Quantitative and specific detection of viable pathogens on a portable microfluidic chip system by combining improved propidium monoazide (PMAxx) and loop-mediated isothermal amplification (LAMP)

Abstract

An accurate and specific detection of viable Candida albicans (C. albicans) in vaginal discharge is crucial for the diagnosis of vulvovaginal candidiasis (VVC) and assessment of antifungal effects. In this study, improved propidium monoazide (PMAxx) and loop-mediated isothermal amplification (LAMP) were used for the first time to distinguish between viable and dead C. albicans. A portable microfluidic chip system was developed to detect multiple viable pathogens in parallel. The consumption of samples and reagents in per reaction cell were only 0.94 μL, less than 1/25 of the conventional 25 μL Eppendorf tubular test method, both significantly reducing testing cost and greatly simplifying the detection of multiple viable pathogens. The concentration of PMAxx was optimized against C. albicans at 4.0 log CFU mL⁻¹ to 5.0 log CFU mL⁻¹, and 1 μM PMAxx was proven to be suitable for the detection of C. albicans in clinical samples. When testing mixtures containing different ratios of viable to dead C. albicans, PMAxx-LAMP could circumvent the signal arising from dead cells and, therefore, reflected the abundance of viable cells precisely. Furthermore, the suitability of this technique to evaluate the effects of antifungal agents, including clotrimazole, miconazole, and tioconazole, was assessed. Finally, the viability of Escherichia coli (E. coli) and C. albicans were detected on the portable microfluidic chip system. PMAxx-LAMP based portable microfluidic chip system was determined to be a feasible technique for assessing the viability of multiple pathogens in gynecology and might provide insights into new VVC treatment strategies.