INTRODUCTION

Many mycotoxins have been shown to be toxic, carcinogenic, mutagenic in nature. Contamination of the food with mycotoxins is a major problem throughout the world and removal of mycotoxins from contaminated food is difficult and costly (Marasas et al., 2001). About 25 to 50% of the crops harvested worldwide, are contaminated with aflatoxigenic fungi. In India 25-50% food and feed have been reported to be contaminated by mycotoxigenic fungi during post harvest operations and storage. The contaminated agricultural commodities will always have a heterogeneous mixture of toxigenic and non-toxigenic species of fungi. The identification and differentiation between toxigenic and non-toxigenic fungi is based on morphological characteristics and cultural characteristics in specific media. This type of identification is tedious and requires extensive training and expertise. Therefore a rapid and reliable assay for routine differential identification of toxigenic and non-toxigenic moulds would benefit the agricultural industry.

Inspite of advances in analytical methods of detection of aflatoxins, these advanced physico-chemical methods have some disadvantages. They need highly elaborate and sophisticated clean-up and/or derivatization procedures (Smith et al., 1994). Much simpler and faster immunochemical methods have the disadvantage to follow the concept of one substance one assay (Smith et al., 1994; Young and Cousin, 2001). The identification of fungi associated with agricultural commodities has been advanced by the development of PCR assay to detect specific sequences of DNA unique to the toxigenic strains. The usefulness of PCR methods to monitor quality and safety of agricultural commodities can be well exploited to differentiate the toxigenic and non-toxigenic strains. Multiplex PCR is used for simultaneous amplification of multiple targets/loci and is applied as a diagnostic tool to detect multiple gene mutations (Uggozzoli et al., 1998) and for simultaneous detection of infectious agents (Jungkind et al., 1996). A single assay combining the detection of genus-specific Aspergilli along with aflatoxigenic species would be of advantage in estimating the extent and type of contamination of any agricultural commodity.

In this communication we report a multiplex PCR assay to detect non-aflatoxigenic and aflatoxigenic species of Aspergilli simultaneously.

MATERIALS AND METHODS

Taq polymerase, 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. The primers were designed specifically for aflatoxin regulatory gene (aflR), o-methyl transferase gene (omt) of aflatoxin biosynthetic pathway (Yu et al., 1993) and 18s small nuclear ribosomal region (NS) (White et al., 1990), using primer 3 software. The primers were designed based on the published sequence strand for A. flavus and A. parasiticus from the NCBI databank by the authors and has been patented (Manonmani et al., 2002). With the assumption that NS is unique to the genera Aspergilli and omt and aflR unique for aflatoxigenic fungi, in present studies, all these primers were used in PCR reaction.

Standard toxin was purchased from Sigma- Aldrich Chemical Company, USA.

Fungal Strains
The fungal species used in this study are shown in Table 1. The isolates were maintained on potato-dextrose agar or on Czapek-Dox Agar media. The cultures were sub cultured periodically and 5 day old slant cultures were used in these studies.

Table 1:	Fungal isolates used for multiplex PCR

Screening of Fungal Strains for Aflatoxins
All the fungal strains belonging to A. flavus and A. parasiticus were screened for the production of aflatoxins using YES medium. YES medium (yeast extract 20 g, sucrose 150 g, distilled water 1 L, pH 4.5) was inoculated with spore suspension of standard/test samples. The flasks were then incubated at room temperature for seven days under dark and stationary conditions. Aflatoxin in the broth medium was extracted and estimated by Pon’s modified method (Rati et al., 1987). Aflatoxin extract was purified on silica gel column, derivatized with trifluoroacetic acid- Aflatoxin B₁, G₁, B₂, G₂, separated by reverse-phase liquid chromatography and detected by fluorescence using a fluorichrom fluorescence detector (varian) with 360 nm excitation and 440 nm emission. The LC column used for separation was Supelcosil LC-18, 15x4.6 mm id, mobile phase was water: acetonitrile: methanol:: 70:17:17. This method can measure 0.1 ng of aflatoxins B₁, B2, G₁ and G₂. The test samples were quantified for aflatoxin by comparative calculation with the standard peak area values (Scott, 1995).

