Review
Polymerase chain reaction-based methods for the detection of heat-resistant ascomycetous fungi
2023 年 64 巻 2 号 p. 47-54
詳細
2023 年 64 巻 2 号 p. 47-54
There is increasing incidence of food spoilage and health hazards caused by heat-resistant fungi belonging to the genera Byssochlamys, Thermoascus, and Neosartorya, among others. Their ascospores cannot be sterilized by heating the food. The microbiological risk assessment studies of these fungi during the production of food and beverages indicated that these fungal species or genera in food are associated with different health risks. Therefore, it is necessary to distinguish Byssochlamys, Thermoascus, and Neosartorya from other fungi in the food industry. These genera can be identified by sequence analysis of housekeeping genes such as β-tubulin, but the process is costly and time-consuming. Therefore, rapid and simple PCR-based methods have been developed using specific primer sets for genus- or species-level identification. PCR amplification products are observed to be specific for each of these genera or species and do not cross-react with other fungi associated with food spoilage and environmental contamination. These identification methods are simple, rapid, and highly specific, making them feasible for use in the quality management of food production plants.
Heat-resistant fungi often cause spoilage of heat-processed products. Since the first incidence in the 1930s (Hull, 1939; Olliver & Rendle, 1934), heat-resistant fungus-induced spoilage of canned food, including pasteurized fruit juices and beverages has been frequently reported (Dos Santos et al., 2018; Dijksterhuis, 2007; Kikoku, Tagashira, & Nakano, 2008; Kikoku, Tagashira, Gabriel, & Nakano, 2009; Rico-Miunoz, 2017; Tremarin, Aragão, Salomão, Brandão, & Silva., 2017; Tournas, 1994). Hyphae and conidia of asexual fungi are susceptible to heat; therefore, these fungi can be killed by heat treatment at 70 °C for 10 min. However, some species belonging to the genera Byssochlamys Westling, Neosartorya Malloch & Cain, Hamigera Stolk & Samson, and Thermoascus Miehe of the order Eurotiales G.W. Martin ex Benny & Kimbr (Plectomycetes Ainsw.) can survive heat sterilization at 85 °C for more than 10 min (Wyatt et al., 2015a). Heat-resistant fungi produce propagules that can tolerate heat treatment for at least 30 min at 75 °C (Samson, Houbraken, Thrane, Frisvad, & Andersen, 2010a). These propagules can be ascospores, sclerotia, chlamydospores, or thick-walled hyphae. These structures can withstand high temperatures (Dijksterhuis, 2007). These fungi are considered to endure adverse environmental changes such as high temperatures to maintain their race. Conidia have thin cell walls with the composition same as that of hyphae; therefore, they can easily disperse and germinate. In contrast, ascospores have thick cell walls and can store nutrients; therefore, they can survive under adverse conditions. Heat resistance is acquired in two phases during ascospore maturation. In the first phase, compatible solutes accumulate and reduce water concentration, which in turn decreases the metabolic rate. In the second phase, the concentration of mannitol decreases and that of trehalose and trehalose-based oligosaccharides increases. Compatible solutes are substances that protect cells from high temperatures, drought, and other stresses. These substances, at any concentration, do not interfere with the functioning of proteins and other biomolecules and the developmental phases of the fungi (Wyatt et al., 2015a, b; Wyatt, Van Leeuwen, Wösten, & Dijksterhuis, 2014). Usually, heat-resistant ascospores inhabit the soil. Breaking of their dormancy and germination requires stimulation, such as heat shock (Warcup & Baker, 1963). Therefore, depending upon the range of heating conditions, heat-processing of food with raw materials contaminated with heat-resistant spores may promote the germination of ascospores rather than sterilization (Rico-Munoz, 2017).
Materials contaminated by heat-resistant fungi can be a risk to consumer health because of the toxic metabolites (mycotoxins) these microbes produce (Fornal, Parfieniuk, Czeczko, Bilinska-Wielgus, & Frąc, 2017; Frąc, Jezierska-Tys & Yaguchi, 2015). Due to the resistance of fungi to high temperatures, they can survive the industrial pasteurization process. Therefore, the only way to prevent the growth of these microorganisms in a product is to select a suitable material by conducting tests for the presence of heat-resistant fungi (Frąc et al., 2015). Food products such as fruits should be processed fresh; therefore, time is a critical factor in assessing the acceptance or rejection of a given batch of raw material. The traditional culture methods to identify contaminating fungi are time-consuming. Moreover, it is difficult to identify fungi at the species level based on morphology alone. Therefore, this method cannot be applied to the selection of raw materials for food production (Hosoya et al., 2012, 2014; Nakayama et al., 2010; Yaguchi et al., 2012).
