Multiplex recombinase polymerase amplification for high-risk and low-risk type HPV detection, as potential local use in single tube

Abstract

High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14–20 high-risk and 13–14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test (Roche Diagnostics, New Jersey, USA) and REBA HPV-ID (Molecules and Diagnostics, Wonju, Republic of Korea) were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35–42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies (equivalent to 2.01 fg) for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15–40 min), less expensive (~ 3–4 USD/reaction) and does not require instrumentation (35–42 °C reaction condition so hand holding or tropical temperature is possible). Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users.

Electrochemical Immunosensor for Ultra-Low Detection of Human Papillomavirus Biomarker for Cervical Cancer

“…0.93 fg) for the HPV-16 L1 genotype). In addition, in 2023, Wongsamart et al 38 reported an advanced form of RPA known as the multiplex recombinase polymerase amplification (mRPA), which gave an LoD of 1000 copies/ reaction (i.e., 2.01 fg) for the HPV-16 L1 genotype. The LoD values of our proposed electrochemical immunosensor are lower than the conventional LBPT, and comparable or even lower than the advanced PCR and mRPA techniques.…”