Isolation of Fungal DNA
Template DNA was extracted from fungal mycelia according to Lee et al. (1998) (individual fungal isolates or their mixtures or from enrichment of food samples) as follows: fungal mycelia grown in Potato Dextrose Broth (PDB) under stationery conditions for 21 days was harvested by filtration. The mycelium was washed twice with phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) followed by centrifugation. The mycelium was transferred to a mortar and ground well. Freshly prepared, sterile 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°C for 1 h. The suspension was centrifuged and supernatant was then extracted twice with phenol: chloroform: isoamylalcohol (25:24:1) and the aqueous layer was washed twice with chloroform and then precipitated with two volumes of isopropanol. The precipitate was resuspended in 200 μL of TE buffer (10 mM Tris-Cl, 1.0 mM EDTA, pH 8.0) (Lee et al., 1998).

Multiplex PCR
All the primers were synthesized by Sigma genosys, UK. The PCR conditions were optimized by varying the concentration of these three primer sets, the number of units of Taq polymerase and annealing temperature of the reaction. The PCR reaction mixture (25 μL) contained 100 ng of genomic DNA, deoxyribonucleoside triphosphates at 0.025 nmol each, primers at 4 nmol each and reaction buffer. Each reaction mixture was heated to 95°C for 10 min before adding 0.3 units of Taq DNA polymerase. Amplification conditions used consisted of 4 min at 94°C followed by 35 cycles at 94°C for 30 sec, 50°C for 45 sec, 72°C for 75 sec. The reaction was completed with incubation for 10 min at 72°C. PCR products were analysed by electrophoresis in a 1% agarose gel in TAE buffer. Ethidium bromide (0.5 μg μL^-1) stained gels were visualized under UV light and documented in a gel-doc system with CCD camera attached to it (Hero-Lab), (Sambrook and Russell, 2001).

RESULTS AND DISCUSSION

PCR Primers
In this study we designed primers from omt gene, which is involved in the conversion of sterigmatocystin to o-methylsterigmatocystin of aflatoxin biosynthetic pathway, alfR gene, which is involved in the regulation of aflatoxin biosynthesis, for use in PCR assay. The NS primer set was from 18s rRNA region uniquely homologous to Aspergillus species (White et al., 1990). The omt and aflR primer sets were obtained from conserved regions reported for omt and aflR genes (Yu et al., 1993). On the basis of the design methodology, a single assay should result in the detection of Aspergillus genus as well as aflatoxin producing species. The main purpose was to combine the three primer sets into a single PCR reaction.

Table 2:	Fungal species analysed by multiplex PCR
+: Positive PCR amplicon, -: Negative for PCR amplification

A key advantage of combining these primers in a single reaction is that the presence or absence of genus specific band at 555 bp corresponding to NS region serves as an internal control for genus specific identification and omt and aflR amplified band serves in the specific detection of aflatoxigenic species. This serves in the detection of the extent of contamination and contaminant type of any food and feed sample. In the earlier work the PCR amplification product of alfR gene primer was confirmed by restriction digestion. PCR amplicons of A. flavus and A. parasiticus were subjected to restriction endonuclease (Hinc II and PvuII) analysis to differentiate the two species with specific RFLP’s (Somashekar et al., 2004).

Amplification of Aflatoxigenic Aspergillus spp.
To test the specificity of the designed primer sets in a PCR reaction, other members of Aspergilli, viz., A. oryzae, A. niger, A. ochraceus and other genera of fungi such as Fusarium spp., Rhizopus spp. and Penicillium spp. were evaluated (Table 2). The NS primer pairs were highly specific for the genus Aspergilli (Fig. 2, lanes 1, 2 and 4). All the aflatoxigenic Aspergilli gave positive amplification with both omt and aflR primer pairs (Fig. 1, lanes 4, 5, 6, 7 and 8). Non-aflatoxigenic Aspergilli screened did not show amplification with omt and aflR primer pairs indicating the absence of aflatoxin producing machinery. However, the Aspergillus genus specific NS primer pair gave positive amplification and other fungal cultures did not show amplification with these primer pairs. This indicated the specificity of NS primer pairs with the genus Aspergillus and omt and aflR primer pairs with aflatoxigenic Aspergilli.

Production of Aflatoxin
All the fungal isolates screened by multiplex PCR were screened for toxin production in YES medium (Table 3). Aflatoxin B₁ was found to be produced by A. flavus while A. parasiticus produced all the four toxins, B₁, B₂, G₁ and G₂. Some of the A. flavus isolates did not produce any of the toxins on YES medium. The HPLC analysis showed a clear differentiation between aflatoxin producing and non-aflatoxin producing strains of A. flavus (data not shown).

