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Detection of aflatoxigenic fungi in selected food commodities by PCR
Process Biochemistry 40 (2005) 2859–2864 www.elsevier.com/locate/procbio
Detection of aflatoxigenic fungi in selected food commodities by PCR H.K. Manonmani a, S. Anand b, A. Chandrashekar c, E.R. Rati b,* a
Fermentation Technology Bio Engineering, Central Food Technological Research Institute, Mysore 570 020, India b Human Resource Development, Central Food Technological Research Institute, Mysore 570 020, India c Plant Cell Bio Technology, Central Food Technological Research Institute, Mysore 570 020, India Received 4 September 2003; received in revised form 27 October 2004; accepted 7 January 2005
Abstract Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. A regulatory gene, aflR, is involved in regulation of aflatoxin biosynthesis and the sequence has been published. In this study, rapid assessment of aflatoxigenic fungi in food was accomplished using an indigenously designed primer pair for the aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure and mixed culture systems using DNA extracted from 28 different fungal strains as PCR template. Positive amplification was achieved only with DNA from aflatoxigenic A. flavus and A. parasiticus. The detection limit for mycelium and spores was determined as 0.05 g and 100 cfu, respectively. Specificity and sensitivity of PCR assay in groundnuts and maize for A. flavus was possible with as few as 100 cfu/g. # 2005 Elsevier Ltd. All rights reserved. Keywords: Aflatoxins; Aspergillus flavus; Aspergillus parasiticus; PCR; aflR; Food commodities
1. Introduction Aflatoxins are secondary metabolites produced by the aflatoxigenic fungi Aspergillus flavus Link and Aspergillus parasiticus Speare [1]. Aflatoxin contamination of agricultural commodities had gained global significance as a result of their deleterious effects on human and animal health as well as their importance to international trade. The contamination of foods by aflatoxigenic fungi, especially in tropical countries may occur during preharvesting, processing, transportation and storage [2]. These fungi mainly infect maize, cotton, peanuts, treenuts [3], figs [4] and spices [5–7]. One of the most important requirements for eliminating aflatoxin is the identification of mycotoxigenic fungal contamination. The detection of the aflatoxigenic fungi is usually performed by traditional dilution plating, use of diagnostic media or by immunological methods. The traditional methods are time consuming, labour-intensive, costly, require mycological expertise and facilities. Immunological methods and diagnostic media have limitation in identifying the aflatoxigenic fungi in that false positives are * Corresponding author. Tel.: +91 821 251430; fax: +91 821 2517233. E-mail address: [email protected] (E.R. Rati). 0032-9592/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.01.004
easy to come by and purifications of samples are a necessary prerequisite. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR) facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for long, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile [8]. Many pathogenic organisms have been detected using PCR [9]. The biosynthetic pathway for aflatoxin production by A. flavus has been deciphered and genes in the aflatoxin biosynthetic pathway have been identified [11,3]. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis [12]. Few other genes of the aflatoxin biosynthetic pathway have been cloned and sequenced [13]. Reports on the detection of aflatoxigenic fungi using PCR are rather scanty [4,10,19]. In our studies, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1) since these genes are nearly identical in A. flavus and A. parasiticus [3], indicating the possibility of detection of both the species with the same PCR system (primers/reaction). However, due to the complexity of the food systems, many food components
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could interfere during PCR interaction while inhibiting the activity of the polymerase [14]. In this study, we describe the use of PCR to distinguish aflatoxigenic fungi present in food samples along with other moulds and microorganisms.
2. Materials and methods
2.4. Primers and PCR chemicals PCR primers were designed using primer 3 software and were procured from Sigma, Genosys Ltd., UK. Taq polymerase and dNTPs were purchased from Bangalore Genei, Bangalore, India. Other chemicals used in these studies were of molecular biology grade and purchased from standard chemical companies.
2.1. Microorganisms 2.5. Isolation of fungal DNA The fungal isolates, their source and data on their mycotoxigenic potential are listed in Table 1. The fungal isolates were maintained on potato dextrose agar or on CzapekDox Agar media. Cultures were sub-cultured periodically and 5-day-old slant cultures were used in these studies. 2.2. Food commodities Raw food ingredients including groundnuts and maize were procured from the local market and powdered to pass through a 20 British Standard Mesh (BSM) sieve (700 mm). 2.3. Assessment of fungal load on commodities Conventional dilution plating techniques were employed to assess the total yeast and mould flora of the raw ingredients [15].
Template DNA was extracted from 0.5 g (wet weight) fungal mycelia harvested from freshly growing cultures in potato dextrose broth (PDB) under stationery conditions. The mycelium was washed twice with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4) followed by centrifugation. The mycelium was transferred to a mortar, frozen in liquid nitrogen and was ground well. Lysis buffer (50 mM Tris, 150 mM EDTA, 1% (w/v) SDS pH 8.0) was added to the pulverized mycelia and incubated at 65 8C for 1 h. The suspension was centrifuged and the supernatant transferred to a new centrifuge tube. The supernatant was then extracted twice with phenol:chloroform:isoamylalcohol (25:24:1) and the aqueous layer was precipitated with 2 volumes of isopropanol. The precipitate was resuspended in 200 ml of TE buffer (10 mM Tris–HCl, 1.0 mM EDTA, pH 8.0) [16].
