Development of cycling probe-based real-time PCR system to detect Fusarium species and Fusarium solani species complex (FSSC)

Abstract

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.

Epidemiological Study of <I>Fusarium</I> Species Causing Invasive and Superficial Fusariosis in Japan

“…All isolates were initially identified by Fusariumspecific real-time PCR using the primer pair Fusa28sF1 and Fusa28sR1 and probe Fprobe1 35) .…”

“…Subsequently, Fusarium isolates were classified as Fusarium solani species complex(FSSC)or non-FSSC Fusarium by FSSC-specific real-time PCR using the primer pair Fusa28sF1 and Fusa28sR1 and probe FSprobe1 35) . Real-time PCR conditions were described in our previous study 35) .…”

“…For fungal identification based on DNA se-quencing analysis, a partial the elongation factor-1 alpha(EF-1α)gene was amplified by PCR using the primers HS392 and HS393, as described in our previous study 35) . The nuclear ribosomal internal transcribed spacer(ITS)region, including ITS1, 5.…”

Detection of Fusarium Species in Clinical Specimens by Probe-Based Real-Time PCR

“…To improve Fusarium identification, DNA detection via polymerase chain reaction (PCR) is a rapid and sensitive tool. There are different methods to detect Fusarium by PCR including conventional and real-time (hybridization or hydrolysis probe based) PCR [6,[13][14][15], which are limited by their high turnaround time, detection of some selected species, or uncommon real-time PCR formats. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.…”

New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections

“…In addition, those protocols based on quantitative real-time PCR (qPCR) have the benefit of quantifying the fungal burden in clinical specimens (5). Several qPCR protocols have been developed for the diagnosis of IFIs, mainly for Aspergillus and Candida species (6) but also for less frequent fungal species (7,8,9,10). However, the utility of a species or genus-specific approach is limited when there is not a clear suspicion of the fungus involved in the IFI.…”