PCR Detection Assay of Fumonisin-Producing Fusarium verticillioides Strains

Abstract

Fusarium verticillioides is considered to be the main source of fumonisins, a group of toxins that contaminate commodities and result in chronic and acute diseases affecting humans and animals. The detection and control of this species is crucial to prevent fumonisins from entering the food chain. The objective of the present research was to develop a specific, sensitive, and robust PCR assay to detect F. verticillioides strains using two pairs of specific primers for F. verticillioides, which have been designed on the basis of the intergenic spacer region of the rDNA units. The first pair of primers was F. verticillioides species specific, whereas the second pair of primers detected fumonisin-producing F. verticillioides strains. This second pair of primers allowed for the discrimination between the major group of F. verticillioides strains, fumonisin-producing strains that are mainly associated with crops, and a minor group of strains, non-fumonisin-producing strains that are associated with bananas. Fifty-four strains of F. verticillioides from different geographical regions and hosts were tested using both sets of primers. Sixteen additional Fusarium species were examined. The specificity of the primer sequences provides the basis for a simple, rapid, accurate, and sensitive detection and identification method of this fungal species that represents a risk for human and animal health.

Detection and quantification of fumonisins from Fusarium verticillioides in maize grown in southern India

“…Morphological identification of F. verticillioides was made following the description given by Nelson et al (1983) as well as Pitt and Hocking (1991). To confirm the morphological identification of F. verticillioides strains, polymerase chain reaction was conducted (Patiño et al 2004). …”

Pathogenic variation and molecular characterization of Fusarium species isolated from wilted Welsh onion in Japan

“…Diagnostic PCR analysis using species-specific primers Identity of F. verticillioides isolates was confirmed by PCR using species-specific primers, VERT-1 (5 0 -GTCAGAA TCCATGCCAGAACG-3 0 ) and VERT-2 (5 0 -CACCCGCA GCAATCCATCAG-3 0 ) for F. verticillioides under the conditions described by Patiño et al (2004), and PRO1 (5 0 -CTTTCCGCCAAGTTTCTTC-3 0 ) and PRO2 (5 0 -TGT CAGTAACTCGACGTTGTTG-3 0 ) for F. proliferatum under the conditions described by Mule et al (2004). PCR was repeated three times and the presence of a single amplicon was confirmed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining.…”

Characterization of Fusarium proliferatum and Fusarium verticillioides based on Species-Specific Gene and Microsatellites Analysis

“…Species-specific PCR amplifications were carried out using primers ProF1 (5'-CTTTCCGCCAAGTTTCTTC-3') and ProR1 (5'-TGTCAGTAACTCGACGTTGTTG-3') for detection of F. proliferatum (Jahan Quazi et al 2013); while VertF1 (5'-GTCAGAATCCATGCCAGAACG-3') and VertR1 (5'-CACCCGCAGCAATCCATCAG-3') for the detection of F. verticillioides (Patino et al 2004). PCR reactions for both primer pairs performed in a final volume of 20 μl consisting of 1 PCR buffer, 0.5 μM primer, 0.2 mM of each deoxynucleotide triphosphate (dNTPs), 2.5 mM magnesium chloride (MgCl 2 ), 0.125 U GoTaq DNA Polymerase, nuclease free water and 20 ng DNA template.…”

“…PCR reactions for both primer pairs performed in a final volume of 20 μl consisting of 1 PCR buffer, 0.5 μM primer, 0.2 mM of each deoxynucleotide triphosphate (dNTPs), 2.5 mM magnesium chloride (MgCl 2 ), 0.125 U GoTaq DNA Polymerase, nuclease free water and 20 ng DNA template. PCR condition for species-specific identification of F. proliferatum follows Jahan Quazi et al (2013); initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 61°C for 30 s, extension at 72°C for 1 min and a single cycle of final extension at 72°C for 5 min while identification of F. verticillioides follows Patino et al (2004); initial denaturation at 94°C for 1.25 min, 25 cycles of denaturation at 95°C for 35 s, annealing at 66°C for 30 s, extension at 72°C for 2 min and final extension at 72°C for 5 min.…”

“…Twenty-one isolates of F. verticillioides have amplified fragments ranging from 700-800 bp. According to Patino et al (2004), amplification of the target DNA from F. verticillioides was successful by using primer VertF1/VertR1 and single fragment of 800 bp was amplified in all strains. Meanwhile, there were no amplifications produced when using another species including a closely related species from the Gibberella fujikuroi species complex.…”