Real-time loop-mediated isothermal amplification (LAMP) assay for group specific detection of important trichothecene producing Fusarium species in wheat.

Trichothecene mycotoxins such as deoxynivaneol (DON), nivalenol (NIV) and T2-Toxin are produced by a variety of Fusarium spp. on cereals in the field and may be ingested by consumption of commodities and products made thereof. The toxins inhibit eukaryotic protein biosynthesis and may thus impair human and animal health. Aimed at rapid and sensitive detection of the most important trichothecene producing Fusarium spp. in a single analysis, a real-time duplex loop-mediated isothermal amplification (LAMP) assay was set up. Two sets of LAMP primers were designed independently to amplify a partial sequence of the tri6 gene in Fusarium (F.) graminearum and of the tri5 gene in Fusarium sporotrichioides, respectively. Each of the two sets detected a limited number of the established trichothecene producing Fusarium-species. However, combination of the two sets in one duplex assay enabled detection of F. graminearum, Fusarium culmorum, Fusarium cerealis, F. sporotrichioides, Fusarium langsethiae and Fusarium poae in a group specific manner. No cross reactions were detected with purified DNA from 127 other fungal species or with cereal DNA. To demonstrate the usefulness of the assay, 100 wheat samples collected from all over the German state of Bavaria were analyzed for the trichothecene mycotoxin DON by HPLC and for the presence of trichothecene producers by the new real-time duplex LAMP assay in parallel analyses. The LAMP assay showed positive results for all samples with a DON concentration exceeding 163ppb. The major advantage of the duplex LAMP assay is that the presence of six of the major trichothecene producing Fusarium spp. can be detected in a rapid and user-friendly manner with only one single assay. To our knowledge this is the first report of the use of a multiplex LAMP assay for fungal organisms.

Accelerated sample preparation and formation of astragaloside IV in Astragali Radix.

Abstract Context: Astragali Radix (Huangqi; Astragalus mongholicus BUNGE, Fabaceae) is used in herbal medicinal products as well as in many food supplements. In traditional Chinese medicine, the roots are used for its Qi tonifying, immunostimulant, cardioprotective, hepatoprotective and hypoglycemic effects. Objective: Astragaloside IV (AGS-IV), a cycloartane-type triterpene glycoside is used as a marker compound for the quality control of Astragali Radix in various pharmacopoeias. Materials and methods: In this study, we analyzed the content of AGS-IV and other astragalosides in various commercial samples of Huangqi by reversed-phase HPLC using evaporative light scattering detection. Results: The analyses revealed that AGS-IV is formed during sample preparation from acylated astragalosides like astragaloside I and astragaloside II, when using the assay method of the European Pharmacopoeia. Discussion and conclusion: For consistent assay results, the extraction methods of the pharmacopoeias should be re-evaluated and optimized. Alternatively, the hydrolysis by ammonia could be omitted and the genuine compounds like astragaloside I, II and malonyl-AGS-I could be considered for the quality control of Astragali Radix.