A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

Abstract

We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings.

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region

“…In the literature, methods for HPV variant detection have been generally designed for testing single HPV types [ 17 , 54 , 55 , 56 , 57 , 58 ]. By using a multiplexed PCR reaction associated with deep sequencing of amplicons, the NGS-based method reported here allowed to detect and genotype all HR-HPV types directly from clinical specimens without prior knowledge on the HPV types present in the sample.…”

Potential applications of polyoxometalates for the discrimination of human papillomavirus in different subtypes

“…20 Herein, we report an improved method that allows not only to detect but also to discriminate different HPV subtypes using each type-specific sequences. In comparison to the reported oligonucleotide microarray-based systems, 21,22 the present study method is simple, low-cost and efficient. The establishment of reliable tools for HPV subtypes differentiation would be crucially important toward the genetic classification of HPV oncogenicity for prospective management.…”

An equipment-free polydimethylsiloxane microfluidic spotter for fabrication of microarrays

“…[12][13][14][15] In addition, with the spread of microarray technology, there is increasing demand for the production of different specialized microarrays with a lower density of probes for use in ordinary laboratories. [16][17][18] Although all of the three above technologies are used to produce commercially available microarrays, there are few methods to economically and flexibly produce small batches of custom microarrays for prototyping experiments and specialized applications. To overcome these limitations posed by the conventional technologies used to manufacture microarray, several groups have recently introduced microfluidic printing techniques for fabricating DNA or protein microarrays in a cost-effective and flexible manner, including lineprinting and spot-printing.…”