Simultaneous polymerase chain reaction detection and restriction typing for the diagnosis of human genital papillomavirus infection.
A polymerase chain reaction method has been developed which allows the simultaneous detection of the majority of clinically relevant HPV types. Degenerate HPV-specific primers direct the one-step amplification of a DNA region spanning E1 and E7 genes. This enables an immediate distinction between the two groups of papillomaviruses, characterized by high or low oncogenic potential, simply from the size of amplified DNA. The PCR product can be subjected to a second round of amplification with internal primers, which are specific for 7 high-risk HPV types, HPV-16, -18, -31, -33, -35, -45 and -58. Precise identification of one-step or two-step amplified DNA is done by endonuclease digestion with one or two enzymes. The detection sensitivity, which has been assessed using cloned HPV genomes and HeLa and CaSki cell lines, varies from a few tens to a few hundreds of viral genome equivalents. The accuracy of the method has been confirmed by examining cervical scrapings of 44 patients.
University of Padua, School of Medicine, Microbiology Department, Italy.
S
S
Pizzighella
G
G
Pisoni
F
F
Bevilacqua
A
A
Vaona
G
G
Palù
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D005831:Genital Diseases, Female / Q000473:pathology / Q000821:virology*; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000473:pathology / Q000821:virology*; D014626:Vaginal Smears
D017931:DNA Primers; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D004262:DNA Restriction Enzymes