Rapid and portable molecular test for Mycobacterium sp based on double-tagged amplification and electrochemical readout

Abstract

Tuberculosis (TB) remains one of the leading causes of death worldwide, with Mycobacterium tuberculosis as the main pathogen responsible. Although several rapid molecular tests endorsed by the World Health Organization (WHO) show high accuracy, their implementation in low-resource settings is still limited by cost and infrastructure requirements. In this work, a rapid and portable molecular test was developed for Mycobacterium detection based on double-tagged polymerase chain reaction (PCR) and electrochemical magneto-genosensing. The method enables amplification of mycobacterial DNA using primers targeting the gyrB and IS6110 genes through a double-tagging PCR completed within approximately one hour, followed by electrochemical detection on a handheld, battery-operated device, yielding a measurable current signal within 15 minutes, including a single-step incubation and electrochemical readout. The test achieved a limit of detection as low as 117 CFU mL⁻¹ for the gyrB target, comparable to the commercial GeneXpert® MTB/RIF assay, while significantly reducing assay time and equipment needs. Total analysis, including amplification and detection, was completed within approximately two hours. In addition, comparable electrochemical responses were obtained when PCR amplification was carried out using either a conventional benchtop thermocycler or a portable battery-operated miniPCR device, further supporting the applicability of the method in decentralized and field settings. Overall, this approach combines high sensitivity, portability, and simplicity, offering a promising solution for point-of-care TB diagnosis and adaptable to other infectious diseases by changing the primer sequences.