Table 1 List of fungal isolates Fungal isolate
Source
Mycotoxin
Aspergillus flavus ATCC 46283 Aspergillus ochraceus CFR 221 Fusarium tricinctum NRRL 32998 Fusarium fujikuori NCIM 665 Fusarium fujikuroi NCIM 1019 Fusarium saubinetti NCIM 851 Fusarium spp. Fusarium spp. Rhizopus spp. Rhizopus chinensis Rhizopus arrhizus NCIM 997 Aspergillus flavus NCIM 645 Aspergillus flavus Aspergillus flavus Aspergillus parasiticus CFR 223 Aspergillus oryzae Aspergillus oryzae Aspergillus oryzae Aspergillus oryzae CFR 225 Aspergillus oryzae Aspergillus flavus MTCC 152 Aspergillus flavus DCS Aspergillus ochraceus CFR 221 Aspergillus glaucus Aspergillus spp. Aspergillus flavus Aspergillus niger CFR 224 Penicillium verrucosum MTCC 2007
Groundnuts seeds Coffee curing premises Standard strain Standard strain Standard strain Standard strain Vegetable (Chow chow) Tomato Traditional starter ‘manapu’ Traditional starter ‘manapu’ Standard strain Standard strain Wheat Maize Groundnuts Curry spice mix Food Meat curry spice mix Soil Japanese isolate Standard strain Neem (Asarichta indica) cake Maize Food Peda-traditional milk sweet Food Soil Peda-traditional milk sweet
Aflatoxin B1 &B2 Ochratoxin A T-2 toxin, DAS DON, T-2 toxin DON, T-2 toxin DON, T-2 toxin Zearalenone T-2 toxin – – – Aflatoxin B1 and G1 – – – – – – – – Aflatoxin B1 and G1 – Ochratoxin A – – – – –
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2.6. DNA isolation at different time periods To assess the relationship between the growth of fungi at different time periods, elaboration of aflatoxins and the positive PCR signal, A. flavus ATCC 46283 and A. parasiticus CFR 223 were grown in PDB and harvested at different time periods from 12 through 144 h. DNA was isolated as mentioned above. Aflatoxin in broth was estimated by Pon’s modified method [17]. 2.7. Isolation of DNA from individual and mixture of fungal isolates To determine the influence of other fungal isolates present in a mixture, DNA from mixed culture of the common foodborne fungal isolates was used. Spore suspensions of various fungi such as Penicillium verrucosum MTCC 2007, Fusarium spp., A. oryzae CFR 225, A. niger CFR 224, Rhizopus arrhizus NCIM 997 and A. ochraceus CFR 221 were prepared and 0.1 ml of spore suspension of each fungal isolate was inoculated into PDB individually into separate flasks and also into a single flask to form a fungal mixture. The flasks were incubated at 30 8C for 48 h under stationery conditions and DNA was extracted. Genomic DNA was also extracted from the fungal cultures listed in Table 1. 2.8. DNA isolation from spores Spores harvested from standard cultures, such as A. flavus ATCC 46283, A. parasiticus CFR 223 (both aflatoxin producers), and spores from food samples from a local market were used in these studies. In one set of experiments, spores from pure cultures were used directly from 101 through 106 levels. In another set of experiments, market samples were diluted (1:10), filtered and known quantity of the supernatant was subjected to centrifugation. The pellet containing spores was ground well and DNA isolated. In another set of experiments, a short enrichment technique was employed. Spores (from 101 through 106 cfu/m) from pure fungal cultures and those from maize and groundnuts were incubated for 12 h in PDB, containing maize or groundnuts extracts prior to isolation of DNA. 2.9. Isolation of fungal DNA directly from food samples Food samples including maize and groundnuts were powdered to pass through a 700-mm screen. Appropriate dilutions were made and known amounts were inoculated into PDB, incubated at 30 8C for 24 h and DNA from the fungal biomass were extracted as above. Spores of A. flavus ATCC 46283 were spiked into maize and groundnuts as positive control and DNA isolated. 2.10. Polymerase chain reaction The polymerase chain reaction was used to amplify the aflatoxin regulatory gene fragments of aflatoxigenic fungal
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genomic DNA. The sequence of the forward and reverse primers aflR1 of the aflatoxin regulatory gene was (50 AACCGCATCCACAATCTCAT-30 ) and (50 -AGTGCAGTTCGCTCAGAACA-30 ), and were designed based on the published sequence strand for A. flavus and A. parasiticus (Accession No. 264763). The primers that cover the region from 540 to 1338 of aflatoxin regulatory gene with product size of 798 base pairs (bp) have been patented [18]. The polymerase chain reaction was performed in 25 ml reaction mixtures containing 100 ng of genomic DNA, 0.025 nm each of deoxyribonucleoside triphosphates, primers at 4 nm each and reaction buffer (10 mM Tris– HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100 and 0.2 mg gelatin per ml). Each reaction mixture was heated to 95 8C for 10 min before adding 0.3 units of Taq DNA polymerase. A total of 30 PCR cycles, each cycle at 0.3 min at 94 8C for denaturation, 0.45 min at 50 8C for annealing, 1.15 min at 72 8C for extension and a 10 min final extension at 72 8C was run on a programmable DNA thermal cycler, model (Perkin-Elmer Applied Biosystems). The PCR products were analysed by electrophoresis on a 1.2% agarose gel in 1 TAE buffer (50 mM Tris–acetate, 1 mM EDTA, pH 8.0) stained in 1 mg/ml ethidium bromide. Environmental controls, along with negative and positive control were included at appropriate places while performing PCR. 2.11. Nested PCR Nested PCR was carried out using the primer set aflR2. The sequence of the primers was 50 -GCACCCTGTCTTCCCTAACA-30 and 50 -ACGACCATGCTCAGCAAGTA30 regulatory gene with product size of 400 bp, and is nested to the primer aflR1. The diluted PCR product of the primer aflR1 was used as the template to carry out PCR using the primer aflR2. PCR was performed under the abovementioned conditions.
3. Results DNA isolated from aflatoxigenic A. flavus ATCC 46283 was first used as template and PCR carried out with primers for portion of the aflatoxin biosynthetic target gene, aflR1. An amplicon corresponding to 798 bp in size was seen after agarose gel electrophoresis (Fig. 1). An amplicon of similar size was formed when the template DNA from A. parasiticus CFR 223, that also produces aflatoxin B1 and aflatoxin G1, was used in PCR (Fig. 1). The primary amplicon when used as template reacted with the aflR2 set of primers provided for the nested amplification of a 400 bp product. PCR was tested with 28 different fungal isolates (Table 1), which included 7 strains of A. flavus; 2 strains of A. ochraceus; 5 strains of A. oryzae, 1 strain each of A. parasiticus; A. glaucus; A. niger, Aspergillus spp. and Penicillium verrucosum MTCC 2007; 6 species
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12 24 48 72 96 144
Fig. 1. Agarose gel analysis of PCR products from A. flavus ATCC 46283. Lanes: (M) DNA molecular size marker; (1) A. flavus ATCC 46283; (3) A. parasiticus CFR 223.
of Fusarium and 3 species of Rhizopus. All aflatoxigenic A. flavus strains reacted positively with the aflR1 primer set (Fig. 2). A. parasiticus, an aflatoxin producer also reacted positively with the aflR1 set of primers. Multiple bands were observed with Fusarium indicating the formation of nonspecific products. No amplification was observed with DNA from strains of Rhizopus spp. or other non-aflatoxigenic Aspergilli. 3.1. Time course of aflatoxin production and PCR analysis A. flavus ATCC 46283 grown on PDB showed aflatoxin accumulation after 48 h of growth (Table 2). The production of aflatoxin increased with incubation time, peaked at 96 h of growth and declining thereafter. The different toxins B1 and B2 were found at a concentration of 260 and 42 ppb, respectively, at 96 h of growth. However, PCR with the genomic DNA isolated at different periods of growth generated amplicons from 24 h mycelia DNA, while toxin production started late at 48 h. A similar trend was recorded with PCR analysis of genomic DNA from A. parasiticus CFR 223 (Fig. 3).
