- Email: [email protected]
PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize
International Journal of Food Microbiology 93 (2004) 101 – 107 www.elsevier.com/locate/ijfoodmicro
PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize D. Somashekar, E.R. Rati, A. Chandrashekar * Food Microbiology Department, Central Food Technological Research Institute, Mysore-570 013, Karnataka, India Received 19 May 2003; received in revised form 21 October 2003; accepted 23 October 2003
Abstract Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products. D 2003 Elsevier B.V. All rights reserved. Keywords: Aspergillus flavus; Aspergillus parasiticus; Maize; PCR; RFLP; aflR
1. Introduction Aflatoxins are the toxic secondary metabolites produced predominantly by Aspergillus flavus and Aspergillus parasiticus (Bennett and Papa, 1988). Food and feeds are susceptible to invasion by aflatoxigenic Aspergillus species during pre-harvesting, processing, transportation or storage. Aflatoxin contaminated food stuffs have also been associated with a * Corresponding author. E-mail address: [email protected] (A. Chandrashekar). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.10.011
high incidence of liver cancer in humans (Berry, 1988; Stark, 1980). The level of mold infestation and the identification of governing species are still important, which could give an indication of the quality of material as well as of the future potential for the presence of mycotoxins (Shapira et al., 1996). Conventional methods for identifying and detection of these fungi in foods rely on microscopic or culture techniques, which are time consuming and laborious. The development of rapid, sensitive method for detection and differentiation of aflatoxigenic species in food are needed to estimate the potential health risk of
102
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
any food. In this direction, DNA-based detection method like PCR is more sensitive, specific and have been employed for the detection of aflatoxigenic fungi (Shapira et al., 1996; Farber et al., 1997; Sweeney et al., 2000; Chen et al., 2002). A. flavus, A. parasiticus, A. oryzae and A. sojae belonging to the Aspergillus flavi group have shown to possess high nuclear cDNA correspondence (69 – 100%) and similar genome size (Kurtzman et al., 1986). The two important fungi A. flavus and A. parasiticus responsible for food spoilage are differentiated by taxonomic identification at the morphological level which needs mycological expertise and is time consuming. The commonly employed Aspergillus differential medium does not differentiate these two species (Pitt et al., 1983) Several genes and enzymes involved in aflatoxin biosynthesis have been identified, cloned and purified; they include a regulatory gene locus aflR from A. flavus and A. parasiticus (Chang et al., 1993; Payne et al., 1993; Bennett et al., 1994). The aflR product is known to regulate the structural genes positively at the level of transcription (Woloshuk and Prieto, 1998). PCR-based methods for Aspergillus species identification have focused on amplification of conserved regions of DNA and sequencing of the gene to differentiate Aspergillus species (Chang et al., 1995; Shapira et al., 1996; Farber et al., 1997; Criseo et al., 2001).
Variation in DNA sequence can be detected by RFLP analysis. The objective of this work was to determine the level of detection of spores of A. flavus and A. parasiticus in spiked maize samples by PCR and to use the PCR amplification of selected regions of aflR gene followed by restriction site analysis of amplified DNA fragments to differentiate these two species.
2. Materials and methods 2.1. Organisms and media A detailed description of the fungal strains used in this study are given in Table 1. All strains were stored as slant cultures using potato dextrose agar medium at 4 jC. 2.2. Detection of fungal spores in maize flour To test the ability of the PCR to detect aflatoxigenic fungi in food, sterilized ground maize (1 g each) were inoculated separately with 101 – 106 spores/g. The maize was then resuspended in 5 ml of Czapekdox-casein medium which contained (g/l) NaNO3 3.0, K2HPO4 1.0, KCl 0.5, MgSO4 0.5, FeSO47H2O 0.01, Casein hydrolysate 3.5 (Hi Media, Mumbai, India) and sucrose 30.0, pH 5.0 and incubated in sterile test
