Robust hybridization-based genotyping probes for HPV 6, 11, 16 and 18 obtained via in vitro selection

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Ivan B. Brukner *
Anne-Marie Larose
Izabella Gorska-Flipot
Maja Krajinovic
Damian Labuda
(*) Corresponding Author:
Ivan B. Brukner | Ibrukner@gmail.com

Abstract

This paper describes the technical and analytical performance of a novel set of hybridization probes for the four GARDASIL® vaccine-relevant HPV types (6, 11, 16 and 18). These probes are obtained through in vitro selection from a pool of random oligonucleotides, rather than the traditional “rational design” approach typically used as the initial step in assay development. The type-specific segment of the HPV genome was amplified using a GP5+/6+ PCR protocol and 39 synthetic oligonucleotide templates derived from each of the HPV types, as PCR targets. The robust performance of the 4 selected hybridization probes was demonstrated by monitoring the preservation of the specificity and sensitivity of the typing assay over all 39 HPV types, using a different spectrum of HPV (genome equivalent: 103-109) and human DNA concentrations (10-100 ng) as well as temperature and buffer composition variations. To the Authors’ knowledge, this is a unique hybridization-based multiplex typing assay. It performs at ambient temperatures, does not require the strict temperature control of hybridization conditions, and is functional with a number of different non-denaturing buffers, thereby offering downstream compatibility with a variety of detection methods. Studies aimed at demonstrating clinical performance are needed to validate the applicability of this strategy.

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Author Biography

Ivan B. Brukner, Jewish General Hospital

Department of Medical Diagnostics
SMBD-Jewish General Hospital, Suite/Room D-133 & D0-132
3755 Cote-Sainte-Catherine Road, H3T 1E2
Montreal, Quebec, Canada
email1: ibrukner@hotmail.com
email2: ibrukner@jgh.mcgill.ca
Tel: 514-3408222 ext 4801 & ext 3699
Cell:514-8038782