Yamashita S, Nakagawa H, Sakaguchi T, Arima T-H, Kikoku Y
Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima, Japan.
R & D Center, Aohata Corporation, Takehara, Hiroshima, Japan.
Lett Appl Microbiol. 2018 Jan;66(1):86-92. doi: 10.1111/lam.12818. Epub 2017 Dec 6.
Heat-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.
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.
耐热真菌偶尔出现，对食品和饮料行业来说一直是个问题。篮状菌属作为一种典型的真菌，能够产生导致加工食品变质的耐热子囊孢子。异柠檬酸裂解酶是乙醛酸循环的标志性酶，对于非发酵性碳化合物（如乙酸盐和乙醇）的代谢是必需的。在此，基于大孢篮状菌和粗糙篮状菌异柠檬酸裂解酶基因的核苷酸序列，设计了用于检测和鉴定源自这两种菌的DNA的种特异性引物组。使用种特异性引物组进行的聚合酶链反应（PCR）扩增出了大孢篮状菌和粗糙篮状菌特有的产物。其他导致食品变质的真菌物种，如黄丝衣霉和条纹哈氏霉，使用篮状菌属特异性引物组未检测到。在不使用巢式PCR方法的情况下，每个种特异性引物组的检测限确定为50 pg模板DNA。在存在1000倍量其他真菌基因组DNA的情况下，每个种特异性引物组的特异性得以保持。该方法还检测到了从接种大孢篮状菌的蓝莓中提取的真菌DNA。这种PCR方法为检测大孢篮状菌和粗糙篮状菌提供了一种快速、简单、强大且可靠的方法。
与传统的耐热真菌检测方法相比，基于聚合酶链反应（PCR）的检测快速、方便且灵敏。在本研究中，开发了一种基于PCR的方法，使用靶向异柠檬酸裂解酶基因的引物组来检测和鉴定大孢篮状菌和粗糙篮状菌的扩增产物。该方法在不久的将来可用于大孢篮状菌和粗糙篮状菌的现场检测，并将有助于原材料以及食品和饮料生产的安全控制。
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