Table 1 Fungal cultures used for PCR-RFLP of aflR gene fragment Name
Origin
Aflatoxin production
Number of sites and size of product (bp) restriction PvuII
HincII
A. parasiticus CFR-223 A. flavus CFS-12
Groundnut
Two 413,239,144
One 546,250
Maize
B1, B2, G1, G2 B1
One 652,144
Two 385,250,161
A. flavus CFS-16
Sorghum
B1
One 652,144
Two 385,250,161
A. A. A. A. A.
flavus CFS-29 flavus CFS-15 flavus CFS-22 flavus CFS-37 flavus ATCC-46283 A. oryzae CCRC-33705
Groundnut Red chilli Groundnut Red chilli Soil
B1 – – – B1, B2
One One One One One
Two Two Two Two Two
Koji
–
No PCR amplicon
A. sojae CCRC-30419
Koji
–
No PCR amplicon
652,144 652,144 652,144 652,144 652,144
385,250,161 385,250,161 385,250,161 385,250,161 385,250,161
Source
Culture Collection Centre, Food Microbiology Department, CFTRI Mysore, India
ATCC, USA Dr. R.Y.Y. Chiou Graduate Institute of Biotechnology Taiwan
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
tubes kept under stationary condition at 30 jC. Uninoculated sterilized maize sample served as the control. After incubation for specific periods of time, from each of the enriched suspension blends, 2-ml portions were taken and DNA was extracted for PCR. 2.3. Isolation of fungal DNA The isolation of DNA from fungal strains was performed according to Farber et al. (1997) with slight modifications. The enriched blend of fungi from maize samples at different time intervals were taken in mortar and ground with sterile glass beads. This was resuspended in lysis buffer (50 mM EDTA, 0.2% SDS, pH 8.0) and was heated to 68 jC for 30 min, and then centrifuged for 15 min at 15,000 g. After centrifugation, the supernatant was taken to a new microfuge tube and 0.3 ml of 4 M sodium acetate was added. This solution was placed on ice for 1 h and centrifuged for 15 min at 15,000 g. Double phenol – chloroform extraction was followed by isopropanol precipitation and resuspension in 30 Al of TE buffer (10 mM Tris –HCl, 1.0 mM EDTA, pH 8.0). 2.4. Polymerase chain reaction PCR was used to amplify two target fragments on A. flavus and A. parasiticus. The primer sequences are for aflatoxin regulatory gene (aflR) and they were designed using ‘Primer-3’ software program (Rozen and Skaletsky, 1996-1997). The synthesised primers were obtained from Sigma, USA. The sequences of the primers used as follows: aflR-1, 5V-AACCGCATCCACAATCTCAT-3V; aflR-2, 5V-AGTGCAGTTCGCTCAGAACA-3V to amplify a fragment of 796 bp; nested primer aflR-1a, 5V-GCACCCTGTCTTCCCTAACA-3V; aflR-2b, 5V-ACGACCATGCTCAGCAAGTA-3V to amplify a fragment of 398 bp. Amplification of fungal DNA was performed in a total reaction volume of 25 Al, which contained 1 Al DNA extracted from spiked maize samples. The reaction mixture consisted of 1 PCR buffer (10 mM Tris –HCl pH 9.0, 1.5 mM MgCl2, 50 mM KCl and 0.01% gelatin), 200 AM of each deoxynucleoside triphosphate, 4 pmol of each primer and 1.0 U of Taq DNA Polymerase (Bangalore Genei, Bangalore, India). Template DNA was initially denatured at 94 jC for 5 min. Subsequently, a total of 30 amplifica-
103
tion cycles were carried out in a programmable thermocycler (GeneAmp. PCR system 9700, PerkinElmer, USA). Each cycle consisted of denaturation for 0.3 min at 94 jC, primer annealing for 1.25 min at 50 jC and extension for 1.40 min at 72 jC. The last cycle was followed by a final extension at 72 jC for 10 min. Nested PCR was carried out by using primers aflR-1a and -2b, using the PCR product of aflR-1 and aflR-2 as template (after dilution 1:1000) under the above conditions. 2.5. Restriction site analysis of PCR products Digestions of PCR products with HincII and PvuII (MBI Fermentas, Lithuania) were performed in separate tubes in a total volume of 25.5 Al. The PCR product was diluted thrice with distilled water. Each restriction reaction contained 22.5 Al diluted PCR product (40 Ag DNA), 10 units of selected enzyme and 2 Al of 1 digestion buffer as recommended by the manufacturer. The reaction mixture was incubated at 37 jC for 4 h. Then the sample was frozen and lyophilized using Edwards lyophiliser, UK. The sample was redissolved in 10 Al T.E. and the resulting fragments were separated by electrophoresis on a 1.5% agarose gel for 1 h 45 min at 100 V. Double digestion of the PCR products from A. flavus and A. parasiticus was also carried out individually. The conditions for double digestion are essentially same except that the 20 units of HincII and 40 units of PvuII was used in 2 Y+/Tangok universal buffer. The sizes of the resulting DNA fragments were estimated in comparison with a commercial 100-bp DNA ladder (Bangalore Genei).
3. Results In order to check the PCR’s sensitivity to detect Aspergillus spores in spiked maize samples, enrichment was done using Czapek-dox medium and aliquots taken at different time intervals were subjected to DNA extraction. The primer pair used for the amplification of aflR gene was tested on aliquots sampled from the enriched cultures after 12 and 24 h of incubation at 101 – 106 spores of A. flavus and A. parasiticus. Positive results were obtained with PCR only after 12 h enrichment with A. flavus at 101 –106
104
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Fig. 1. PCR-based detection of A. flavus in spiked maize samples after enrichment. Lane M—100-bp DNA ladder standard; 1 – 4: 101, 102, 104, 106 spores/g and incubation for 12 h; 5 – 8: 101, 102, 104, 106 spores/g and incubation for 24 h; 9—control; 10—nested PCR product of aflR.
concentrations (Fig. 1). The spores of A. parasiticus were germinated to produce mycelial cells in Czapekdox medium for 12 h. The enriched samples generated a PCR product of 796 bp with aflR primers at the spore concentration of 104 and 106, but not at 101 and 102. No PCR products were observed in the uninoculated maize sample, which served as control (Fig. 2). The samples taken at 0 and 6 h did not give amplicons and samples taken at 36 h showed amplification of aflR gene in both the cultures (data not shown). There was a steady increase in the intensity of amplicons with the increasing cell number. For both A. flavus and A. parasiticus, the amplicon of 796 bp was reconfirmed by using it as a template in nested PCR. The nested PCR primers generated an expected size amplicon of 398 bp (Figs. 1 and 2). The DNA of A. oryzae and A. sojae were also subjected to PCR using aflR primers, but no amplicons were observed (data not shown).
Fig. 2. PCR-based detection of A. parasiticus in spiked maize samples after enrichment. Lane M—100-bp DNA ladder standard; 1 – 4: 101, 102, 104, 106 spores/g and incubation for 12 h; 5 – 8: 101, 102, 104, 106 spores/g and incubation for 24 h; 9—control; 10— nested PCR product of aflR.