Microbiological risk assessment during the production of foods and beverages requires a detailed understanding of the microbiological profiles for the capacity of mycotoxin production and the degree of heat resistance. These profiles indicate that the presence of each of these species or genera in foods is with different health risks.
In this review, we discuss the necessity to discriminate heart-resistant fungi, and the development of rapid and simple PCR-based methods using specific primer sets to detect them at the genus or species level. Byssocholamys, which has been previously reported as a food-hazard mold, and Thermoascus, which is highly heat-resistant, were phylogenetically closely related. We evaluated their heat resistance and ability to produce patulin, and developed the methods to discriminate between these genera (Hosoya et al., 2012, 2014; Nakayama et al., 2010). Furthermore, we conducted a study to discriminate Neosartorya and its anamorph, Aspergillus fumigatus Fresen., important as a clinical fungus (Yaguchi et al., 2012).
The scientific names of fungi were derived from the International Code of Nomenclature for Algae, Fungi, and Plants (formerly the International Code of Botanical Nomenclature, referred to as the ‘Code of Nomenclature’). Amendments to the Code of Nomenclature are discussed by the Nomenclature Subcommittee of the International Congress of Plant Sciences, which meets every six years. In 2011, a symposium ‘One Fungus = One Name’ held in Amsterdam, Netherlands included the deletion of Article 59 (a dual nomenclature for the fungi) of the Code of Nomenclature (Hawksworth et al., 2011; Wingfield, 2011). This was followed by discussions at the International Congress of Plant Sciences, held in Shenzhen, China, in 2017. Here, article 57.2 of the Nomenclature Code, which stipulates the ‘priority of teleomorph names’, was deleted. It suggested that ‘teleomorph and anamorph names of Ascomycetes and Basidiomycetes should be integrated according to the priority of publication.
The integration of the scientific names of sexual forms, which include the industrially important fungi Penicillium Link and Aspergillus P. Micheli ex Haller and the major heat-resistant molds, was discussed mainly by the International Commission of Penicillium and Aspergillus (ICPA) and is available online (http://www.aspergilluspenicillium.org/). An overview of this (Fig. 1) is provided below (Houbraken & Samson, 2011; Samson et al., 2011).
Aspergillus species and their related teleomorphs, Eurotium Link, Emericella Berk., Neosartorya and etc. are divided into six subgenera. Based on molecular phylogenetic studies, these species belong to a single phylum; therefore, the name Aspergillus is used for all Aspergillus species (including teleomorphs) (Samson et al., 2014). However, in some cases, such as heat-resistant fungi, names such as Neosartorya are optionally used. The genus Neoartorya is equivalent to the teleomorph species in Aspergillus section Fumigati, and cannot to be describe in one word. Therefore, following to this proposal of ICPA, teleomorphic names, such as Neosartorya are presented in this review, although the official name is Aspergillus.
In contrast, Penicillium species and their related teleomorphs, Eupenicillium F. Ludw. and Talaromyces C.R. Benj. are broadly divided into three subgenera, Aspergilloides Dierckx, Biverticillium Dierckx and Penicillium Link. Among these, Eupenicillium and two subgenera, Aspergilloides and Penicillium are combined into a monophyletic group; therefore, the name Penicillium is used for these genera. Talaromyces, on the other hand, has been regarded as a polyphyletic genus, but the name Talaromyces is used for the other subgenus Biverticillium and the related teleomorph, and Rasamsonia Houbraken & Frisvad for the anamorph Geosmithia Pitt (Visagie et al., 2014).
The genera Byssochlamys, Hamigera, and Thermoascus are heat-resistant fungi, each representing a monophyletic group; therefore, these generic names are used as current names.