Fig. 2. Agarose gel analysis of PCR products obtained from different fungal species. Lanes: (1) A. flavus NCIM 645; (2) Fusarium spp.; (3) A. flavus MTCC 152; (4) Penicillium verrucosum MTCC 2007; (5) A. ochraceus CFR 221; (6) A. parasiticus CFR 223; (7) A. flavus ATCC 46283; (8) environmental control.
Aflatoxin (ppb) B1
B2
– Nil 160 160 260 160
– Nil 20 30 42 20
3.2. Quantity of biomass and PCR reaction Genomic DNA isolated from 0.05 through 0.5 g of freshly harvested mycelia mass was subjected to PCR to assess the minimum quantity of mycelium required to obtain a positive amplification for visible electrophoretic separation of PCR product. PCR amplification was observed from 0.05 g (wet weight) mycelia indicating that this was the minimal quantity of mycelia required to get sufficient quantity of DNA for positive PCR (Fig. 4). 3.3. Isolation of DNA from spores and PCR Genomic DNA isolated from spores of 7-day-old sporulating aflatoxigenic culture (101 through 106) was used in PCR using the aflR1 primer set. Positive reaction was obtained even in DNA isolated from 102 spores (data not shown). Thus, initial enrichment steps could be avoided if the sample contained at least 100 spores of aflatoxigenic fungi. 3.4. Aflatoxigenic fungal detection in mixed flora The result of PCR using DNA isolated from mixed fungal flora and from pure cultures of aflatoxigenic strains as template with aflR1 primer set is presented in Fig. 5. Plate count of the mixed flora was also carried out to assess the growth status of individual inoculated culture and the result
Fig. 3. PCR product of A. parasiticus CFR 223 isolated at different time interval. Lanes: (1) 24 h; (2) 48 h; (3) 72 h; (4) 96 h; (5) 120 h; (M) 3000 bp DNA ladder.
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Table 3 Detection of aflatoxigenic fungi by PCR in pure culture system
Fig. 4. PCR amplification of A. parasiticus CFR 223 from different quantities of biomass. Lanes: (1) 0.05 g; (2 and 3) environmental and negative control; (4) 0.1 g; (5) 0.2 g; (6) 0.4 g; (7) 0.5 g.
Genomic DNA 48 h
Aflatoxin regulatory gene aflR1*
Total plate count (cfu/ml)
A. flavus ATCC 46283 A. parasiticus CFR 223 Mixed culture A. flavus ATCC 46283 + mixed cultures
+ + +
26 102 32 102 266 102 300 102
Mixed culture—Penicillium verrucosum MTCC 2007; Fusarium spp., A. oryzae CFR 225; A. niger CFR 225; Rhizopus arrhizus NCIM 997; A. ochraceus CFR221. * PCR amplification.
4. Discussion is presented in Table 3. The positive signal was seen only with aflatoxigenic Aspergilli. There was no formation of PCR product with DNA from mixtures of fungal biomass (where aflatoxigenic A. flavus was absent). 3.5. Aflatoxigenic fungal detection in raw food samples Raw food ingredients were spiked with spores of A. flavus ATCC 46283 and studies carried out to assess the interference of the food matrix in isolation of genomic DNA and subsequent PCR. Food samples groundnuts and maize spiked with A. flavus ATCC 46283 spores were used for DNA isolation. The counts of the native fungal flora of groundnuts and maize and that of A. flavus are presented in Table 4. PCR with primer aflR1 set showed specific amplification as seen after electrophoresis. There was no interference from the DNA of either food samples or the other microbial contaminants. From the Table it is clear that the amplification of template DNA of fungi was possible even at 30 102 to 50 102 cfu/g range of contamination in mixed flora.
DNA isolated from fungi belonging to the genus Penicillium, Rhizopus, Fusarium and other species of the genus Aspergillus either individually or in the mixture showed no positive reaction indicating the high specificity of aflR primer and thus avoiding any false positives. Shapira et al. [10] have described PCR approach for the detection of aflatoxigenic fungi, using the genes ver-1 and omtA as targets. Clear results were obtained with DNA from A. parasiticus, only weak signals were obtained with those of A. flavus with these primer pairs [10]. However, in our studies strong signals were obtained with aflatoxigenic A. flavus and A. parasiticus. This difference could be due to the different primer pairs used for amplification. The results of this study demonstrated that aflR primer based PCR method had high sensitivity and specificity in detecting aflatoxigenic Aspergilli in pure and mixed culture systems. Farber et al. [19] and Rossen et al. [14] have described the interference of food components such as fats, proteins and even high amounts of carbohydrates. They showed greater inhibition to PCR with increasing ratio between DNA from figs and fungal DNA. The problem of inhibition of PCR reaction by certain food components may be overcome by using adapted PCR or DNA preparation protocols [19]. Using very diluted DNA in first PCR reaction followed by a second reaction with nested PCR reduced inhibitory activity Table 4 Detection of A. flavus in food commodities
Fig. 5. PCR products of DNA of pure and mixed cultures. Lanes: (1) DNA from mixed fungal flora; (2) A. flavus ATCC 46283; (3) A. parasiticus CFR 221; (4) A. flavus ATCC 46283 + mixture of fungi; (5) A. parasiticus + mixture of fungi.