Fig. 3. Schematic representation of restriction sites for PvuII and *HincII on aflR gene fragment (796 bp) of A. flavus and A. parasiticus (not to scale).
Variation in DNA sequence can be detected by PCR restriction fragment length polymorphism. Therefore in order to discriminate between A. flavus and A. parasiticus PCR-RFLP was carried out. A detailed comparison of the restriction maps of the PCR product of aflR gene fragment (GenBank accession number: A. flavus AY197608 and A. parasiticus AF110766) allowed the identification of at least two restriction endonucleases (HincII and PvuII) that could be used to differentiate above mentioned species (Fig. 3). According to the sequence analysis, there are two restriction sites for PvuII in the sequence of A. parasiticus that should cleave the PCR products into three fragments of 413, 239 and 144 bp. However, there is only one restriction site for this enzyme in the sequence of A. flavus that should produce two fragments of 652 and 144 bp (Table 1 and Fig. 4).
Fig. 4. Electrophoretic analysis showing the restriction profiles of the aflR gene PCR product digested with PvuII. Lane M—100-bp DNA marker; 1—uncut PCR product; 2—A. flavus ATCC 46283; A. flavus strains; 3—CFS-12; 4—CFS-15; 5—CFS-16; 6—CFS-22; 7—CFS-29; 8—CFS-37; 9—A. parasiticus CFR-223.
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Fig. 5. Electrophoretic analysis showing the restriction profiles of the aflR gene PCR product digested with HincII. Lane M—100-bp DNA marker; 1—uncut PCR product; 2—A. flavus ATCC 46283, A. flavus strains: 3—CFS-12; 4—CFS-15; 5—CFS-16; 6—CFS-22; 7—CFS-29; 8—CFS-37; 9—A. parasiticus CFR-223.
The restriction enzyme HincII was able to cut PCR product of A. flavus at two sites resulting in three fragments of 385, 250 and 161 bp. On the other hand, there was only one restriction site in the sequence of A. parasiticus yielding two fragments of 546 and 250 bp (Table 1 and Fig. 5). By doing this PCR-RFLP, one can differentiate these two most commonly occurring fungi in contaminated foods, feeds and identify them without involving long procedures of taxonomical classification. Double digestion of the PCR products from A. flavus and A. parasiticus did not show any difference in the RFLP pattern (data not shown). Most of the work in literature cited involve monomeric or multiplex PCR, which detect aflatoxigenic strains of A. flavus and A. parasiticus, but it does not always permit differentiation between them and non-aflatoxigenic strains (Criseo et al., 2001). Hence, we examined whether the PCR-RFLP could differentiate between aflatoxigenic and non-aflatoxigenic A. flavus strains. They were subjected to restriction analysis by using HincII and PvuII, but there was no difference in restriction fragment lengths between aflatoxigenic and non-aflatoxigenic cultures (Figs. 4 and 5).
4. Discussion The conventional method of detection of Aspergillus species takes about 44 – 48 h, but it does not
105
differentiate between A. flavus and A. parasiticus, which are known to produce aflatoxin. PCR for pure cultures is much easier, when compared to the detection of these organisms in food samples. It is known that food components can interfere with Taq polymerase giving false negative results (Rossen et al., 1992). Farber et al. (1997) detected aflatoxin-producing strains of A. flavus in contaminated figs by performing a monomeric PCR. Chen et al. (2002) were able to detect Aspergillus by multiplex PCR after spiking and incubating the peanuts after 7 days. Shapira et al. (1996), by using the primers omt-1 and ver-1, were able to detect Aspergillus species in corn flour after 24-h incubation in enriched media. In the abovementioned reports, liquid nitrogen has been used for extraction of DNA and the detection of Aspergillus species had taken longer time (>24 h). The method employed in this study is simple and does not involve the use of liquid nitrogen for extraction of DNA. We were able to detect the A. flavus and A. parasiticus in much shorter time at 12 h and at the level of 101 for A. flavus and 104 for A. parasiticus. This difference in level of detection may be due to the fragility of cell wall for DNA extraction. A. flavus cell wall may be more fragile compared to A. parasiticus for lysis and it is possible that the amplicons generated below 104 spore concentration in A. parasiticus could not be detected on agarose gels. PCR protocols have been developed for the pure culture systems, but detection of the same in food samples is limiting. Moreover, molds are found on dry food mostly as asexual spores or dried mycelia which contain only small amounts of DNA and are resistant to cellular disruption for DNA extraction (Shapira et al., 1996). The primers were specific for aflR gene fragment and the size of the amplicons corresponded to the expected size and no additional or non-specific bands were observed. Nested PCR was used mainly to confirm the authenticity of the primary PCR. The detection limit and incubation time to detect A. flavus and A. parasiticus in our study was less than the reported by other workers. No amplification was observed with A. oryzae and A. sojae tested, which may be due to lack of complimentary regions in the primer binding regions. Uninoculated samples of maize failed to give any amplification, indicating that the PCR product was the result of spiked samples infected with Aspergillus spores.