To analyze the risks associated with Byssochlamys spp., we first evaluated the heat resistance of the ascospores of each species. In microbiology, the decimal reduction time values (D-values) is an indicator for heat resistance and the time required to kill 90% of the heated microorganisms at a prescribed temperature. D80 and D85 of B. fulva were 90 and 17 min, respectively, in apple juice and D80 and D85 were 71 and 12 min, respectively, in saline solution (Table 1). Other Byssochlamys spp. showed lower levels of heat resistance than B. fulva. All species and strains examined showed higher D-values in apple juice than in saline solution, which was possibly due to the protective effects of the juice components. Of the species examined, B. fulva showed the greatest heat resistance. Under standard sterilization conditions involving exposure of vegetative cells to a temperature of 80 °C for 10 min, three Byssochlamys species (B. fulva Olliver & G. Sm., B. lagunculariae (C. Ram) Samson, Houbraken & Frisvad, and B. nivea Westling) showed high D80s (min) indicating that commercial pasteurization is insufficient to kill these organisms. On the other hand, B. zollerniae C. Ram did not form ascospores under our experimental conditions, indicating a very low probability of survival after heat sterilization. These findings indicated that there are marked differences in heat resistance among three species of Byssochlamys spp. that have been reported to act as causative agents of food spoilage (Nakayama et al., 2010, Ueda, Kawara, Yaguchi, & Udagawa, 2010). Moreover, D90s of H. avellanea Stolk & Samson and H. striata (Raper & Fennell) Stolk & Samson were calculated to be 6.5-7.6 and 10 min, and this species showed higher levels of heat resistance than all species of Byssochlamys (Scaramuzza & Berni, 2014; Kikoku et al., 2008).
| D-value (min)a) | Patulin production (PPB)a, b) | PCR with specific primer for idh genea, c) | Genus specific primera) | Species specific primera) | |||
| Heating temperature (°C) | |||||||
| 80 | 85 | 90 | |||||
| Thermoascus aegyptiacus | - | - | 56 | N.D.d) | x | RPBF2/RPB2R | - |
| T. aurantiacus | 57-288 | 11-16 | - | N.D. | + | - | |
| T. crustaceus | - | - | 90 (survival) | N.D. | x | - | |
| T. thermophilus | - | - | 21 | N.D. | x | - | |
| T. verrcosa | - | - | - | N.D. | + | - | |
| Byssochlamys fulva | 71-90 | 12-17 | - | N.D. | x | B1F/B1R | B.fulva1F/R |
| B. nivea | 14-15 | 1.4-2 | - | 46 | + | B.nivea1F/R | |
| B. lagunculariae | 4.5-5 | 0.4 | - | 1.3 | + | B.lag1F/R | |
| B. zollerniae | - | - | - | N.D. | x | B.zol3F/R | |
| B. spectabilis/Pae. variotii | Conidial survival >10% after a 10-min-heat treatment at 59 °Ce) | - | - | - | Pae4F/Pae4-1R | ||
| Hamigera avellanea | - | - | 6.5-7.6f) | - | - | H2F/H2R | - |
| H. striata | 286g) | 43g) | 10g) | - | - | - | |
b)Cultivation on Czapek-glucose broth, for 14d, extracted with ethyl acetate, alkaline washing and detection by HPLC.
c)Gene encoding dehydrogenase of Isoepoxydon, detected by PCR with specific primers.
d)Not detected.
e)van Brule et al. (2020)
Patulin is a mycotoxin produced by species belonging to genera of Penicillium, Aspergillus and heat resistant Byssochlamys (Moake et al., 2005). The toxicity concerns the acute and sub-acute toxicity, geno-toxicity, embryo-toxicity and teratogenicity (Puel, Galtier, & Oswald, 2010). The highest risk of contamination by patulin is connected with apple industry producing juices and other apples-derived products (Puel et al., 2010). The maximum level of patulin in food products and juices allowed by European Union is 50 µg/kg (CodexAlimentarius, 2003) and Japan also limit patulin to 50 µg/kg in apple juice.
Luque et al. (2011) reported a PCR-based method to detect Penicillium and Aspergillus species that produce patulin, but did not discuss patulin production by Byssochlamys spp. In our study, PCR was performed using the primers idhBPF/R (Hosoya et al., 2012). The PCR products revealed the amplification of the idh gene in B. nivea and B. lagunculariae, indicating that they are potential producer of patulin. No amplification product was obtained for B. fulva or B. zollerniae, suggesting that these species lack the idh gene. B. nivea and B. lagunculariae showed patulin production in Czapek-glucose liquid medium and potato dextrose broth, whereas B. fulva and B. zollerniae did not (Hosoya et al., 2012).
