Development of a Novel Liquid Bead Array Human Papillomavirus Genotyping Assay (PGMY-LX) and Comparison with Linear Array for Continuity in Longitudinal Cohort Studies

Abstract

Studies of the natural history of human papillomavirus (HPV) infection require reproducible, type-specific testing of the viral types that infect cervical tissue; Linear Array (LA) is one method that has been widely used. We sought to develop a cost-effective, high-throughput alternative using the same PGMY09/11 primer/probe system and offering sensitivity and specificity comparable to those with LA to ensure continuity in longitudinal studies. We report here on a Luminex-based approach, PGMY-LX, that offers type-specific detection of 33 oncogenic and nononcogenic types. Detection of HPV type-specific plasmid DNA was highly specific, with high signal-to-noise ratios for all types except nononcogenic type 40 and no cross-reactivity between types. H uman papillomavirus (HPV) is the most common sexually transmitted infection among sexually active individuals.More than 40 genotypes infect the mucosal epithelium and have been divided into two main groups, "high-risk" (HR) and "lowrisk" (LR), according to their oncogenic potential (1). Persistent infection with one or more HR types is a prerequisite for the development of cervical cancer or precancerous lesions.Studies seeking to understand the natural history of HPV infection and associated risk factors require type-specific testing of the HR and LR types that infect cervical tissue. Such studies have historically relied on a combination of in-house methods and commercial tests, such as Inno-LiPA and Roche Molecular Diagnostics Linear Array (LA), to detect type-specific HPV DNA sequences. The latter are both line blot assays and distinguish 28 and 37 specific HPV genotypes, respectively. LA, with its ability to individually identify the greatest number of HPV types is well established and widely used. It is a PCR-driven method employing the PGMY09/11 primer system, which uses pooled, nondegenerate primers to amplify part of the highly conserved L1 gene of the viral capsid. Detection is based on solid-phase hybridization of amplified HPV sequences to a slot blot membrane.The introduction of Luminex technology, allowing multiplexed testing of up to 100 unique assays within a single, smallvolume sample, brought researchers the possibility of developing a high-throughput HPV genotyping assay suitable for cohort studies. In the past decade, researchers have combined Luminex technology with GP5ϩ/6ϩ (2, 3, 4, 5), MY09/11 (6), PGMY09/11 (7), and other primer systems. These various primer systems all have extensive histories of use for HPV typing in the research setting predating their employment in Luminex-based approaches. Because the various primer systems have been shown to have different performance characteristics in type-specific HPV detection (8, 9), continuity within a prospective study generally is best facilitated by using the same primer set throughout.Since 2000, we have used the PGMY09/11-based LA approach, as well as its precommercial predecessor, reverse line blot (10), to prospectively characterize the natural history of cervical HPV infection in adolescents...

Detection of Nucleic Acids with a Novel Stem-Loop Primer Rolling Circle Amplification Technique

“…There are around 100 types of HPV with different genetic variations (23). With respect to oncogenic potential, HPVs are divided into high-risk HPV (hrHPV) and low-risk HPV (lrHPV) (24). The most common worldwide hrHPVs are HPV-16 and 18 (2527), which lead to approximately 70% of cervical cancers (28).…”

Cervical-Vaginal Microbiome and Associated Cytokine Profiles in a Prospective Study of HPV 16 Acquisition, Persistence, and Clearance

“…Two ml were removed for HPV DNA testing and the remaining 3 ml were divided into 3 cryovials for −80° C storage. HPV testing was performed based on PCR amplification using Roche Linear Array and an in-house Luminex platform (Moscicki et al, 1998 , 2001 ; Farhat et al, 2015 ). The primary study was approved by the Committee on Human Research, University of California, San Francisco and patients consented to participate and to place their samples in repository.…”

The effect of intrauterine devices on acquisition and clearance of human papillomavirus

“…HPV testing was done by Linear Array (Roche Molecular Diagnostics, Alameda, CA) and a novel Luminex-based approach, PGMY-LX, which was shown to have a sensitivity and specificity comparable to Linear Array in this population and is described elsewhere in detail. 9 Sexual and substance use behavior and contraceptive use, including IUD use, were assessed by interview-administered questionnaire. IUD use was verified at the time of speculum examination but the type of IUD strings observed were not recorded.…”