Design and Development of a Target Amplification-Free Microfluidic Platform for Human Papillomavirus Genotyping: A New Generation of Hybrid Capture Immunoassay.

Hybrid capture nucleic acids detection is a universal target amplification-free technology with the ease of sample manipulation shared by traditional immunoassays. However, with the increasing demand for multiple tests on a single sample in clinical practice, multiplex nucleic acid detection has become a focus of attention. In this study, we developed a novel microfluidic chip that can perform full genotyping detection of 14 high-risk human papillomavirus (hrHPV) types using hybrid capture technology without nucleic acids amplification. This microfluidic chip integrates seven chambers and three pneumatic valve channels, which can achieve four levels of liquid directional transfer and complete the entire hybrid capture reaction process. We verified that this chip has good detection linearity by detecting 14 hrHPV plasmids at different concentrations. To assess the clinical performance, we conducted a comparative analysis between the results derived from the microfluidic chip and the results from clinical laboratory using the same cohort of 427 HPV positive and 500 HPV negative samples. The HPV genotyping agreement was 91.91%, and kappa coefficient was 0.87. The consistency between the microfluidic HPV test and DH3 HPV test on primary cervical cancer screening triage strategy was 93.31%. The kappa coefficient was 0.89 in clinical triage. This microfluidic platform offers researchers and clinicians an effective, standardized, and accurate tool for pathogen detection. Hybrid capture immunoassay without target amplification has the potential to carry out multiplex nucleic acid detections, which may significantly improve patient care in the world including the resource-limited areas.