3.3. PCR using species-specific primers for Byssochlamys spp.At first, the genus specific primer sets were designed to specifically detect strains of Byssochlamys, Hamigera and related genera, Paecilomyces variotii Bainier (teleomorph: B. spectabilis (Udagawa & Shoji Suzuki) Houbraken & Samson), respectively, using the sequences of β-tubulin gene (Fig. 2; Nakayama et al., 2010). Moreover, it is necessary to distinguish the species of Byssochlamys, because species of Byssochlamys exhibit differences in thermostability and patulin-producing capacity. The PCR-based assay using the B.fulva1F/1R primer set was designed to specifically detect strains of B. fulva based on the sequences of β-tubulin gene. The primers did not yield any PCR product for B. nivea, B. lagunculariae, B. zollerniae, Paecilomyces spp., Hamigera spp., Talaromyces spp., or any other environmental fungal isolates (Fig. 2; Hosoya et al., 2012).
Similarly, PCR with the B.nivea1F/1R, B.lag1F/1R, and B.zol3F/3R primer sets yielded products only for B. nivea, B. lagunculariae, and B. zollerniae, respectively, indicating the specificity of the used primers (Fig. 2; Hosoya et al., 2012) , supporting the discovery above.
The species of Thermoascus can grow at high temperatures and have been detected in a variety of agricultural products, including maize stored in sub-Sahara Africa and olive and olive cake in Morocco, and in food-related environments (Roussos et al., 2006). The production of high amounts of amylase and cellulase by Thermoascus spp. can markedly alter the properties of food products and reduces the commercial value of food. The high thermostability of these enzymes prevents their heat-inactivation. Recently, B. verrucosa Samson & Tansey was reclassified as T. verrucosus (Samson & Tansey) Houbraken, Frisvad & Samson (Houbraken et al., 2020).
4.1. Heat resistance of ascospores formed by Thermoascus spp.This genus poses a risk to the food industry because of the presence of heat-resistant ascospores. For instance, ascospores of T. aurantiacus can survive at 88 °C for 60 min (King, Michener, & Ito, 1969). In a study to detect heat-resistance of Thermoascus spp., D90s of T. egyptiacus S. Ueda & Udagawa and T. thermophilus (Sopp) Arx were 56 min and 21 min, respectively, and the D90 of T. aurantiacus Miehe at 85 °C was 11-16 min. The D-value for T. crustaceus (Apinis & Chesters) Stolk could not be determined accurately; however, their survival was confirmed even after heating at 90 °C for 90 min. These species showed greater degrees of heat resistance than other heat-resistant fungi, such as B. fulva (D85 = 17 min) and N. fischeri (Wehmer) Malloch & Cain (current name: A. fischeri Wehmer) (D85 = 10-30 min), which have highly heat-resistant ascospores. Thermoascus aegyptiacus showed the highest degree of heat resistance (D90 = 56 min) among all the studied species. In addition, all examined species of Thermoascus grew at temperatures > 50 °C (Table 1; Hosoya et al, 2014).
4.2. Rapid detection based on idh gene for patulin production for Thermoascus.Byssochlamys nivea and B. lagunculariae, which are related to the genus Thermoascus, were shown to possess the idh and produce patulin. Byssochlamys and some species of Thermoascus have the same anamorph, Paecilomyces Bainier. As T. aurantiacus is toxic to chicken embryos and weanling rats, it was hypothesized that Thermoascus spp. may carry patulin mycotoxin. Both T. aurantiacus and T. verrucosa possess the idh gene, and each showed 99.9% identity with that of B. nivea at the nucleotide level. However, T. aegyptiacus, T. crustaceus, and T. thermophiles did not possess the idh gene and form a monophyletic clade separate from T. aurantiacus and T. verrucosa in the phylogenetic tree. Despite abundant fungal growth, patulin was not detected in potato dextrose broth or Czapek-glucose liquid medium. It may be due to the defect in a gene, other than idh, involved in patulin biosynthesis, such as 6-MSA synthase (Puel et al., 2010) or the deactivation of idh (Puel et al., 2007). These findings indicated a low risk of patulin production by Thermoascus spp. As this genus is used to produce heat-resistant enzymes, such studies are important not only for food safety but also for industrial enzyme production (Hosoya et al., 2014).
4.3. Evaluation of Thermoascus genus-specific primers.All species of Thermoascus showed high levels of heat resistance, therefore, it is necessary to distinguish those species in the genus level. PCR using the RPB_F2 and RPB_R2 primer sets based on the sequences of DNA-directed RNA polymerase II gene, produced amplification products for all the Thermoascus species. However, no amplification products were detected for Byssochlamys or Hamigera species, or other fungi associated with food spoilage (Fig. 2). In addition, gene amplification products were produced through PCR conducted using DNA extracted from T. aurantiacus that had been added to an acidic beverage. Therefore, this method is unaffected to detect the causative fungi in acidic beverages, and even in cases in which a causative fungus is not isolated, if the DNA can be extracted, the causative agents can be detected (Hosoya et al., 2014).