106
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Differentiation of A. flavus from A. parasiticus is important because of the differences in metabolite production. A. parasiticus makes ‘B’ and ‘G’ type toxins, whereas A. flavus makes only ‘B’ type toxins. Taxonomically, A. flavus has finely roughened conidia mostly produced from heads bearing both metulae and phialides, while conidia of A. parasiticus are usually conspicuously roughened and most heads bear phialides alone (Pitt and Hocking, 1985). RFLPs result specific differences in DNA sequences, which alter the fragment sizes that are generated by digestion with type II restriction endonucleases. RFLPs that correlate with particular morphological and biochemical characters may be identified and used to fingerprint individuals (Michelmore and Hulbert, 1987). In this study, based on the PCR-RFLP, the two species A. flavus and A. parasiticus can be differentiated. PCRRFLP patterns obtained with HincII and PvuII showed enough differences to distinguish these two species. Double digestion of the PCR products could not be used to distinguish the two species. This was due to the similar sized restriction digestion products produced as a result of similar restriction sites (Fig. 3). Since we could not get several strains of A. parasiticus strains from the food samples screened for aflatoxigenic fungi (data not shown), the study was restricted to the comparison of a single strain of A. parasiticus with several A. flavus strains. However, this method can be employed to differentiate other strains of A. parasiticus. The present study did not permit discrimination between aflatoxigenic and nonaflatoxigenic strains. This indicates that the mere presence of gene responsible for aflatoxin biosynthesis need not confer the status of aflatoxigenicity in Aspergillus species. It is always the environmental influences that dictate the genes whether to express or remain non-functional at any time. Hence, the presence or lack of mRNAs could allow direct differentiation between aflatoxigenic and non-aflatoxigenic strains. The PCR-RFLP, to differentiate A. flavus and A. parasiticus used in this study, is simpler, cost effective and quicker than conventional sequencing of PCR products followed by comparison of individual sequences. In spite of several methods described in literature, a convincing method of differentiating between the members belonging to the A. flavus group is still lacking. Hence there is lot of scope to develop
simpler and rapid methods to differentiate the species belonging to A. flavi group.
Acknowledgements The author DS is thankful to Dr. V. Prakash, Director, CFTRI Mysore and CSIR, New Delhi for giving him an opportunity to work as a Scientist Fellow.
References Bennett, J.W., Papa, K.E., 1988. The aflatoxigenic Aspergillus spp. Adv. Plant Pathol. 6, 263 – 280. Bennett, J.W., Bhatnagar, D., Chang, P.K., 1994. The molecular genetics of aflatoxin biosynthesis. In: Powell, K.A., Renwick, A., Peberdy, J.F. (Eds.), The Genus Aspergillus. Plenum, New York, pp. 51 – 58. Berry, C., 1988. The pathology of mycotoxins. J. Pathol. 154, 301 – 311. Chang, P.K., Cary, W.J., Bhatnagar, D., Cleveland, T.E., Bennett, J.W., Linz, J.E., Woloshuk, C.P., Payne, G.A., 1993. Cloning of the Aspergillus apa-2 gene associated with regulation of aflatoxin biosynthesis. Appl. Environ. Microbiol. 59, 3273 – 3279. Chang, P.K., Bhatnagar, D., Cleveland, T.E., Bennett, J.W., 1995. Sequence variability in homologs of the aflatoxin pathway gene aflR distinguish species in the Aspergillus section Flavi. Appl. Environ. Microbiol. 61, 40 – 43. Chen, R.S., Tsay, J.G., Huang, Y.F., Chiou, R.Y.Y., 2002. Polymerase chain reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A. parasiticus in peanut kernels by multiplex polymerase chain reaction. J. Food Prot. 65, 840 – 844. Criseo, G., Bagnara, A., Bisignano, G., 2001. Differentiation of aflatoxin producing and non-producing strains of Aspergillus flavus group. Lett. Appl. Microbiol. 33, 291 – 295. Farber, P., Geisen, R., Holzapfel, W.H., 1997. Detection of aflatoxigenic fungi in figs by a PCR reaction. Int. J. Food Microbiol. 36, 215 – 220. Kurtzman, C.P., Smiley, M.J., Robnett, C.J., Wicklow, D.T., 1986. DNA relatedness among wild and domesticated species in the Aspergillus flavus group. Mycologia 78, 955 – 959. Michelmore, R.M., Hulbert, S.H., 1987. Molecular markers for genetic analysis of phytopathogenic fungi. Annu. Rev. Phytopathol. 25, 383 – 404. Payne, G.A., Nystrom, G.J., Bhatnagar, D., Cleaveland, T.E., Woloshuk, C.P., 1993. Cloning of the aflR-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl. Environ. Microbiol. 59, 156 – 162. Pitt, J.I., Hocking, A.D., 1985. Fungi and Food spoilage. Academic Press, Australia, pp. 259 – 311. Pitt, J.I., Hocking, A.D., Glenn, D.R., 1983. An improved medium
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107 for the detection of Aspergillus flavus and Aspergillus parasiticus. J. Appl. Bacteriol. 54, 109 – 114. Rossen, L., Nosrskov, P., Holmstrom, K., Rasmussen, O.F., 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA extraction solutions. Int. J. Food Microbiol. 17, 37 – 45. Rozen, E., Skaletsky, H.J., 1996 – 1997. Primer 3, http://wwwgenome.wi.mit.edu/genome-software/other/primer3.html. Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A., Salomon, R., 1996. Detection of aflatoxigenic molds in grains by PCR. Appl. Environ. Microbiol. 62, 3270 – 3273.
107
Stark, A.A., 1980. Mutagenecity and carcinogenicity of mycotoxins: DNA binding as possible mode of action. Annu. Rev. Microbiol. 34, 235 – 262. Sweeney, M.J., Pamies, P., Dobson, A.D.W., 2000. The use of reverse transcription – polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439. Int. J. Food Microbiol. 56, 97 – 103. Woloshuk, C.P., Prieto, R., 1998. Genetic organization and function of the aflatoxin B1. FEMS Microbiol. Lett. 160, 169 – 176.
PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize D. Somashekar, E.R. Rati, A. Chandrashekar * Food Microbiology Department, Central Food Technological Research Institute, Mysore-570 013, Karnataka, India Received 19 May 2003; received in revised form 21 October 2003; accepted 23 October 2003
Abstract Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products. D 2003 Elsevier B.V. All rights reserved. Keywords: Aspergillus flavus; Aspergillus parasiticus; Maize; PCR; RFLP; aflR
1. Introduction Aflatoxins are the toxic secondary metabolites produced predominantly by Aspergillus flavus and Aspergillus parasiticus (Bennett and Papa, 1988). Food and feeds are susceptible to invasion by aflatoxigenic Aspergillus species during pre-harvesting, processing, transportation or storage. Aflatoxin contaminated food stuffs have also been associated with a * Corresponding author. E-mail address: [email protected] (A. Chandrashekar). 0168-1605/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2003.10.011
high incidence of liver cancer in humans (Berry, 1988; Stark, 1980). The level of mold infestation and the identification of governing species are still important, which could give an indication of the quality of material as well as of the future potential for the presence of mycotoxins (Shapira et al., 1996). Conventional methods for identifying and detection of these fungi in foods rely on microscopic or culture techniques, which are time consuming and laborious. The development of rapid, sensitive method for detection and differentiation of aflatoxigenic species in food are needed to estimate the potential health risk of
102
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
any food. In this direction, DNA-based detection method like PCR is more sensitive, specific and have been employed for the detection of aflatoxigenic fungi (Shapira et al., 1996; Farber et al., 1997; Sweeney et al., 2000; Chen et al., 2002). A. flavus, A. parasiticus, A. oryzae and A. sojae belonging to the Aspergillus flavi group have shown to possess high nuclear cDNA correspondence (69 – 100%) and similar genome size (Kurtzman et al., 1986). The two important fungi A. flavus and A. parasiticus responsible for food spoilage are differentiated by taxonomic identification at the morphological level which needs mycological expertise and is time consuming. The commonly employed Aspergillus differential medium does not differentiate these two species (Pitt et al., 1983) Several genes and enzymes involved in aflatoxin biosynthesis have been identified, cloned and purified; they include a regulatory gene locus aflR from A. flavus and A. parasiticus (Chang et al., 1993; Payne et al., 1993; Bennett et al., 1994). The aflR product is known to regulate the structural genes positively at the level of transcription (Woloshuk and Prieto, 1998). PCR-based methods for Aspergillus species identification have focused on amplification of conserved regions of DNA and sequencing of the gene to differentiate Aspergillus species (Chang et al., 1995; Shapira et al., 1996; Farber et al., 1997; Criseo et al., 2001).
Variation in DNA sequence can be detected by RFLP analysis. The objective of this work was to determine the level of detection of spores of A. flavus and A. parasiticus in spiked maize samples by PCR and to use the PCR amplification of selected regions of aflR gene followed by restriction site analysis of amplified DNA fragments to differentiate these two species.
2. Materials and methods 2.1. Organisms and media A detailed description of the fungal strains used in this study are given in Table 1. All strains were stored as slant cultures using potato dextrose agar medium at 4 jC. 2.2. Detection of fungal spores in maize flour To test the ability of the PCR to detect aflatoxigenic fungi in food, sterilized ground maize (1 g each) were inoculated separately with 101 – 106 spores/g. The maize was then resuspended in 5 ml of Czapekdox-casein medium which contained (g/l) NaNO3 3.0, K2HPO4 1.0, KCl 0.5, MgSO4 0.5, FeSO47H2O 0.01, Casein hydrolysate 3.5 (Hi Media, Mumbai, India) and sucrose 30.0, pH 5.0 and incubated in sterile test
