Guidelines for the Use of Molecular Tests for the Detection and Genotyping of Human Papilloma Virus from Clinical Specimens
Accurate genotyping of a human papilloma virus (HPV) isolated from clinical specimens depends on molecular identification of the unique and exclusive nucleotide base sequence in the hypervariable region of a highly conserved segment of the HPV L1 gene. Among other options, a heminested (nested) polymerase chain reaction (PCR) technology using two consecutive PCR replications of the target DNA in tandem with three consensus general primers may be used to detect a minute quantity of HPV DNA in crude proteinase K digestate of cervicovaginal cells, and to prepare the template for genotyping by automated direct DNA sequencing. A short target sequence of 40–60 bases excised from the computer-generated electropherogram is sufficient for BLAST determination of all clinically relevant HPV genotypes, based on the database stored in the GenBank. This chapter discusses the principle and the essential technical elements in performing nested PCR DNA amplification for the detection of HPV from clinical specimens and short target sequence genotyping for HPV, using standard molecular biology laboratory equipment and commercially available reagents.
Key wordsHPV DNA Nested PCR Genotyping DNA sequencing
American Type Culture Collection
Basic Local Alignment Search Tool
Human papilloma virus
National Center for Biotechnology Information
Polymerase chain reaction