Commodity
Aflatoxin regulatory gene aflR1*
Count of A. flavus ATCC 46283 (cfu/g)
Fungal flora (cfu/g)
Groundnuts control Groundnuts + A. flavus ATCC 46283 Maize control Maize + A. flavus ATCC 46283
+
<102 9 102
41 102 30 102
+
<102 46 102
3 102 50 102
Note: Yeast, Rhizopus, Aspergilli, A. niger were predominant in the commodities. * PCR amplification.
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from environmental samples [20]. The PCR in the present study did not show any negative or false negative results due to the presence of food components. Quantitation of phytopathogens such as Verticillium dahliae and Verticillium albo-atrum and the detection and identification of obligate biotrophic vesicular arbuscular mycorrhizal fungi has been demonstrated by PCR [21,22]. Usually fungi are found on dry food samples as asexual spores or dried mycelia. It is a well known fact that the amount of DNA that could be extracted from these would be very small and more over fungal spores are dormant structures that are resistant to many of the DNA extraction procedures. Enrichment has been used as one of the techniques for the detection of Pseudomonas syringae pv. phaseolieola [22]. However, in our studies, DNA could be extracted from dry spores in foods and the PCR signal was positive, thus enabling the detection in shorter time period. It is clearly demonstrated that if the spore count is less than 102 then, enrichment technique has to be followed. Positive amplification was obtained with 0.05 g of 24 h-old mycelium of aflatoxigenic fungi. Similar observation has been made by Farber et al. [19]. However, Shapira et al. [10] obtained results from 48 h-old mycelia. Thus, in our studies using primer designed to aflatoxin regulatory pathway gene aflR detection of aflatoxigenic fungi is attempted. This approach presents a rapid method of detecting the aflatoxigenic fungi in selected raw food samples, compared to conventional plating techniques.
Acknowledgements The authors wish to thank Dr. V. Prakash, Director, CFTRI and Dr. M.S. Prasad, Former Head, Food Microbiology for their constant encouragement. One of the authors, S. Anand, thanks CSIR for the grant of Senior Research Fellowship.
References [1] Roehuck BD, Maxuitenko YY. Biochemical mechanisms and biological implication of the toxicity of aflatoxins as related to aflatoxin carcinogenesis. In: Eaton DL, Groopman JD, editors. The toxicology of aflatoxins: human health, veternary and agricultural significance. San Diego, CA: Academic Press Inc.; 1994. p. 27–41.
[2] Ellis WO, Smith JP, Simpson BK. Aflatoxins in food—occurrence, biosynthesis, effects in organisms, detection and methods of control. Crit Rev Food Sci Nutr 1991;30:403–39. [3] Yu J, Chang PH, Cary JW, Wright M, Bhatnagar D, Cleveland TE, et al. Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl Environ Microbiol 1995;61(6):2365–71. [4] Doster MA, Michailides TJ, Morgan DP. Aspergillus species and mycotoxins in figs from California orchards. Plant Dis 1996;80:484–9. [5] Llewellyn GC, Mooney RI, Chearle TF, Flannigan B. Mycotoxin contamination of spices, an update. Biotechnol Bioeng 1992;29:111–21. [6] Jelinek CF, Pohland AE, Wood G. Worldwide occurrence of mycotoxins in foods and feeds. J Assoc Anal Chem 1989;F2:223–30. [7] Vasanthi S, Bhat RV. Mycotoxins in foods—occurrence, health and economic significance and food control measures. Indian J Med Res 1998;108:212–24. [8] Arnheim N, Erlich H. Polymerase chain reaction strategy. Ann Rev Biochem 1992;61:131–56. [9] Miller SA, Martin RR. Molecular diagnosis of plant disease. Ann Rev Phytopathol 1988;26:409–32. [10] Shapira R, Pasti NR, Fyai O, Minastherov M, Mett A, Salomon R. Detection of aflatoxigenic moulds in grains by PCR. Appl Environ Microbiol 1996;62:3270–3. [11] Payne GE, Woloshuk CP. Transformation of Aspergillus flavus to study aflatoxin biosynthesis. Mycopathology 1989;107:139–44. [12] Payne GE, Nystrum GJ, Bhatnagar B, Cleveland TE, Woloshuk CP. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl Environ Microbiol 1993;59:156–62. [13] Trail F, Muhami N, Mehigh R, Zhou R, Link I. Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in aflatoxin pathway. Appl Environ Microbiol 1995;61:2665–73. [14] Rossen L, Nesrskov P, Holmstrom K, Rasmussen OP. Inhibition of PCR by Components of food samples, microbial diagnostics assays and DNA extraction solution. Int J Food Microbiol 1992;17:37–45. [15] Speck ML. Compendium of methods for the microbiological examination of foods. Washington, DC: American Public Health Association, 1984. [16] Lee. et al. Fungal Genet Newslett 1998;35:23–4. [17] Rati ER, Prema V, Shantha T. Modification of Pon’s method for estimating aflatoxin B1 in maize, groundnuts and groundnuts cake. J Food Sci Technol 1987;24(2):90–1. [18] Manonmani HK, Chandrashekar A, Rati ER. An improved method for detection of aflatoxigenic Aspergilli; 2002 (Patent No. NF-120/ 2002). [19] Farber P, Geisen R, Holzapfe WH. Detection of aflatoxigenic fungi in figs by a PCR reaction. Int J Food Microbiol 1997;36:215–20. [20] Krentzinger N, Podere R, Gruber F, Gobl F, Kabicek CP. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their spd (glyceroldehyde-3-phosphate dehydrogenase) genes. Appl Environ Microbiol 1996;62:3432–8. [21] Hu X, Nazar RN, Robb J. Quantification of Verticillium biomass in wilt disease. Physiol Mol Plant Pathol 1993;42:23–36. [22] Wyss P, Bonfante P. Amplification of genomic DNA of arbuscular– mycorrhizal (AM) fungi by PCR using short arbitrary primers. Mycol Res 1993;97:1351–7.