Neosartorya species, used as optional heat-resistant fungi in Aspergillus section fumigati, are commonly found in soil and pasteurized food products worldwide. They can be distinguished by their ascospore ornamentation. Neosartorya fischeri, N. glabra (Fennell & Raper) Kozak. (current name: A. neoglaber Kozak.), N. hiratsukae Udagawa, Tsub. & Y. Horie (current name: A. hiratsukae Udagawa, Tsub. & Y. Horie), N. pseudofischeri S.W. Peterson (current name: A. thermomutatus (Paden) S.W. Peterson), N. spinosa (Raper & Fennell) Kozak. (current name: A. spinosa (Raper & Fennell) Kozak.), and other species can cause spoilage of heat-processed food (Samson, Houbraken, Thrane, Frisvad, & Andersen, 2010c). In contrast, N. fischeri is phylogenetically close to A. fumigatus, which can be inactivated by heating at approximately 70 °C for 10 min. Therefore, it was considered to be difficult to design primer sets that can discriminate between them.
A phylogenetic study based on sequence analysis of the β-tubulin, calmodulin and actin genes was performed in A. fumigatus and related species, including Neosartorya species. At the results, those sequences were able to clearly identify A. fumigatus and Neosartorya spp. at the species level. (Yaguchi et al., 2007).
5.1. Rapid detection on Neosartorya genus-specific and species-specific primersA study was conducted to develop a method to discriminate Neosartorya from other genera and identify different species in N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri, and N. spinosa-complex (Yaguchi et al., 2012). As the first step, N2F/2R primer sets were designed to identify the genus of Neosartorya based on the sequences of β-tubulin gene. These primers were designed using a region that is conserved only in Neosartorya species; therefore, theoretically, it appeared possible to detect only Neosartorya species using this primer, although this was not observed in the study. PCR products were detected not only for Neosartorya but also for A. fumigatus, which has a similar morphology to the anamorph of Neosartorya and clusters with it on the phylogenetic tree. Recently, a teleomorph of A. fumigatus, N. fumigata, was identified by crossing experiments using combinations of opposite mating types. At the sexual stage, this species forms cleistothecia, producing viable ascospores that germinate to form the anamorph A. fumigatus (O'Gorman, Fuller, & Dyer, 2009). However, A. fumigatus has never been reported as a spoiling agent of heat-processed food products, and it is not considered to be distributed at the sexual stage in nature. Therefore, it is extremely important to discriminate between A. fumigatus and the other Neosartorya species in the food industry (Yaguchi et al., 2012; Pertile et al., 2020).
Similarly, A. lentulus Balajee & K.A. Marr and A. udagawae Y. Horie, Miyaji & Nishim. were detected using the primer set N2F/2R. Although these three species are not the agents of food spoilage, they are the causative agents of mycoses (de Hoog et al., 2021a). As all these species are harmful to humans, the detection of these species does not affect the discrimination of Neosartorya species as food spoilage agents.
D95 and D85 for N. pseudofischeri are 0.33 min and 26-27 min, respectively; therefore, N. psuedofischeri is considered to have high heat resistance (Dijksterhuis, 2007; Ueda, Kawara, Yaguchi, & Udagawa, 2009). Furthermore, the detection frequency of Neosartorya spp. in food and soil varies according to the species. These species produce various toxins; for example, N. fischeri produces neurotrophic mycotoxins fumitremorgin A and verruculogen, and N. pseudofischeri produces gliotoxins that cause fungal infections (de Hoog et al., 2021b; Samson, Hong, Peterson, Frisvad, & Varga, 2007). Therefore, the development of a rapid method for species identification is essential for the food industry. Recently, based on phylogeny, strains such as N. spinosa have been identified to have new species, N. coreana Hong, Frisvad, & Samson (current name: A. coreanus Hong, Frisvad, & Samson) (Hong, Cho, Shin, Frisvad, & Samson, 2006) and N. paulistensis Y. Horie, Miyaji & Nishim. (current name: A. paulistensis Y. Horie, Miyaji & Nishim.) (Horie, Miyaji, Nishimura, Franco, & Coelho, 1995). As these species formed a monophyletic clade, a significant difference among them has not been established. Additionally, D85s of N. spinosa and N. paulistensis are almost the same in grape juice, 15-20 min and 16 min, respectively (Ueda et al., 2009). Therefore, these three species are recognized as N. spinosa complex.
