Work

Establishment of a multiplex real-time quantitative PCR assay using TaqMan fluorescent probes to detect Mucorales species

Year: 2025

Type: article

Abstract: To establish a specific and sensitive TaqMan multiplex real-time quantitative PCR (RT-qPCR) assay to rapidly detect Mucorales. Customized oligonucleotide primers and corresponding detection probes were developed using the 18S ribosomal RNA gene sequences of Rhizomucor spp., Lichtheimia spp., and Mucor spp./ Rhizopus spp.. A multiplex RT-qPCR assay was established using TaqMan fluorescent probes, and its specificity, repeatability, and sensitivity were evaluated. The method was also used for the detection of simulated Mucorales-positive clinical samples and on samples from clinical patients possibly having fungal infections. The established TaqMan fluorescent probe-based multiplex RT-qPCR assay accurately detected Mucorales with high sensitivity, showing a minimum detection limit of 1 fg/μL. The assay also showed high specificity and accurately detected organisms representing the four main genera in Mucorales (i.e., Rhizomucor miehei , Lichtheimia corymbifera , Mucor circinelloides , and Rhizopus oryzae ). Additionally, no cross-reactivity was observed with 21 non-targeted strains (including viruses, bacteria, fungi, and Cunninghamella bertholletiae ). The reproducibility of the assay was confirmed, with a relative standard deviation within groups of less than 1%. The assay was able to detect Mucorales pathogens (i.e., Rhizomucor spp., Lichtheimia spp., Mucor spp., and Rhizopus spp.) in simulated positive clinical specimens of blood, urine, lavage fluid, and sputum. Among 56 specimens from clinical patients with suspected fungal infections that were tested using the assay, one positive specimen was detected, consistent with the findings from sequencing and fungal culture identification. A highly specific and sensitive TaqMan fluorescent probe-based multiplex RT-qPCR detection method for Mucorales was established that has promising applications in the early and rapid diagnosis of Mucorales pathogens in clinical specimens. • Firstly, Rapid diagnosis and treatment at an early stage of Mucorales infection will help reduce the fatality rate among patients, it is crucial to establish prompt and accurate methods for early detection. • Secondly, the epidemiology of mucormycosis exhibits regional variation, and some studies on PCR detection techniques for mucormycosis, both domestically and internationally, lack standardization. In this study, specific TaqMan-based primers and probes were designed against conserved regions of the 18S ribosomal RNA gene, and a multiplex real-time qPCR assay was established. • Thirdly, in this study, the established TaqMan fluorescent probe-based multiplex real-time quantitative PCR assay with high specificity, sensitivity, and good reproducibility that could realize synchronous rapid amplification of DNA of multiple pathogens of Mucorales. • Finally, the TaqMan multiplex real-time qPCR assay developed in this study was successfully applied for the detection of Mucorales pathogens in simulated clinical samples of blood, urine, and lavage fluid, and clinical patient specimens with suspected fungal infection.

Source: Indian Journal of Medical Microbiology

DOI: 10.1016/j.ijmmb.2025.100977