Rapid and selective detection of macrocyclic trichothecene producing Stachybotrys chartarum strains by loop-mediated isothermal amplification (LAMP)

Abstract

Cytotoxic macrocyclic trichothecenes such as satratoxins are produced by chemotype S strains of Stachybotrys chartarum. Diseases such as stachybotryotoxicosis in animals and the sick building syndrome as a multifactorial disease complex in humans have been associated with this mold and its toxins. Less toxic non-chemotype S strains of S. chartarum are morphologically indistinguishable from chemotype S strains, which results in uncertainties in hazard characterization of isolates. To selectively identify macrocyclic trichothecene producing S. chartarum isolates, a set of sat14 gene-specific primers was designed and applied in a loop-mediated isothermal amplification (LAMP) assay using neutral red for visual signal detection. The assay was highly specific for S. chartarum strains of the macrocyclic trichothecene producing chemotype and showed no cross-reaction with non-macrocyclic trichothecene producing S. chartarum strains or 152 strains of 131 other fungal species. The assay's detection limit was 0.635 pg/rxn (picogram per reaction) with a reaction time of 60 min. Its high specificity and sensitivity as well as the cost-saving properties make the new assay an interesting and powerful diagnostic tool for easy and rapid testing.

Keywords: Diagnostic test kit; Indoor air quality; Sick building syndrome; Stachybotryotoxicosis; Water damage; sat14 gene.

Fig. 1

Comparison of the sat14 gene partial sequence with a consensus sequence of the PCR product obtained with primers F2/B2 on the smallest DNA fragment produced during reactions with the sat14 gene specific LAMP assay

Fig. 2

Influence of loop primers on reaction speed of the sat14 gene specific LAMP reaction using V13 as fluorescent indicator. Graph-pair 1: Both LF + LB loop primers added to the reaction. Graph-pair 2: only LB primer added to the reaction. Graph-pair 3: only LF primer added to the reaction. Graph-pair 4: no loop primers added to the reaction

Fig. 3

Sat14 gene specific LAMP reaction with a serial dilution of gDNA of reference strain CBS 414.95 using neutral red as pH sensitive indicator (positive reaction indicated by color change from yellow to purple). Cap with 10 ^-1 holds 1.27 ng/μl (equivalent to 6.35 ng per reaction) as initial template DNA concentration, NTC = No template control