We examined to identify Neosartorya and A. fumigatus at the species level, and succeeded in developing the PCR method of differentiating and identifying Neosartorya and A. fumigatus using specific primer sets (Yaguchi et al., 2012). Moreover, we developed specific primer sets for the species-level identification of N. fischeri, N. glabra, N. hiratsukae, N. pseudofischeri and N. spinosa-complex, which are important in food spoilage and have different heat resistance properties and productivities of mycotoxins depending on the species (Table 2; Yaguchi et al., 2012).
| D-value (min)a) | Mycotoxin production | Genus specific primerb) | Species specific primerb) | |||
| Heating temperature (°C) | ||||||
| 80 | 85 | 95 | ||||
| Neosartorya fischeri | - | 10-30 | - | Fumitremorgin A, B, verruculogen | N2F/N2R | Nfi3F/3R |
| N. glabra | - | 10-21 | - | - | Ngl1F/1R | |
| N. hiratsukae | 74 | - | - | - | Nhi1F/2R | |
| N. pseudofischeri | - | 26-27 | 0.33 | Gliotoxin | Npf2F/2R | |
| N. spinosa | - | 15-20 | - | - | Nsp3F/3R | |
| Aspergillus fumigatus | - | - | - | Gliotoxin | Af1F/1R | |
The primers specific for N. fischeri and A. fumigatus work very well not only for pure cultures but also for fungal DNA extracted from strawberry fruit and juice. First, the mycotoxin presence in the fruits and juices is detected. If positive results are found, N. fischeri is detected directly in environmental samples with specific primers (Pertile et al., 2020). This method can be applied for the quick diagnosis of the presence of these fungi in agricultural environments.
5.2. Detection limits and effects of contamination by other fungal DNAPCR and nested PCR using primers N2F/2R have a DNA template detection limit of 40 pg/µL and 4 pg/µL, respectively, to identify Neosartorya. The specificity of the PCR assay has been maintained even if the sample was contaminated 100-fold with A. fumigatus DNA (Yaguchi et al., 2012).
The primers specific for Neosartrya, Byssochlamys, and Thermoascus exhibited the same results. For example, the specificity of the PCR assays for Byssochlamys and Thermoascus was maintained even if the sample was contaminated 1000-fold with other fungal DNA for all the primer sets tested (Hosoya et al., 2012, 2014; Nakayama et al., 2010).
The detection and identification of fungi are generally based on morphological examination; however, there is a need for the development of more rapid and versatile methods. The PCR-based detection method has several advantages over sequence analysis of the ITS region and housekeeping genes. First, it can be performed rapidly with high sensitivity and requires no special expertise. Second, the equipment involved is inexpensive, making it feasible to perform quality management at production plants. Third, the causative fungi of food spoilage can be determined by the extracted DNA, without the isolation of fungi from the food product. Using those methods, even dead fungi can be detected, if the DNA can be extract. Lastly, this method is not affected by acidic beverages.
The discrimination between heat-resistant fungi and others are important to perform microbiological risk assessment during the production of acidic foods and beverages. Therefore, simple, rapid, and highly specific PCR-based methods are expected to be beneficial for the surveillance of raw materials used in food production (Hosoya et al., 2012, 2014; Nakayama et al., 2010; Yaguchi et al., 2012).
We developed a rapid method in genus or species level of detecting Byssochlamys, Neosartorya, Hamigera, and Thermoascus, which are important heat-resistant fungi in food products. PCR methods with specific primers are very useful for the rapid identification of fungi other than heat-resistant ones (Hirano & Arie, 2006; Nakayama, Hosoya, Shimizu-Imanishi, Chibana, Yaguchi, 2016; Susca, Stea, Mule, & Perrone, 2007). For example, a method of detecting acetic acid and ethanol resistant species of Moniliella using a primer targeting the D1/D2 domain of 28S rDNA was recently reported (Nakayama et al., 2016). However, if the primers are not specific enough, they may cause false positive result. The need for the development and application of more specific primers will be increased in the future.
This work was supported in part by the Global R&D-Safety Science, Kao Corporation, and the National Bioresource Project-Pathogenic microbes in Japan (http://www.nbrp.jp/).
