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Minerva Biotecnologica 2020 September;32(3):101-5

DOI: 10.23736/S1120-4826.20.02621-X


lingua: Inglese

Real time qPCR TaqMan method for detection of Fusarium solani

Elena ZANIOL 1, Valentina DAPRÀ 1, 2, Rebecca FILOMENA 1, Carla ALLIAUDI 1, Cristina CALVI 1, Paola MONTANARI 1, Ilaria GALLIANO 1, Massimiliano BERGALLO 1, 2

1 Department of Public Health and Pediatrics, School of Medicine, University of Turin, Turin, Italy; 2 BioMole srl., Turin, Italy

BACKGROUND: Since no routine tests for the fusariosis diagnosis are currently available and DNA-based techniques for detection are not yet fully standardized or commercially available, the aim of this study was to develop new procedures to perform a rapid fungal identification vital to patient management.
METHODS: Polymerase chain reaction (PCR) was set up in a volume of 20 μL, containing 100 ng of DNA (20 ng/μL) and 15 μL of amplification mix Fusarium solani PCR real time.
RESULTS: Efficiency and sensitivity, variability, specificity and dynamic range were tested. The reproducibility was expressed as the coefficient of variation (CV) in the log10 values of the concentration. Quantification of Fusarium solani was highly reproducible for as few as 102 copies. The RT-PCR assay was able to quantify Fusarium solani from 109 to 102 copies/reaction.
CONCLUSIONS: The performance of our real time PCR assay using samples from culture was excellent, with 100% sensitivity and no cross-reactivity demonstrated with the tested species of other microbes. Real time PCR offers significant improvements to microbial load quantitation because of its wide dynamic range, which can accommodate at least eight log10 copies of nucleic acid template. The RT-qPCR TaqMan assay described herein can be utilized to provide an objective result either as a separate test or as a complement to microscopy.

KEY WORDS: Fusarium; Real-time polymerase chain reaction; Microscopy

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