Table 1 Fungal cultures used for PCR-RFLP of aflR gene fragment Name
Origin
Aflatoxin production
Number of sites and size of product (bp) restriction PvuII
HincII
A. parasiticus CFR-223 A. flavus CFS-12
Groundnut
Two 413,239,144
One 546,250
Maize
B1, B2, G1, G2 B1
One 652,144
Two 385,250,161
A. flavus CFS-16
Sorghum
B1
One 652,144
Two 385,250,161
A. A. A. A. A.
flavus CFS-29 flavus CFS-15 flavus CFS-22 flavus CFS-37 flavus ATCC-46283 A. oryzae CCRC-33705
Groundnut Red chilli Groundnut Red chilli Soil
B1 – – – B1, B2
One One One One One
Two Two Two Two Two
Koji
–
No PCR amplicon
A. sojae CCRC-30419
Koji
–
No PCR amplicon
652,144 652,144 652,144 652,144 652,144
385,250,161 385,250,161 385,250,161 385,250,161 385,250,161
Source
Culture Collection Centre, Food Microbiology Department, CFTRI Mysore, India
ATCC, USA Dr. R.Y.Y. Chiou Graduate Institute of Biotechnology Taiwan
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
tubes kept under stationary condition at 30 jC. Uninoculated sterilized maize sample served as the control. After incubation for specific periods of time, from each of the enriched suspension blends, 2-ml portions were taken and DNA was extracted for PCR. 2.3. Isolation of fungal DNA The isolation of DNA from fungal strains was performed according to Farber et al. (1997) with slight modifications. The enriched blend of fungi from maize samples at different time intervals were taken in mortar and ground with sterile glass beads. This was resuspended in lysis buffer (50 mM EDTA, 0.2% SDS, pH 8.0) and was heated to 68 jC for 30 min, and then centrifuged for 15 min at 15,000 g. After centrifugation, the supernatant was taken to a new microfuge tube and 0.3 ml of 4 M sodium acetate was added. This solution was placed on ice for 1 h and centrifuged for 15 min at 15,000 g. Double phenol – chloroform extraction was followed by isopropanol precipitation and resuspension in 30 Al of TE buffer (10 mM Tris –HCl, 1.0 mM EDTA, pH 8.0). 2.4. Polymerase chain reaction PCR was used to amplify two target fragments on A. flavus and A. parasiticus. The primer sequences are for aflatoxin regulatory gene (aflR) and they were designed using ‘Primer-3’ software program (Rozen and Skaletsky, 1996-1997). The synthesised primers were obtained from Sigma, USA. The sequences of the primers used as follows: aflR-1, 5V-AACCGCATCCACAATCTCAT-3V; aflR-2, 5V-AGTGCAGTTCGCTCAGAACA-3V to amplify a fragment of 796 bp; nested primer aflR-1a, 5V-GCACCCTGTCTTCCCTAACA-3V; aflR-2b, 5V-ACGACCATGCTCAGCAAGTA-3V to amplify a fragment of 398 bp. Amplification of fungal DNA was performed in a total reaction volume of 25 Al, which contained 1 Al DNA extracted from spiked maize samples. The reaction mixture consisted of 1 PCR buffer (10 mM Tris –HCl pH 9.0, 1.5 mM MgCl2, 50 mM KCl and 0.01% gelatin), 200 AM of each deoxynucleoside triphosphate, 4 pmol of each primer and 1.0 U of Taq DNA Polymerase (Bangalore Genei, Bangalore, India). Template DNA was initially denatured at 94 jC for 5 min. Subsequently, a total of 30 amplifica-
103
tion cycles were carried out in a programmable thermocycler (GeneAmp. PCR system 9700, PerkinElmer, USA). Each cycle consisted of denaturation for 0.3 min at 94 jC, primer annealing for 1.25 min at 50 jC and extension for 1.40 min at 72 jC. The last cycle was followed by a final extension at 72 jC for 10 min. Nested PCR was carried out by using primers aflR-1a and -2b, using the PCR product of aflR-1 and aflR-2 as template (after dilution 1:1000) under the above conditions. 2.5. Restriction site analysis of PCR products Digestions of PCR products with HincII and PvuII (MBI Fermentas, Lithuania) were performed in separate tubes in a total volume of 25.5 Al. The PCR product was diluted thrice with distilled water. Each restriction reaction contained 22.5 Al diluted PCR product (40 Ag DNA), 10 units of selected enzyme and 2 Al of 1 digestion buffer as recommended by the manufacturer. The reaction mixture was incubated at 37 jC for 4 h. Then the sample was frozen and lyophilized using Edwards lyophiliser, UK. The sample was redissolved in 10 Al T.E. and the resulting fragments were separated by electrophoresis on a 1.5% agarose gel for 1 h 45 min at 100 V. Double digestion of the PCR products from A. flavus and A. parasiticus was also carried out individually. The conditions for double digestion are essentially same except that the 20 units of HincII and 40 units of PvuII was used in 2 Y+/Tangok universal buffer. The sizes of the resulting DNA fragments were estimated in comparison with a commercial 100-bp DNA ladder (Bangalore Genei).
3. Results In order to check the PCR’s sensitivity to detect Aspergillus spores in spiked maize samples, enrichment was done using Czapek-dox medium and aliquots taken at different time intervals were subjected to DNA extraction. The primer pair used for the amplification of aflR gene was tested on aliquots sampled from the enriched cultures after 12 and 24 h of incubation at 101 – 106 spores of A. flavus and A. parasiticus. Positive results were obtained with PCR only after 12 h enrichment with A. flavus at 101 –106
104
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Fig. 1. PCR-based detection of A. flavus in spiked maize samples after enrichment. Lane M—100-bp DNA ladder standard; 1 – 4: 101, 102, 104, 106 spores/g and incubation for 12 h; 5 – 8: 101, 102, 104, 106 spores/g and incubation for 24 h; 9—control; 10—nested PCR product of aflR.
concentrations (Fig. 1). The spores of A. parasiticus were germinated to produce mycelial cells in Czapekdox medium for 12 h. The enriched samples generated a PCR product of 796 bp with aflR primers at the spore concentration of 104 and 106, but not at 101 and 102. No PCR products were observed in the uninoculated maize sample, which served as control (Fig. 2). The samples taken at 0 and 6 h did not give amplicons and samples taken at 36 h showed amplification of aflR gene in both the cultures (data not shown). There was a steady increase in the intensity of amplicons with the increasing cell number. For both A. flavus and A. parasiticus, the amplicon of 796 bp was reconfirmed by using it as a template in nested PCR. The nested PCR primers generated an expected size amplicon of 398 bp (Figs. 1 and 2). The DNA of A. oryzae and A. sojae were also subjected to PCR using aflR primers, but no amplicons were observed (data not shown).
Fig. 2. PCR-based detection of A. parasiticus in spiked maize samples after enrichment. Lane M—100-bp DNA ladder standard; 1 – 4: 101, 102, 104, 106 spores/g and incubation for 12 h; 5 – 8: 101, 102, 104, 106 spores/g and incubation for 24 h; 9—control; 10— nested PCR product of aflR.
Fig. 3. Schematic representation of restriction sites for PvuII and *HincII on aflR gene fragment (796 bp) of A. flavus and A. parasiticus (not to scale).
