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Applied and Environmental Microbiology, May 2008, p. 3306-3309, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02082-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Rapid Species Identification of Cooked Poisonous Mushrooms by Using Real-Time PCR{triangledown}

Kazuhiko Maeta,1,3 Tomoya Ochi,2 Keisuke Tokimoto,4 Norihiro Shimomura,2 Nitaro Maekawa,2 Nobuhisa Kawaguchi,5 Makoto Nakaya,6 Yutaka Kitamoto,7 and Tadanori Aimi2*

The United Graduate School of Agricultural Sciences, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan,1 Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori-shi, Tottori 680-8553, Japan,2 Scientific Crime Investigation Laboratory, Tottori Police Headquarters, 2-12 Chiyomi, Tottori 680-0911, Japan,3 The Tottori Mycological Institute, 211 Kokoge, 689-1125 Tottori, Japan,4 Laboratory, Biological Business Department, Ichimasa Kamaboko Co., Ltd., 77-1 Junishin, Agano-shi, Niigata 959-1936, Japan,5 Hokkaido Forest Products Research Institute, Nishikagura, Asahikawa, Hokkaido 071-0198, Japan,6 Science Laboratory, Asano Industry Co. Ltd., 3-20-6 Tamahara, Tamano, Okayama 706-0014, Japan7

Received 12 September 2007/ Accepted 21 March 2008


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ABSTRACT
 
Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.


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INTRODUCTION
 
Mushrooms are identified by morphological characteristics; however, the morphological characteristics are inconsistent and unstable criteria because they are strongly influenced by the environmental conditions (2, 9). For this reason, poisonous wild mushrooms can be misidentified as edible mushrooms, and they can be eaten by mushroom hunters.

In general, rapid identification of poisonous mushroom species eaten by patients is required for proper medical treatment. However, cooked and eaten mushrooms do not retain their original shape. Therefore, techniques such as DNA-based identification, which do not depend on morphology, are required. Identification of wild and cultivated mushrooms and determination of genetic differences among basidiomycetes by using DNA techniques are usually performed by comparing the nucleotide sequences of amplified DNA fragments from nuclear and mitochondrial ribosomal DNAs (rDNAs) (1, 4, 6, 9, 10, 12, 13).

In this study, we tried to develop a rapid system of poisonous mushroom identification using a real-time PCR system. We herein propose a new strategy for mushroom identification in medical facilities.


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Strains and culture conditions.
 
In this study, 4 poisonous mushroom species that are frequently eaten by mistake in Japan and 27 popular edible mushroom species (Tables 1 and 2) were used. The mycelia of most strains were grown on GP medium (7); the mycelia of Clitocybe and Lactarius strains were grown on modified Hamada's medium (14); and the mycelia of the Armillaria strains were grown on malt extract medium (5) for later DNA extraction.


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  		TABLE 1. Poisonous mushroom strains used in this study


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  		TABLE 2. Edible mushroom strains used in this study

The fruiting body of Omphalotus japonicus was cultivated on sawdust medium (2.5 kg wet weight, 80% sawdust, 20% wheat bran, 65% final moisture content) in polypropylene bags. O. japonicus mycelia were inoculated to the medium and grown at room temperature for 2 months. At the end of this period, the bags were removed and the colonized substrates were irradiated with visible light (12 h dark and 12 h light) at room temperature for 2 months.


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Cooking of fruiting bodies.
 
Fruiting bodies were cooked as follows. For baking, the pilei of fruiting bodies were cut into 3-cm square pieces and baked on both sides in an indoor electric grill (HPS-39G-T; Sanyo Electric Co., Osaka, Japan) without oil at 240°C for 2 min each. For stir-frying, the pilei of fruiting bodies were cut into 3-mm-thick slices and stir-fried with mixing on the oiled electric grill at 240°C for 2 min. For tempura-style cooking (deep-frying in tempura batter), the pilei of fruiting bodies were cut into 3-cm square pieces and battered. The battered mushroom pieces were deep-fried in oil at 180°C for 2 min. For boiling, the pilei of fruiting bodies were cut into 3-cm square pieces and boiled in water for 30, 60, 120, or 180 min.


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DNA extraction from fruiting bodies.
 
Fruiting body tissues and mycelia were frozen in liquid nitrogen and ground to a fine powder in a mortar and pestle. DNAs were extracted from 300 mg of frozen powder (3). Figure 1 compares the size distributions of DNA isolated from fresh and cooked mushrooms. A broad range of DNA sizes up to 23 kbp was consistently present in the fresh, baked, stir-fried, and tempura-style samples, and the sizes of DNAs extracted from boiled mushroom samples were smaller than those in the other samples. For DNA isolated from O. japonicus boiled for 120 and 180 min, only DNA of less than 500 bp in size was observed.