Detection of aflatoxigenic fungi in selected food commodities by PCR H.K. Manonmani a, S. Anand b, A. Chandrashekar c, E.R. Rati b,* a
Fermentation Technology Bio Engineering, Central Food Technological Research Institute, Mysore 570 020, India b Human Resource Development, Central Food Technological Research Institute, Mysore 570 020, India c Plant Cell Bio Technology, Central Food Technological Research Institute, Mysore 570 020, India Received 4 September 2003; received in revised form 27 October 2004; accepted 7 January 2005
Abstract Aflatoxins are toxic secondary metabolites produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius. A regulatory gene, aflR, is involved in regulation of aflatoxin biosynthesis and the sequence has been published. In this study, rapid assessment of aflatoxigenic fungi in food was accomplished using an indigenously designed primer pair for the aflatoxin regulatory gene aflR in polymerase chain reaction (PCR). Specificity was assayed in pure and mixed culture systems using DNA extracted from 28 different fungal strains as PCR template. Positive amplification was achieved only with DNA from aflatoxigenic A. flavus and A. parasiticus. The detection limit for mycelium and spores was determined as 0.05 g and 100 cfu, respectively. Specificity and sensitivity of PCR assay in groundnuts and maize for A. flavus was possible with as few as 100 cfu/g. # 2005 Elsevier Ltd. All rights reserved. Keywords: Aflatoxins; Aspergillus flavus; Aspergillus parasiticus; PCR; aflR; Food commodities
1. Introduction Aflatoxins are secondary metabolites produced by the aflatoxigenic fungi Aspergillus flavus Link and Aspergillus parasiticus Speare [1]. Aflatoxin contamination of agricultural commodities had gained global significance as a result of their deleterious effects on human and animal health as well as their importance to international trade. The contamination of foods by aflatoxigenic fungi, especially in tropical countries may occur during preharvesting, processing, transportation and storage [2]. These fungi mainly infect maize, cotton, peanuts, treenuts [3], figs [4] and spices [5–7]. One of the most important requirements for eliminating aflatoxin is the identification of mycotoxigenic fungal contamination. The detection of the aflatoxigenic fungi is usually performed by traditional dilution plating, use of diagnostic media or by immunological methods. The traditional methods are time consuming, labour-intensive, costly, require mycological expertise and facilities. Immunological methods and diagnostic media have limitation in identifying the aflatoxigenic fungi in that false positives are * Corresponding author. Tel.: +91 821 251430; fax: +91 821 2517233. E-mail address: [email protected] (E.R. Rati). 0032-9592/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2005.01.004
easy to come by and purifications of samples are a necessary prerequisite. Hence, it is imperative to develop methodologies that are relatively rapid, highly specific and as an alternative to the existing methods. The polymerase chain reaction (PCR) facilitates the in vitro amplification of the target sequence. The main advantages of PCR is that organisms need not be cultured, at least not for long, prior to their detection, target DNA can be detected even in a complex mixture, no radioactive probes are required, it is rapid, sensitive and highly versatile [8]. Many pathogenic organisms have been detected using PCR [9]. The biosynthetic pathway for aflatoxin production by A. flavus has been deciphered and genes in the aflatoxin biosynthetic pathway have been identified [11,3]. The gene afl-2 has been isolated and shown to regulate aflatoxin biosynthesis [12]. Few other genes of the aflatoxin biosynthetic pathway have been cloned and sequenced [13]. Reports on the detection of aflatoxigenic fungi using PCR are rather scanty [4,10,19]. In our studies, the PCR reaction was targeted against aflatoxin synthesis regulatory gene (aflR1) since these genes are nearly identical in A. flavus and A. parasiticus [3], indicating the possibility of detection of both the species with the same PCR system (primers/reaction). However, due to the complexity of the food systems, many food components
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could interfere during PCR interaction while inhibiting the activity of the polymerase [14]. In this study, we describe the use of PCR to distinguish aflatoxigenic fungi present in food samples along with other moulds and microorganisms.
2. Materials and methods
2.4. Primers and PCR chemicals PCR primers were designed using primer 3 software and were procured from Sigma, Genosys Ltd., UK. Taq polymerase and dNTPs were purchased from Bangalore Genei, Bangalore, India. Other chemicals used in these studies were of molecular biology grade and purchased from standard chemical companies.
2.1. Microorganisms 2.5. Isolation of fungal DNA The fungal isolates, their source and data on their mycotoxigenic potential are listed in Table 1. The fungal isolates were maintained on potato dextrose agar or on CzapekDox Agar media. Cultures were sub-cultured periodically and 5-day-old slant cultures were used in these studies. 2.2. Food commodities Raw food ingredients including groundnuts and maize were procured from the local market and powdered to pass through a 20 British Standard Mesh (BSM) sieve (700 mm). 2.3. Assessment of fungal load on commodities Conventional dilution plating techniques were employed to assess the total yeast and mould flora of the raw ingredients [15].
Template DNA was extracted from 0.5 g (wet weight) fungal mycelia harvested from freshly growing cultures in potato dextrose broth (PDB) under stationery conditions. The mycelium was washed twice with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4 and 1.5 mM KH2PO4) followed by centrifugation. The mycelium was transferred to a mortar, frozen in liquid nitrogen and was ground well. Lysis buffer (50 mM Tris, 150 mM EDTA, 1% (w/v) SDS pH 8.0) was added to the pulverized mycelia and incubated at 65 8C for 1 h. The suspension was centrifuged and the supernatant transferred to a new centrifuge tube. The supernatant was then extracted twice with phenol:chloroform:isoamylalcohol (25:24:1) and the aqueous layer was precipitated with 2 volumes of isopropanol. The precipitate was resuspended in 200 ml of TE buffer (10 mM Tris–HCl, 1.0 mM EDTA, pH 8.0) [16].