Variation in DNA sequence can be detected by PCR restriction fragment length polymorphism. Therefore in order to discriminate between A. flavus and A. parasiticus PCR-RFLP was carried out. A detailed comparison of the restriction maps of the PCR product of aflR gene fragment (GenBank accession number: A. flavus AY197608 and A. parasiticus AF110766) allowed the identification of at least two restriction endonucleases (HincII and PvuII) that could be used to differentiate above mentioned species (Fig. 3). According to the sequence analysis, there are two restriction sites for PvuII in the sequence of A. parasiticus that should cleave the PCR products into three fragments of 413, 239 and 144 bp. However, there is only one restriction site for this enzyme in the sequence of A. flavus that should produce two fragments of 652 and 144 bp (Table 1 and Fig. 4).
Fig. 4. Electrophoretic analysis showing the restriction profiles of the aflR gene PCR product digested with PvuII. Lane M—100-bp DNA marker; 1—uncut PCR product; 2—A. flavus ATCC 46283; A. flavus strains; 3—CFS-12; 4—CFS-15; 5—CFS-16; 6—CFS-22; 7—CFS-29; 8—CFS-37; 9—A. parasiticus CFR-223.
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Fig. 5. Electrophoretic analysis showing the restriction profiles of the aflR gene PCR product digested with HincII. Lane M—100-bp DNA marker; 1—uncut PCR product; 2—A. flavus ATCC 46283, A. flavus strains: 3—CFS-12; 4—CFS-15; 5—CFS-16; 6—CFS-22; 7—CFS-29; 8—CFS-37; 9—A. parasiticus CFR-223.
The restriction enzyme HincII was able to cut PCR product of A. flavus at two sites resulting in three fragments of 385, 250 and 161 bp. On the other hand, there was only one restriction site in the sequence of A. parasiticus yielding two fragments of 546 and 250 bp (Table 1 and Fig. 5). By doing this PCR-RFLP, one can differentiate these two most commonly occurring fungi in contaminated foods, feeds and identify them without involving long procedures of taxonomical classification. Double digestion of the PCR products from A. flavus and A. parasiticus did not show any difference in the RFLP pattern (data not shown). Most of the work in literature cited involve monomeric or multiplex PCR, which detect aflatoxigenic strains of A. flavus and A. parasiticus, but it does not always permit differentiation between them and non-aflatoxigenic strains (Criseo et al., 2001). Hence, we examined whether the PCR-RFLP could differentiate between aflatoxigenic and non-aflatoxigenic A. flavus strains. They were subjected to restriction analysis by using HincII and PvuII, but there was no difference in restriction fragment lengths between aflatoxigenic and non-aflatoxigenic cultures (Figs. 4 and 5).
4. Discussion The conventional method of detection of Aspergillus species takes about 44 – 48 h, but it does not
105
differentiate between A. flavus and A. parasiticus, which are known to produce aflatoxin. PCR for pure cultures is much easier, when compared to the detection of these organisms in food samples. It is known that food components can interfere with Taq polymerase giving false negative results (Rossen et al., 1992). Farber et al. (1997) detected aflatoxin-producing strains of A. flavus in contaminated figs by performing a monomeric PCR. Chen et al. (2002) were able to detect Aspergillus by multiplex PCR after spiking and incubating the peanuts after 7 days. Shapira et al. (1996), by using the primers omt-1 and ver-1, were able to detect Aspergillus species in corn flour after 24-h incubation in enriched media. In the abovementioned reports, liquid nitrogen has been used for extraction of DNA and the detection of Aspergillus species had taken longer time (>24 h). The method employed in this study is simple and does not involve the use of liquid nitrogen for extraction of DNA. We were able to detect the A. flavus and A. parasiticus in much shorter time at 12 h and at the level of 101 for A. flavus and 104 for A. parasiticus. This difference in level of detection may be due to the fragility of cell wall for DNA extraction. A. flavus cell wall may be more fragile compared to A. parasiticus for lysis and it is possible that the amplicons generated below 104 spore concentration in A. parasiticus could not be detected on agarose gels. PCR protocols have been developed for the pure culture systems, but detection of the same in food samples is limiting. Moreover, molds are found on dry food mostly as asexual spores or dried mycelia which contain only small amounts of DNA and are resistant to cellular disruption for DNA extraction (Shapira et al., 1996). The primers were specific for aflR gene fragment and the size of the amplicons corresponded to the expected size and no additional or non-specific bands were observed. Nested PCR was used mainly to confirm the authenticity of the primary PCR. The detection limit and incubation time to detect A. flavus and A. parasiticus in our study was less than the reported by other workers. No amplification was observed with A. oryzae and A. sojae tested, which may be due to lack of complimentary regions in the primer binding regions. Uninoculated samples of maize failed to give any amplification, indicating that the PCR product was the result of spiked samples infected with Aspergillus spores.