The multiplex PCR developed using three sets of primers for omt, alfR and NS showed positive correlation for aflatoxin production where a complete pattern with three bands was obtained on agarose gel (Fig. 1). For non-aflatoxigenic producing Aspergilli, only one band corresponding to Aspergillus specific 18s rRNA region was obtained (Fig. 2). The multiplex PCR could be used as a marker to clearly differentiate between the aflatoxin-producing and non-aflatoxigenic Aspergilli. In a similar kind of work (Criseo et al., 2001) studied the differentiation between aflatoxin producing and non-producing A. flavus group. They studied quadruplex-PCR using aflR, nor-1, ver-1 and omt4 gene primers.

Fig. 1:	PCR amplification of omt and afl R genes from different fungal species. Lane No. (1) Marker 1000 bp, (2) Rhizopus, (3) Environmental control, (4) A. flavus ATCC 46283, (5) A. flavus NCIM 645, (6) A. flavus MTCC 152, (7) A. parasiticus and (8) A. flavus (Millet isolate)

Fig. 2:	PCR amplification of 18s rRNA region of different fungal species. Lanes (1) A. ochraceus CFR 221, (2) A. flavus (Millet), (3) Fusarium (Tomato isolate), (4) A. oryzae CFR 225, (5) Fusarium NCIM 665, (6) Fusarium NCIM 851, (7) Fusairum spp. and (8) Environmental Control

Table 3:	Aflatoxin production by fungi

They obtained a four band pattern for all producers and a variable pattern for non-producers of aflatoxin. They argue that the lack of aflatoxin production apparently need not only be related to an incomplete pattern obtained in quadrauplex PCR. Different types of mutations may be responsible in inactivating the aflatoxin biosynthetic pathway genes in other A. flavus strains (Geisen, 1996). Yet, in another study (Liu and Chu, 1998), they studied quadruplex PCR using avfA, omtA, ver-1 and ITS primers. Their multiplex PCR assay gave positive results (tetrad banding pattern) for fermented foods. The ITS Universal primers anneal to the flanking Internal Transcribed Spacer (ITS) regions of the fungus 5.8s rDNA and amplify an approximately 600-bp amplicon and they are not specific for Aspergilli. Here only the aflatoxin biosynthetic pathway genes were used for the detection of aflatoxigenic Aspergilli, but ITS primer specific for the identification of genus Aspergillus was not used in their study.

In the present study, aflR and omt gene primers were used because aflR gene regulates the expression of omt gene, a structural gene enclosed in the aflatoxin biosynthetic pathway and omt gene is necessary for almost the final formalities of aflatoxin biosynthesis (Liu and Chu, 1998). The presence of these two genes clearly indicates the aflatoxin production machinery. With the use of NS primer pairs, NS indicate the presence of Aspergillus contamination in any commodity. The detection of aflatoxigenic strains with three bands corresponding to aflR, omt and NS genes confirm the identity of the invading genus.

Conventional methods using Aspergillus Differential Medium (ADM) however, could differentiate aflatoxin producers and non-producers. But this method is time consuming. Sometimes it may fail to identify the aflatoxin production because of instability of aflatoxin-producing strains growing on culture media (Abarca et al., 1988).

RT-PCR could be another biomolecular technique as a tool to differentiate aflatoxin-producing strains from non-aflatoxin producing strains of A. flavus, A. parasiticus and other Aspergilli, where in the presence or the absence of mRNA could help in direct differentiation between these strains. An RT-PCR assay using tri-5 gene provided a screening tool for trichothecene producing Fusarium species (Doohan et al., 1999).

PCR/RT-PCR are time-consuming and involve multiple steps thereby increasing the chances of contamination with exogenous DNA. The simultaneous identification of these agents in one step PCR procedure would offer a number of advantages over the conventional methods and would save both time and cost without compromising on the efficiency. Thus, the identification of a single toxigenic species is precise, simple, rapid and cost-effective detection assay which can be applied in food industry as an integral part of quality control programme for the assurance of raw products and clinical diagnosis. Thus, the multiplex PCR will be an even more powerful diagnostic tool for detecting aflatoxigenic Aspergilli.

ACKNOWLEDGMENTS

The authors are thankful to Dr. V. Prakash, Director, CFTRI for supporting the work. We greatly acknowledge DBT (Department of Biotechnology), Government of India, New Delhi for funding this project.