Table 1 List of fungal isolates Fungal isolate
Source
Mycotoxin
Aspergillus flavus ATCC 46283 Aspergillus ochraceus CFR 221 Fusarium tricinctum NRRL 32998 Fusarium fujikuori NCIM 665 Fusarium fujikuroi NCIM 1019 Fusarium saubinetti NCIM 851 Fusarium spp. Fusarium spp. Rhizopus spp. Rhizopus chinensis Rhizopus arrhizus NCIM 997 Aspergillus flavus NCIM 645 Aspergillus flavus Aspergillus flavus Aspergillus parasiticus CFR 223 Aspergillus oryzae Aspergillus oryzae Aspergillus oryzae Aspergillus oryzae CFR 225 Aspergillus oryzae Aspergillus flavus MTCC 152 Aspergillus flavus DCS Aspergillus ochraceus CFR 221 Aspergillus glaucus Aspergillus spp. Aspergillus flavus Aspergillus niger CFR 224 Penicillium verrucosum MTCC 2007
Groundnuts seeds Coffee curing premises Standard strain Standard strain Standard strain Standard strain Vegetable (Chow chow) Tomato Traditional starter ‘manapu’ Traditional starter ‘manapu’ Standard strain Standard strain Wheat Maize Groundnuts Curry spice mix Food Meat curry spice mix Soil Japanese isolate Standard strain Neem (Asarichta indica) cake Maize Food Peda-traditional milk sweet Food Soil Peda-traditional milk sweet
Aflatoxin B1 &B2 Ochratoxin A T-2 toxin, DAS DON, T-2 toxin DON, T-2 toxin DON, T-2 toxin Zearalenone T-2 toxin – – – Aflatoxin B1 and G1 – – – – – – – – Aflatoxin B1 and G1 – Ochratoxin A – – – – –
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2.6. DNA isolation at different time periods To assess the relationship between the growth of fungi at different time periods, elaboration of aflatoxins and the positive PCR signal, A. flavus ATCC 46283 and A. parasiticus CFR 223 were grown in PDB and harvested at different time periods from 12 through 144 h. DNA was isolated as mentioned above. Aflatoxin in broth was estimated by Pon’s modified method [17]. 2.7. Isolation of DNA from individual and mixture of fungal isolates To determine the influence of other fungal isolates present in a mixture, DNA from mixed culture of the common foodborne fungal isolates was used. Spore suspensions of various fungi such as Penicillium verrucosum MTCC 2007, Fusarium spp., A. oryzae CFR 225, A. niger CFR 224, Rhizopus arrhizus NCIM 997 and A. ochraceus CFR 221 were prepared and 0.1 ml of spore suspension of each fungal isolate was inoculated into PDB individually into separate flasks and also into a single flask to form a fungal mixture. The flasks were incubated at 30 8C for 48 h under stationery conditions and DNA was extracted. Genomic DNA was also extracted from the fungal cultures listed in Table 1. 2.8. DNA isolation from spores Spores harvested from standard cultures, such as A. flavus ATCC 46283, A. parasiticus CFR 223 (both aflatoxin producers), and spores from food samples from a local market were used in these studies. In one set of experiments, spores from pure cultures were used directly from 101 through 106 levels. In another set of experiments, market samples were diluted (1:10), filtered and known quantity of the supernatant was subjected to centrifugation. The pellet containing spores was ground well and DNA isolated. In another set of experiments, a short enrichment technique was employed. Spores (from 101 through 106 cfu/m) from pure fungal cultures and those from maize and groundnuts were incubated for 12 h in PDB, containing maize or groundnuts extracts prior to isolation of DNA. 2.9. Isolation of fungal DNA directly from food samples Food samples including maize and groundnuts were powdered to pass through a 700-mm screen. Appropriate dilutions were made and known amounts were inoculated into PDB, incubated at 30 8C for 24 h and DNA from the fungal biomass were extracted as above. Spores of A. flavus ATCC 46283 were spiked into maize and groundnuts as positive control and DNA isolated. 2.10. Polymerase chain reaction The polymerase chain reaction was used to amplify the aflatoxin regulatory gene fragments of aflatoxigenic fungal
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genomic DNA. The sequence of the forward and reverse primers aflR1 of the aflatoxin regulatory gene was (50 AACCGCATCCACAATCTCAT-30 ) and (50 -AGTGCAGTTCGCTCAGAACA-30 ), and were designed based on the published sequence strand for A. flavus and A. parasiticus (Accession No. 264763). The primers that cover the region from 540 to 1338 of aflatoxin regulatory gene with product size of 798 base pairs (bp) have been patented [18]. The polymerase chain reaction was performed in 25 ml reaction mixtures containing 100 ng of genomic DNA, 0.025 nm each of deoxyribonucleoside triphosphates, primers at 4 nm each and reaction buffer (10 mM Tris– HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100 and 0.2 mg gelatin per ml). Each reaction mixture was heated to 95 8C for 10 min before adding 0.3 units of Taq DNA polymerase. A total of 30 PCR cycles, each cycle at 0.3 min at 94 8C for denaturation, 0.45 min at 50 8C for annealing, 1.15 min at 72 8C for extension and a 10 min final extension at 72 8C was run on a programmable DNA thermal cycler, model (Perkin-Elmer Applied Biosystems). The PCR products were analysed by electrophoresis on a 1.2% agarose gel in 1 TAE buffer (50 mM Tris–acetate, 1 mM EDTA, pH 8.0) stained in 1 mg/ml ethidium bromide. Environmental controls, along with negative and positive control were included at appropriate places while performing PCR. 2.11. Nested PCR Nested PCR was carried out using the primer set aflR2. The sequence of the primers was 50 -GCACCCTGTCTTCCCTAACA-30 and 50 -ACGACCATGCTCAGCAAGTA30 regulatory gene with product size of 400 bp, and is nested to the primer aflR1. The diluted PCR product of the primer aflR1 was used as the template to carry out PCR using the primer aflR2. PCR was performed under the abovementioned conditions.
3. Results DNA isolated from aflatoxigenic A. flavus ATCC 46283 was first used as template and PCR carried out with primers for portion of the aflatoxin biosynthetic target gene, aflR1. An amplicon corresponding to 798 bp in size was seen after agarose gel electrophoresis (Fig. 1). An amplicon of similar size was formed when the template DNA from A. parasiticus CFR 223, that also produces aflatoxin B1 and aflatoxin G1, was used in PCR (Fig. 1). The primary amplicon when used as template reacted with the aflR2 set of primers provided for the nested amplification of a 400 bp product. PCR was tested with 28 different fungal isolates (Table 1), which included 7 strains of A. flavus; 2 strains of A. ochraceus; 5 strains of A. oryzae, 1 strain each of A. parasiticus; A. glaucus; A. niger, Aspergillus spp. and Penicillium verrucosum MTCC 2007; 6 species
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H.K. Manonmani et al. / Process Biochemistry 40 (2005) 2859–2864 Table 2 Aflatoxin produced by A. flavus ATCC 46283 in potato dextrose broth Incubation period (h)
12 24 48 72 96 144
Fig. 1. Agarose gel analysis of PCR products from A. flavus ATCC 46283. Lanes: (M) DNA molecular size marker; (1) A. flavus ATCC 46283; (3) A. parasiticus CFR 223.
