Indian journal of medical microbiology · 2025
Establishment of a multiplex real-time quantitative PCR assay using TaqMan fluorescent probes to detect Mucorales species.
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Pan Yongsheng, Pu Dan, Xu Hongren, Jiang Kerong, Wang Jiajie, Bai Minfeng, Xu Xiaoliang, Zhao Yunping, Yin Limin
Аннотация
PURPOSE: To establish a specific and sensitive TaqMan multiplex real-time quantitative PCR (RT-qPCR) assay to rapidly detect Mucorales.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.