Specific Detection of Fusarium Species in Blood and Tissues by a PCR Technique

Abstract

Fusarium species are opportunistic nosocomial pathogens that often cause fatal invasive mycoses. We designed a primer pair that amplifies by PCR a fragment of a gene coding for the rRNA ofFusarium species. The DNAs of the main Fusariumspecies and Neocosmospora vasinfecta but not the DNAs from 11 medically important fungi were amplified by these primers. The lower limit of detection of the PCR system was 10 fg of Fusarium solani DNA by ethidium bromide staining. To test the ability of this PCR system to detect Fusarium DNA in tissues, we developed a mouse model of disseminated fusariosis. Using the PCR, we detected Fusarium DNA in mouse tissues and in spiked human blood. Furthermore, F. solani, Fusarium moniliforme, and Fusarium oxysporum were testing by random amplified polymorphic DNA (RAPD) analysis. The bands produced by RAPD analysis were purified, cloned, and sequenced. The information was used to design primer pairs that selectively amplified one or severalFusarium species. The method developed may be useful for the rapid detection and identification of Fusarium species both from culture and from clinical samples.

Mycotic keratitis: an overview of diagnosis and therapy

“…Polymerase chain reaction has emerged as a new promising tool, which has been shown to be useful for the culture-independent diagnosis of various microbial infections, including mycoses. [105][106][107][108] This method has been used for the diagnosis of keratitis in rabbit model of Fusarium keratitis using specific primers. 109 As described above, there are more than 56 fungal genera known to cause mycotic keratitis, thus, there is a need to design fungal specific primers, which anneal only with fungal DNA but not with other microbes including bacteria, viruses and Acanthamoeba DNA that are other most common causal agents of keratitis.…”

Development of a bipartite method for Fusarium identification based on cellobiohydrolase-C: CAPS and Western blot analysis

“…The ampli¢cation of a 329-bp fragment from genomic DNA with primers P58SL and P28SL was shown to be nearly exclusive to the Fusarium genus [6]. All genomic DNAs of our Fusarium showed ampli¢cations of the expected size ( Fig.…”

“…Speci¢c primers for rDNA ampli¢cation [6] were used to verify that our strains belonged to the genus Fusarium. The preliminary species determination was then carried out [7].…”

“…PCR ampli¢cation, followed by restriction fragment length polymorphism (the combination of these two methods is also known as cleaved ampli¢ed polymorphic sequence (CAPS)) di¡erentiates sapstain fungi [4] and Aspergillus species [5]. PCR on rDNA permits the speci¢c detection of fungi belonging to the genus Fusarium [6] and rDNA CAPS allows identi¢cation of 23 haplotypes de¢ning 18 Fusarium species [7]. Antibody techniques have also been used for identi¢cation of fungi [8].…”

Identification of Fusarium graminearum in cereal samples by DNA Detection Test StripsTM

“…(Buglova et al 1983). Other PCR protocols for detection of several other Fusarium species in clinical samples exist (Hue and Huerre 1999;Jaeger et al 2000). With regard to the problem of mycotoxin production in fusaria, a PCR protocol was developed for the detection of potentially trichothecene producing Fusarium species .…”