106
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107
Differentiation of A. flavus from A. parasiticus is important because of the differences in metabolite production. A. parasiticus makes ‘B’ and ‘G’ type toxins, whereas A. flavus makes only ‘B’ type toxins. Taxonomically, A. flavus has finely roughened conidia mostly produced from heads bearing both metulae and phialides, while conidia of A. parasiticus are usually conspicuously roughened and most heads bear phialides alone (Pitt and Hocking, 1985). RFLPs result specific differences in DNA sequences, which alter the fragment sizes that are generated by digestion with type II restriction endonucleases. RFLPs that correlate with particular morphological and biochemical characters may be identified and used to fingerprint individuals (Michelmore and Hulbert, 1987). In this study, based on the PCR-RFLP, the two species A. flavus and A. parasiticus can be differentiated. PCRRFLP patterns obtained with HincII and PvuII showed enough differences to distinguish these two species. Double digestion of the PCR products could not be used to distinguish the two species. This was due to the similar sized restriction digestion products produced as a result of similar restriction sites (Fig. 3). Since we could not get several strains of A. parasiticus strains from the food samples screened for aflatoxigenic fungi (data not shown), the study was restricted to the comparison of a single strain of A. parasiticus with several A. flavus strains. However, this method can be employed to differentiate other strains of A. parasiticus. The present study did not permit discrimination between aflatoxigenic and nonaflatoxigenic strains. This indicates that the mere presence of gene responsible for aflatoxin biosynthesis need not confer the status of aflatoxigenicity in Aspergillus species. It is always the environmental influences that dictate the genes whether to express or remain non-functional at any time. Hence, the presence or lack of mRNAs could allow direct differentiation between aflatoxigenic and non-aflatoxigenic strains. The PCR-RFLP, to differentiate A. flavus and A. parasiticus used in this study, is simpler, cost effective and quicker than conventional sequencing of PCR products followed by comparison of individual sequences. In spite of several methods described in literature, a convincing method of differentiating between the members belonging to the A. flavus group is still lacking. Hence there is lot of scope to develop
simpler and rapid methods to differentiate the species belonging to A. flavi group.
Acknowledgements The author DS is thankful to Dr. V. Prakash, Director, CFTRI Mysore and CSIR, New Delhi for giving him an opportunity to work as a Scientist Fellow.
References Bennett, J.W., Papa, K.E., 1988. The aflatoxigenic Aspergillus spp. Adv. Plant Pathol. 6, 263 – 280. Bennett, J.W., Bhatnagar, D., Chang, P.K., 1994. The molecular genetics of aflatoxin biosynthesis. In: Powell, K.A., Renwick, A., Peberdy, J.F. (Eds.), The Genus Aspergillus. Plenum, New York, pp. 51 – 58. Berry, C., 1988. The pathology of mycotoxins. J. Pathol. 154, 301 – 311. Chang, P.K., Cary, W.J., Bhatnagar, D., Cleveland, T.E., Bennett, J.W., Linz, J.E., Woloshuk, C.P., Payne, G.A., 1993. Cloning of the Aspergillus apa-2 gene associated with regulation of aflatoxin biosynthesis. Appl. Environ. Microbiol. 59, 3273 – 3279. Chang, P.K., Bhatnagar, D., Cleveland, T.E., Bennett, J.W., 1995. Sequence variability in homologs of the aflatoxin pathway gene aflR distinguish species in the Aspergillus section Flavi. Appl. Environ. Microbiol. 61, 40 – 43. Chen, R.S., Tsay, J.G., Huang, Y.F., Chiou, R.Y.Y., 2002. Polymerase chain reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A. parasiticus in peanut kernels by multiplex polymerase chain reaction. J. Food Prot. 65, 840 – 844. Criseo, G., Bagnara, A., Bisignano, G., 2001. Differentiation of aflatoxin producing and non-producing strains of Aspergillus flavus group. Lett. Appl. Microbiol. 33, 291 – 295. Farber, P., Geisen, R., Holzapfel, W.H., 1997. Detection of aflatoxigenic fungi in figs by a PCR reaction. Int. J. Food Microbiol. 36, 215 – 220. Kurtzman, C.P., Smiley, M.J., Robnett, C.J., Wicklow, D.T., 1986. DNA relatedness among wild and domesticated species in the Aspergillus flavus group. Mycologia 78, 955 – 959. Michelmore, R.M., Hulbert, S.H., 1987. Molecular markers for genetic analysis of phytopathogenic fungi. Annu. Rev. Phytopathol. 25, 383 – 404. Payne, G.A., Nystrom, G.J., Bhatnagar, D., Cleaveland, T.E., Woloshuk, C.P., 1993. Cloning of the aflR-2 gene involved in aflatoxin biosynthesis from Aspergillus flavus. Appl. Environ. Microbiol. 59, 156 – 162. Pitt, J.I., Hocking, A.D., 1985. Fungi and Food spoilage. Academic Press, Australia, pp. 259 – 311. Pitt, J.I., Hocking, A.D., Glenn, D.R., 1983. An improved medium
D. Somashekar et al. / International Journal of Food Microbiology 93 (2004) 101–107 for the detection of Aspergillus flavus and Aspergillus parasiticus. J. Appl. Bacteriol. 54, 109 – 114. Rossen, L., Nosrskov, P., Holmstrom, K., Rasmussen, O.F., 1992. Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA extraction solutions. Int. J. Food Microbiol. 17, 37 – 45. Rozen, E., Skaletsky, H.J., 1996 – 1997. Primer 3, http://wwwgenome.wi.mit.edu/genome-software/other/primer3.html. Shapira, R., Paster, N., Eyal, O., Menasherov, M., Mett, A., Salomon, R., 1996. Detection of aflatoxigenic molds in grains by PCR. Appl. Environ. Microbiol. 62, 3270 – 3273.
107
Stark, A.A., 1980. Mutagenecity and carcinogenicity of mycotoxins: DNA binding as possible mode of action. Annu. Rev. Microbiol. 34, 235 – 262. Sweeney, M.J., Pamies, P., Dobson, A.D.W., 2000. The use of reverse transcription – polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439. Int. J. Food Microbiol. 56, 97 – 103. Woloshuk, C.P., Prieto, R., 1998. Genetic organization and function of the aflatoxin B1. FEMS Microbiol. Lett. 160, 169 – 176.