Development and evaluation of a capillary electrophoresis-based multiplex RT-PCR kit set for detection of upper respiratory tract pathogens

Abstract

Respiratory tract infections (RTIs) are a major global public health problem, impacting both health and economics significantly, and have long been a concern for governments and research institutions worldwide. Although numerous related products are available, many of them only offer a limited panel of targets. In this study, we describe a set of multiplex RT-PCR kits based on capillary electrophoresis. Kit A can simultaneously detect ten viruses, two atypical pathogens, and one bacterium, covering most upper respiratory tract pathogens. Kit B can classify eight pathogens into a total of 22 subtypes. To obtain suitable primers, we used bioinformatics methods and applied different conditions to assess the conservation of primers and amplification products. For the 10 targets included in Kit A, the minimum concentration that could be reliably detected did not exceed 0.5 copies/μL. Compared to the commercial kit (Kit Health), it offers broader assay coverage and higher positivity rates. Kit B can differentiate subtypes at the detection limit concentration of Kit A and Kit B showed no cross-interference between subtypes during supersaturated concentration testing. Using both kits in combination can enhance their performance, offering better support for pathogen research and clinical applications.