of Fusarium and 3 species of Rhizopus. All aflatoxigenic A. flavus strains reacted positively with the aflR1 primer set (Fig. 2). A. parasiticus, an aflatoxin producer also reacted positively with the aflR1 set of primers. Multiple bands were observed with Fusarium indicating the formation of nonspecific products. No amplification was observed with DNA from strains of Rhizopus spp. or other non-aflatoxigenic Aspergilli. 3.1. Time course of aflatoxin production and PCR analysis A. flavus ATCC 46283 grown on PDB showed aflatoxin accumulation after 48 h of growth (Table 2). The production of aflatoxin increased with incubation time, peaked at 96 h of growth and declining thereafter. The different toxins B1 and B2 were found at a concentration of 260 and 42 ppb, respectively, at 96 h of growth. However, PCR with the genomic DNA isolated at different periods of growth generated amplicons from 24 h mycelia DNA, while toxin production started late at 48 h. A similar trend was recorded with PCR analysis of genomic DNA from A. parasiticus CFR 223 (Fig. 3).
Fig. 2. Agarose gel analysis of PCR products obtained from different fungal species. Lanes: (1) A. flavus NCIM 645; (2) Fusarium spp.; (3) A. flavus MTCC 152; (4) Penicillium verrucosum MTCC 2007; (5) A. ochraceus CFR 221; (6) A. parasiticus CFR 223; (7) A. flavus ATCC 46283; (8) environmental control.
Aflatoxin (ppb) B1
B2
– Nil 160 160 260 160
– Nil 20 30 42 20
3.2. Quantity of biomass and PCR reaction Genomic DNA isolated from 0.05 through 0.5 g of freshly harvested mycelia mass was subjected to PCR to assess the minimum quantity of mycelium required to obtain a positive amplification for visible electrophoretic separation of PCR product. PCR amplification was observed from 0.05 g (wet weight) mycelia indicating that this was the minimal quantity of mycelia required to get sufficient quantity of DNA for positive PCR (Fig. 4). 3.3. Isolation of DNA from spores and PCR Genomic DNA isolated from spores of 7-day-old sporulating aflatoxigenic culture (101 through 106) was used in PCR using the aflR1 primer set. Positive reaction was obtained even in DNA isolated from 102 spores (data not shown). Thus, initial enrichment steps could be avoided if the sample contained at least 100 spores of aflatoxigenic fungi. 3.4. Aflatoxigenic fungal detection in mixed flora The result of PCR using DNA isolated from mixed fungal flora and from pure cultures of aflatoxigenic strains as template with aflR1 primer set is presented in Fig. 5. Plate count of the mixed flora was also carried out to assess the growth status of individual inoculated culture and the result
Fig. 3. PCR product of A. parasiticus CFR 223 isolated at different time interval. Lanes: (1) 24 h; (2) 48 h; (3) 72 h; (4) 96 h; (5) 120 h; (M) 3000 bp DNA ladder.
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Table 3 Detection of aflatoxigenic fungi by PCR in pure culture system
Fig. 4. PCR amplification of A. parasiticus CFR 223 from different quantities of biomass. Lanes: (1) 0.05 g; (2 and 3) environmental and negative control; (4) 0.1 g; (5) 0.2 g; (6) 0.4 g; (7) 0.5 g.
Genomic DNA 48 h
Aflatoxin regulatory gene aflR1*
Total plate count (cfu/ml)
A. flavus ATCC 46283 A. parasiticus CFR 223 Mixed culture A. flavus ATCC 46283 + mixed cultures
+ + +
26 102 32 102 266 102 300 102
Mixed culture—Penicillium verrucosum MTCC 2007; Fusarium spp., A. oryzae CFR 225; A. niger CFR 225; Rhizopus arrhizus NCIM 997; A. ochraceus CFR221. * PCR amplification.
4. Discussion is presented in Table 3. The positive signal was seen only with aflatoxigenic Aspergilli. There was no formation of PCR product with DNA from mixtures of fungal biomass (where aflatoxigenic A. flavus was absent). 3.5. Aflatoxigenic fungal detection in raw food samples Raw food ingredients were spiked with spores of A. flavus ATCC 46283 and studies carried out to assess the interference of the food matrix in isolation of genomic DNA and subsequent PCR. Food samples groundnuts and maize spiked with A. flavus ATCC 46283 spores were used for DNA isolation. The counts of the native fungal flora of groundnuts and maize and that of A. flavus are presented in Table 4. PCR with primer aflR1 set showed specific amplification as seen after electrophoresis. There was no interference from the DNA of either food samples or the other microbial contaminants. From the Table it is clear that the amplification of template DNA of fungi was possible even at 30 102 to 50 102 cfu/g range of contamination in mixed flora.
DNA isolated from fungi belonging to the genus Penicillium, Rhizopus, Fusarium and other species of the genus Aspergillus either individually or in the mixture showed no positive reaction indicating the high specificity of aflR primer and thus avoiding any false positives. Shapira et al. [10] have described PCR approach for the detection of aflatoxigenic fungi, using the genes ver-1 and omtA as targets. Clear results were obtained with DNA from A. parasiticus, only weak signals were obtained with those of A. flavus with these primer pairs [10]. However, in our studies strong signals were obtained with aflatoxigenic A. flavus and A. parasiticus. This difference could be due to the different primer pairs used for amplification. The results of this study demonstrated that aflR primer based PCR method had high sensitivity and specificity in detecting aflatoxigenic Aspergilli in pure and mixed culture systems. Farber et al. [19] and Rossen et al. [14] have described the interference of food components such as fats, proteins and even high amounts of carbohydrates. They showed greater inhibition to PCR with increasing ratio between DNA from figs and fungal DNA. The problem of inhibition of PCR reaction by certain food components may be overcome by using adapted PCR or DNA preparation protocols [19]. Using very diluted DNA in first PCR reaction followed by a second reaction with nested PCR reduced inhibitory activity Table 4 Detection of A. flavus in food commodities
Fig. 5. PCR products of DNA of pure and mixed cultures. Lanes: (1) DNA from mixed fungal flora; (2) A. flavus ATCC 46283; (3) A. parasiticus CFR 221; (4) A. flavus ATCC 46283 + mixture of fungi; (5) A. parasiticus + mixture of fungi.
