Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC).
In the present study, we developed a new real-time PCR system based on the cycling probe technology (CPT), which is composed of two single tube real-time PCR assays: the Fusarium genus-specific assay and the Fusarium solani species complex (FSSC)-specific assay with primers targeting the 28s ribosomal RNA gene. The Fusarium genus-specific assay was shown to be highly specific, detecting all reference Fusarium strains with no cross-reaction with other reference fungal strains, such as Aspergillus spp. and human DNA. The FSSC-specific assay also reacted very specifically with FSSC, except for a cross-reaction with Fusarium lunatum. To validate the real-time PCR system, we tested 87 clinical isolates of Fusarium spp. Identification results from the real-time PCR system were found to be 100% concordant with those from DNA sequencing of EF-1α gene. The sensitivity testing also demonstrated high sensitivity, enabling detection of one copy of standard DNA with good reproducibility. Furthermore, both assays were shown to be extremely sensitive even when fungal cells were mixed with human cells, detecting 3 germinated conidia spiked in 3mL of human blood. To apply our new real-time PCR system to the molecular diagnosis of fusariosis, we evaluated its efficacy using a mouse model of invasive F. solani infection. Plasma and whole blood samples of infected mice were tested using the real-time PCR system. The sensitivity of the real-time PCR system was found to be 100% (n=4) in plasma samples. In contrast, no amplification signal was detected in whole blood samples. This system could provide a rapid and precise diagnostic tool for early diagnosis, which is necessary for appropriate treatment and improvement of prognosis of disseminated fusariosis.
Medical Mycology Research Center, Chiba University, Chiba, Japan. Electronic address: y.muraosa@faculty.chiba-u.jp.
Yasunori
Y
Muraosa
Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil.
Angelica Zaninelli
AZ
Schreiber
Infectious Diseases Division, Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil.
Plínio
P
Trabasso
Medical Mycology Research Center, Chiba University, Chiba, Japan.
Tetsuhiro
T
Matsuzawa
Medical Mycology Research Center, Chiba University, Chiba, Japan.
Hideaki
H
Taguchi
Infectious Diseases Division, Department of Internal Medicine, Faculty of Medical Sciences, State University of Campinas, São Paulo, Brazil.
Maria Luiza
ML
Moretti
Medical Mycology Research Center, Chiba University, Chiba, Japan.
Yuzuru
Y
Mikami
Medical Mycology Research Center, Chiba University, Chiba, Japan.
Katsuhiko
K
Kamei
D000818:Animals; D017931:DNA Primers / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D060585:Fusariosis / Q000175:diagnosis* / Q000382:microbiology*; D005670:Fusarium / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008813:Mice, Inbred ICR; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009172:Mycology / Q000379:methods*; D012331:RNA, Fungal / Q000235:genetics; D012339:RNA, Ribosomal, 28S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D012331:RNA, Fungal; D012339:RNA, Ribosomal, 28S
Cycling probe technology; Fusariosis; Fusarium solani species complex (FSSC); Real-time PCR