Figure 1
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  		FIG. 1. Electrophoretic analysis of extracted DNA. Samples were electrophoresed on 2.0% agarose gels and stained with ethidium bromide. Panels A, B, and C correspond to DNA purified from the fruiting bodies of O. japonicus, E. rhodopolius, and T. ustale, respectively. In each panel, lanes and samples are lane 1, fresh; lane 2, baked for 4 min; lane 3, stir-fried for 2 min; lane 4, tempura-style (deep-fried in tempura batter for 2 min); lanes 5 to 8, boiled for 30, 60, 120, or 180 min, respectively. The size markers are EcoRI/HindIII double digests of lambda DNA and 100-bp ladders.


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Primer design for species-specific primers.
 
DNA sequencing of the rDNA ITS region (12) was carried out as previously described (8). The nucleotide sequences of the rDNA ITS region of four poisonous mushrooms and of edible mushrooms that are morphologically similar were aligned, and four oligonucleotide primer pairs were designed for the specific amplification of O. japonicus, Entoloma rhodopolius, Clitocybe acromelalga, and Tricholoma ustale rDNA (Table 3), because these poisonous mushrooms have been responsible for approximately 85% of the mushroom poisoning events in Japan over the last five years.


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  		TABLE 3. Species-specific primers for each poisonous mushroom species


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Species-specific detection.
 
PCR amplifications were performed using four species-specific primer pairs, using genomic DNAs from twenty-seven mushroom species as templates. DNA was also extracted from 300 mg of frozen powder using a GeneAll plant SV mini kit (GeneAll Biotechnology, Seoul, Korea) in accordance with the manufacturer's recommendations. Real-time PCR was carried out using Sybr green real-time PCR master mix (Toyobo Co., Osaka, Japan) with 2 µM forward and reverse primers for detection of O. japonicus and T. ustale and 0.4 µM forward and reverse primers for detection of E. rhodopolius and C. acromelalga. Real-time PCR was carried out in a 10-µl reaction mixture volume containing 5 µl of the master mix, about 2.5 ng of extracted genomic DNA, and appropriate primers. Thermocycling was performed using a LineGene real-time thermal cycler (BioFlux, Tokyo, Japan). The reaction was performed for 20 cycles, and the following cycling profile was used. The first denaturing step was at 95°C for 1 min, and then the PCR cycles were 5 s of denaturation at 95°C, 5 s of annealing at 65°C, and then 15 s of extension at 72°C. Fluorescence data were acquired during the elongation step in every cycle.

When the specific primer pairs were used, increases in fluorescence intensity were detected only for the targeted species (data not shown). Thus, we concluded that the designated primers were species specific for each poisonous mushroom. In order to confirm the universality of the four species-specific primer pairs for each species, PCR amplification was performed using genomic DNAs from 11 strains of O. japonicus, 5 strains of E. rhodopolius, and 4 strains of T. ustale (data not shown).

To confirm the amplification of specific DNA fragments of cooked mushrooms, real-time PCR was carried out. The results are shown in Fig. 2. For each primer set used for amplifying DNA from the poisonous mushroom species, species-specific detection was successful. In our proposed system, less than 1 h is required for detection via real-time PCR. However, in order to perfect the system we developed in this study, more specific primers should be designed in further studies.


Figure 2
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  		FIG. 2. Fluorescence intensity profile during real-time PCR with species-specific primer pairs using genomic DNAs from cooked and uncooked mushrooms as templates. (A) Amplified with OJSP-F/OJSP-R using genomic DNA of O. japonicus. (B) Amplified with ERSP-F/ERSP-R using genomic DNA of E. rhodopolius. (C) Amplified with CASP-F/CASP-R using genomic DNA of T. ustale. Blank, no genomic DNA; Fresh, genomic DNA from fresh fruiting body; Baked, baked for 4 min; Stir-fried, stir-fried for 2 min; Tempura, deep-fried in tempura batter for 2 min.


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Nucleotide sequence accession numbers.
 
The nucleotide sequence data obtained in this study have been deposited in the DDBJ nucleotide sequence database under accession numbers AB301601 to AB301613.


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ACKNOWLEDGMENTS
 
This research was partially supported by the Japanese Ministry of Education, Science, Sports, and Culture via a grant-in-aid for scientific research (C), 18580164, 2006-2007.


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FOOTNOTES
 
* Corresponding author. Mailing address: Faculty of Agriculture, Tottori University, 4-101 Koyama-cho Minami, Tottori 690-8553, Japan. Phone and fax: 81-857-31-5372. E-mail: taimi{at}muses.tottori-u.ac.jp Back

{triangledown} Published ahead of print on 31 March 2008. Back


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Applied and Environmental Microbiology, May 2008, p. 3306-3309, Vol. 74, No. 10
0099-2240/08/$08.00+0     doi:10.1128/AEM.02082-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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