Commodity
Aflatoxin regulatory gene aflR1*
Count of A. flavus ATCC 46283 (cfu/g)
Fungal flora (cfu/g)
Groundnuts control Groundnuts + A. flavus ATCC 46283 Maize control Maize + A. flavus ATCC 46283
+
<102 9 102
41 102 30 102
+
<102 46 102
3 102 50 102
Note: Yeast, Rhizopus, Aspergilli, A. niger were predominant in the commodities. * PCR amplification.
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from environmental samples [20]. The PCR in the present study did not show any negative or false negative results due to the presence of food components. Quantitation of phytopathogens such as Verticillium dahliae and Verticillium albo-atrum and the detection and identification of obligate biotrophic vesicular arbuscular mycorrhizal fungi has been demonstrated by PCR [21,22]. Usually fungi are found on dry food samples as asexual spores or dried mycelia. It is a well known fact that the amount of DNA that could be extracted from these would be very small and more over fungal spores are dormant structures that are resistant to many of the DNA extraction procedures. Enrichment has been used as one of the techniques for the detection of Pseudomonas syringae pv. phaseolieola [22]. However, in our studies, DNA could be extracted from dry spores in foods and the PCR signal was positive, thus enabling the detection in shorter time period. It is clearly demonstrated that if the spore count is less than 102 then, enrichment technique has to be followed. Positive amplification was obtained with 0.05 g of 24 h-old mycelium of aflatoxigenic fungi. Similar observation has been made by Farber et al. [19]. However, Shapira et al. [10] obtained results from 48 h-old mycelia. Thus, in our studies using primer designed to aflatoxin regulatory pathway gene aflR detection of aflatoxigenic fungi is attempted. This approach presents a rapid method of detecting the aflatoxigenic fungi in selected raw food samples, compared to conventional plating techniques.
Acknowledgements The authors wish to thank Dr. V. Prakash, Director, CFTRI and Dr. M.S. Prasad, Former Head, Food Microbiology for their constant encouragement. One of the authors, S. Anand, thanks CSIR for the grant of Senior Research Fellowship.
References [1] Roehuck BD, Maxuitenko YY. Biochemical mechanisms and biological implication of the toxicity of aflatoxins as related to aflatoxin carcinogenesis. In: Eaton DL, Groopman JD, editors. The toxicology of aflatoxins: human health, veternary and agricultural significance. San Diego, CA: Academic Press Inc.; 1994. p. 27–41.
[2] Ellis WO, Smith JP, Simpson BK. Aflatoxins in food—occurrence, biosynthesis, effects in organisms, detection and methods of control. Crit Rev Food Sci Nutr 1991;30:403–39. [3] Yu J, Chang PH, Cary JW, Wright M, Bhatnagar D, Cleveland TE, et al. Comparative mapping of aflatoxin pathway gene clusters in Aspergillus parasiticus and Aspergillus flavus. Appl Environ Microbiol 1995;61(6):2365–71. [4] Doster MA, Michailides TJ, Morgan DP. Aspergillus species and mycotoxins in figs from California orchards. Plant Dis 1996;80:484–9. [5] Llewellyn GC, Mooney RI, Chearle TF, Flannigan B. Mycotoxin contamination of spices, an update. Biotechnol Bioeng 1992;29:111–21. [6] Jelinek CF, Pohland AE, Wood G. Worldwide occurrence of mycotoxins in foods and feeds. J Assoc Anal Chem 1989;F2:223–30. [7] Vasanthi S, Bhat RV. Mycotoxins in foods—occurrence, health and economic significance and food control measures. Indian J Med Res 1998;108:212–24. [8] Arnheim N, Erlich H. Polymerase chain reaction strategy. Ann Rev Biochem 1992;61:131–56. [9] Miller SA, Martin RR. Molecular diagnosis of plant disease. Ann Rev Phytopathol 1988;26:409–32. [10] Shapira R, Pasti NR, Fyai O, Minastherov M, Mett A, Salomon R. Detection of aflatoxigenic moulds in grains by PCR. Appl Environ Microbiol 1996;62:3270–3. [11] Payne GE, Woloshuk CP. Transformation of Aspergillus flavus to study aflatoxin biosynthesis. Mycopathology 1989;107:139–44. [12] Payne GE, Nystrum GJ, Bhatnagar B, Cleveland TE, Woloshuk CP. Cloning of the afl-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl Environ Microbiol 1993;59:156–62. [13] Trail F, Muhami N, Mehigh R, Zhou R, Link I. Physical and transcriptional map of an aflatoxin gene cluster in Aspergillus parasiticus and functional disruption of a gene involved early in aflatoxin pathway. Appl Environ Microbiol 1995;61:2665–73. [14] Rossen L, Nesrskov P, Holmstrom K, Rasmussen OP. Inhibition of PCR by Components of food samples, microbial diagnostics assays and DNA extraction solution. Int J Food Microbiol 1992;17:37–45. [15] Speck ML. Compendium of methods for the microbiological examination of foods. Washington, DC: American Public Health Association, 1984. [16] Lee. et al. Fungal Genet Newslett 1998;35:23–4. [17] Rati ER, Prema V, Shantha T. Modification of Pon’s method for estimating aflatoxin B1 in maize, groundnuts and groundnuts cake. J Food Sci Technol 1987;24(2):90–1. [18] Manonmani HK, Chandrashekar A, Rati ER. An improved method for detection of aflatoxigenic Aspergilli; 2002 (Patent No. NF-120/ 2002). [19] Farber P, Geisen R, Holzapfe WH. Detection of aflatoxigenic fungi in figs by a PCR reaction. Int J Food Microbiol 1997;36:215–20. [20] Krentzinger N, Podere R, Gruber F, Gobl F, Kabicek CP. Identification of some ectomycorrhizal basidiomycetes by PCR amplification of their spd (glyceroldehyde-3-phosphate dehydrogenase) genes. Appl Environ Microbiol 1996;62:3432–8. [21] Hu X, Nazar RN, Robb J. Quantification of Verticillium biomass in wilt disease. Physiol Mol Plant Pathol 1993;42:23–36. [22] Wyss P, Bonfante P. Amplification of genomic DNA of arbuscular– mycorrhizal (AM) fungi by PCR using short arbitrary primers. Mycol Res 1993;97:1351–7.