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Research Article

Polymerase chain reaction amplification and restriction enzyme typing as an accurate and simple way to detect and identify human papillomaviruses

Pizzighella, S., Rassu, M., Piacentini, I., Maschera, B., Palu, G.

Journal of Medical Microbiology 1993; 39(1):33

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Summary auto-generated

This study describes a simple and economical method for detecting and identifying human papillomaviruses (HPVs) using polymerase chain reaction (PCR) amplification followed by restriction enzyme analysis. The researchers used degenerate consensus primers to amplify genomic sequences from six common genital HPV types (HPV-6, 11, 16, 18, 31, and 33) from both cloned DNA and clinical samples. The amplified fragments, located in the conserved E7-E1 region, were then digested with various restriction enzymes including AccI, TaqI, HincII, SspI, NdeI, and HinfI to generate type-specific digestion patterns that serve as a fingerprint for identification. Testing with clinical condylomata specimens confirmed the method's effectiveness. The approach proved sensitive to approximately 10 femtograms of template DNA for most HPVs tested, and offered significant advantages over traditional Southern blot hybridization methods, including easier execution, better specificity due to direct sequence recognition, and reduced false positive results from cross-hybridization. The restriction typing approach also allowed flexible discrimination between oncogenic and non-oncogenic HPVs or specific typing within groups, making it clinically practical for routine screening.

Key findings

  • Degenerate PCR primers successfully amplified HPV DNA from six common genital types (HPV-6, 11, 16, 18, 31, 33) with sensitivity around 10 femtograms of template DNA.
  • Restriction enzyme digestion of PCR products generates unique digestion patterns for each HPV type, enabling accurate identification without Southern blotting.
  • The method is more specific and practical than hybridization-based approaches, avoiding false positives from cross-hybridization between similar HPV genotypes.
  • Single restriction enzymes can discriminate between oncogenic versus non-oncogenic HPVs or provide complete typing of all six HPV types tested.
  • The approach was validated with both cloned HPV DNA and clinical condylomata samples, demonstrating clinical utility.

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Abstract

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.