol (Ngamkham et al, 2013). Genomic DNA extraction was performed using QIAamp DNA Mini kit (QIAGEN, Germany) according to the manufacturer's instruction and genomic DNA quality was evaluated by polymerase-chain reaction with β-globin primers (Jacob et al, 1996); sense 5'-ACA CAA CTG TGT TCA CTA GC-3' and antisense 5'-GAA ACC CAA GAG TCT TCT CT-3'. Total volume of 25 μl PCR mixture was consisted of 5 μl extracted DNA, 0.4μM of sense and antisense primers, 2.5U Taq polymerase, 200nM of each dNTPs and 1.5mM MgCl 2 .…”