Rapid Genotyping of Human Papillomavirus by Post-PCR Array-Based Hybridization Techniques

Abstract

Kinetic hybridization measurements on a microarray are expected to become a valuable tool for genotyping applications. A method has been developed that enables kinetic hybridization measurements of PCR products on a low-density microarray. This is accomplished by pumping a solution containing PCR products up and down through a porous microarray substrate. After every pumping cycle, the fluorescently labeled PCR products hybridized to capture probes immobilized on the solid surface of the porous microarray substrate are measured. By this method, both binding curves and high-resolution melting curves are obtained instead of the single endpoint hybridization intensities as with commonly used post-PCR array-based hybridization techniques. We used 20 subtypes of the human papillomavirus (HPV) as a model system to test our detection method and blindly analyzed 216 clinical samples. We compared our microarray flowthrough method with a reference method, PCR followed by a reverse line blot (RLB). Real-time hybridization measurements followed by high-resolution melting curves of low concentrations of fluorescently labeled HPV targets on a microarray were successfully carried out without any additional chemical signal amplification. The results of our new method were in good agreement (93%, with a kappa coefficient of ‫؍‬ 0.88 [95% CI, 0.81 to 0.94]) with the RLB results. All discrepant samples were analyzed by a third method, enzyme immunoassay (EIA). Furthermore, in a number of cases, we were able to identify false-positive samples by making use of the information contained in the kinetic binding and melting curves. This clearly demonstrates the added value of the use of kinetic measurements and high-resolution melting curves, especially for highly homologous targets.

Low prevalence of HPV detection and genotyping in non-muscle invasive bladder cancer using single-step PCR followed by reverse line blot

“…As post-PCR hybridization assays are a suitable alternative for the parallel detection of a larger number of different PCR products, we performed RLB, which captures and detects specific subtypes on a membrane support. RLB has the advantage that specific HPV genotypes and multiple infections can be detected, but has the disadvantage of being time consuming and laborious [19]. To the best of our knowledge, this is the first study using post-PCR RLB hybridization technique for HPV-DNA detection in FFPE bladder cancer tissue.…”

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

“…In the meantime, oncogenicity might keep continue in a high frequency until we changes in mortality rates may diminished. Although array assays for the detection of HPV types, particularly high vs. low oncogenic risk are already available [ 37 , 38 ], these assay types cannot distinguish between different oncological behaviors.…”

PCR-reverse blot hybridization assay for fast and accurate identification of causative species in superficial fungal infections

“…4,5 To date, PCR-REBA has been used in genotyping of human papilloma virus, diagnosis and identification of mycobacterial infections, and identification of sepsis pathogens. 4,19,20 Some studies have used PCR-REBA for the identification of fungal species, and reported that it is a sensitive, specific and rapid method for the diagnosis of various fungal infections. [21][22][23][24][25] However, these previous studies detected only a limited number of species such as Candida or Aspergillus spp., which might cause an invasive fungal infection in immunosuppressed patients, [21][22][23][24][25] whereas in the current study, we simultaneously detected and identified most of the pathogenic species that commonly cause superficial fungal infections, including Trichophyton, Candida and Aspergillus spp.…”

“…PCR‐REBA is a molecular tool using a single membrane to which multiple probes are bound, thus it allows simultaneous detection and identification of multiple pathogens in multiple samples . To date, PCR‐REBA has been used in genotyping of human papilloma virus, diagnosis and identification of mycobacterial infections, and identification of sepsis pathogens . Some studies have used PCR‐REBA for the identification of fungal species, and reported that it is a sensitive, specific and rapid method for the diagnosis of various fungal infections .…”