Design of a species-specific PCR method for the detection of the heat-resistant fungi Talaromyces macrosporus and Talaromyces trachyspermus

Abstract

Significance and Impact of the Study: Polymerase chain reaction (PCR)-based detection is rapid, convenient and sensitive compared with traditional methods of detecting heat-resistant fungi. In this study, a PCR-based method was developed for the detection and identification of amplification products from Talaromyces macrosporus and Talaromyces trachyspermus using primer sets that target the isocitrate lyase gene. This method could be used for the on-site detection of T. macrosporus and T. trachyspermus in the near future, and will be helpful in the safety control of raw materials and in food and beverage production. AbstractHeat-resistant fungi occur sporadically and are a continuing problem for the food and beverage industry. The genus Talaromyces, as a typical fungus, is capable of producing the heat-resistant ascospores responsible for the spoilage of processed food products. Isocitrate lyase, a signature enzyme of the glyoxylate cycle, is required for the metabolism of non-fermentable carbon compounds, like acetate and ethanol. Here, species-specific primer sets for detection and identification of DNA derived from Talaromyces macrosporus and Talaromyces trachyspermus were designed based on the nucleotide sequences of their isocitrate lyase genes. Polymerase chain reaction (PCR) using a species-specific primer set amplified products specific to T. macrosporus and T. trachyspermus. Other fungal species, such as Byssochlamys fulva and Hamigera striata, which cause food spoilage, were not detected using the Talaromyces-specific primer sets. The detection limit for each species-specific primer set was determined as being 50 pg of template DNA, without using a nested PCR method. The specificity of each species-specific primer set was maintained in the presence of 1,000-fold amounts of genomic DNA from other fungi. The method also detected fungal DNA extracted from blueberry inoculated with T. macrosporus. This PCR method provides a quick, simple, powerful and reliable way to detect T. macrosporus and T. trachyspermus.

Detection of Talaromyces macrosporus and Talaromyces trachyspermus by a PCR assay targeting the hydrophobin gene

“…The PCR detection limit for each species‐specific primer set based on the hydrophobin gene was similar to the corresponding results of an earlier study involving species‐specific primer sets targeting the isocitrate lyase gene (Yamashita et al . ). Thus, conventional PCR assays specific for the hydrophobin gene may represent a highly sensitive method for detecting Talaromyces species.…”

“…The inoculation of blueberries with mycelia and the subsequent extraction of DNA were completed as previously described (Yamashita et al 2018). The extracted DNA samples for inoculated and non-inoculated blueberries were used for the specific detection of T. macrosporus.…”

“…Additionally, species‐specific PCR methods have recently been developed for the accurate detection of heat‐resistant fungi, including Talaromyces flavus , T. macrosporus and T. trachyspermus (Panek and Frac ; Yamashita et al . ).…”

“…Among the Byssochlamys species, PCR assays using species-specific primers designed based on the b-tubulin gene have produced amplicons specific for B. fulva, B. lagunculariae, B. nivea and B. zollerniae . Additionally, species-specific PCR methods have recently been developed for the accurate detection of heat-resistant fungi, including Talaromyces flavus, T. macrosporus and T. trachyspermus (Panek and Frac 2018;Yamashita et al 2018).…”

Current PCR-based methods for the detection of mycotoxigenic fungi in complex food and feed matrices

“…Niessen (2007) reported that mycotoxigenic fungi detection technologies based on the specific DNA sequences amplification are easy to use, safe to use, rapid, sensitive, and capable of replacing the conventional mycotoxigenic fungi detection technologies. Yamashita et al (2018) developed a PCR-based method for the detection and identification of amplification products from Talaromyces macrosporus and Talaromyces trachyspermus using primer sets that target the isocitrate lyase gene. PCR-based technologies make it…”