1. Use of sulfonated primers to detect and type papillomavirus in cell cultures and cervical biopsies
- Author
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Michael Friedman, Israel Nur, and Thierry Paper
- Subjects
Sexually transmitted disease ,Biopsy ,Molecular Sequence Data ,Cervix Uteri ,Biology ,Polymerase Chain Reaction ,Sensitivity and Specificity ,law.invention ,chemistry.chemical_compound ,law ,Primer dimer ,Genetics ,Humans ,DNA Probes, HPV ,Papillomaviridae ,Polymerase chain reaction ,Electroblotting ,Cells, Cultured ,Base Sequence ,DNA–DNA hybridization ,General Medicine ,Virology ,Molecular biology ,Blotting, Southern ,Poly C ,chemistry ,Oligodeoxyribonucleotides ,Recombinant DNA ,Female ,Primer (molecular biology) ,Ethidium bromide ,Sulfur ,Plasmids - Abstract
Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.
- Published
- 1991