Ann Microbiol (2014) 64:1597–1605 DOI 10.1007/s13213-014-0803-5

ORIGINAL ARTICLE

Detection of flavus and Aspergillus parasiticus from aflatoxin-contaminated and their differentiation using PCR-RFLP

Malik Mobeen Ahmad & Mahboob Ahmad & Athar Ali & Rifat Hamid & Saleem Javed & Malik Zainul Abdin

Received: 16 September 2013 /Accepted: 7 January 2014 /Published online: 4 February 2014 # Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract The possibility of using PCR to specify the detec- Introduction tion of aflatoxigenic fungi in food and feeds was investigated. The method, based on amplification of the aflP gene encoding Fungi are ingenious producers of secondary extrolites that the biosynthesis of aflatoxins, was optimized for the detection show a broad range of biological activities (Bohnert et al. of aflatoxin-producing molds (Aspergillus flavus MTCC 277, 2010). -contaminated food and feed poses a high A. flavus JH 11 and A. parasiticus MTCC 2796). The speci- risk to both human as well as animal health (Bennett and Klich ficity of the optimized PCR method was proved with the 2003). Most agriculture raw materials are generally found to amplification of genomes from aflatoxin-producing be colonized by a broad range of mycotoxigenic molds, Aspergillus strains. Amplification of DNA from serially dilut- including Aspergillus flavus, Aspergillus parasiticus, ed revealed the detection of A. flavus, A. flavus JH 11 Penicillium, Fusarium,andAspergillus niger (Aldred et al. and A. parasiticus with as low as 104 fungal spores mL−1, 2004). Globally, about 25–50 % of harvested crops are con- whereas -contaminated peanuts showed a threshold limit taminated with aflatoxigenic fungi. In tropical countries like of 108 spores g−1 at 0 h. Determination of the spore limit from India, where molds thrives best, 25–50 % of food and feeds samples was followed by differentiation of aflatoxin- are damaged during post harvest and storage operations producing A. flavus strains from A. parasiticus by the restric- (Abdin et al. 2010). Many have been shown to tion digestion of the partially amplified aflP gene product. be highly toxic, carcinogenic as well as mutagenic in nature This method allows not only conclusive detection of (Latha et al. 2008). Consumption of infected foodstuffs has aflatoxin-producing species, but also simultaneous species been shown to cause liver cancer in humans (European differentiation in contaminated agricultural and commercial Commission 2006;Giornietal.2007). products. Aflatoxins (AFs) are secreted by number of Aspergillus species, particularly A. flavus, A. parasiticus, A. nomius, A. pseudotamarii, A. parvisclerotigenus,andA. bombycis of Keywords Aspergillus . Aflatoxin contamination . aflP . section Flavi. A. ochraceoroseus and A. rambellii from section Polymerase chain reaction . PCR-RFLP Ochraceorosei and Emericella astellata, Emericella venezuelensis from section Nidulatans also produce extrolites (Abdin et al. 2010). The naturally occurring aflatoxins desig- M. M. Ahmad : A. Ali : M. Z. Abdin nated as , B2, G1, and G2. B1 have been identi- Centre for Transgenic Plant Development, Department of fied as the most potent genotoxic and hepatocarcinogenic Biotechnology, Jamia Hamdard, New Delhi 110062, India among the mycotoxins (Ellinger-Ziegelbauer et al. 2004). : : AFs belong to the Group I type of most carcinogenic myco- M. Ahmad R. Hamid S. Javed – Molecular Biology and Biotechnology Laboratory, Department of toxins, while OT and F have been placed in Group 2B (WHO Biochemistry, Jamia Hamdard, New Delhi 110062, India IARC 1993a; b). Contamination with AFs has been reported mostly in peanuts, cotton seed, corn, pea, sorghum, rice, * M. Z. Abdin ( ) , maize, oilseed rape, spices, meat and meat products, Department of Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi 110062, India fig, fruit juices, etc. (Abdin et al. 2010). AF contamination in e-mail: [email protected] food/feeds is a major problem throughout the world and, 1598 Ann Microbiol (2014) 64:1597–1605 therefore, its removal is a challenging and costly matter Materials and methods (Marasas et al. 2001). Scientific advancements and a particular interest in Fungal isolates and culture conditions this field have combined to build up and develop pre- ventive strategies to reduce exposure to these toxins Fungal strains used in the experimental procedures, along with (Aldred et al. 2004). Molecular techniques decrease the their procurement site, are listed in Table 1. Aflatoxigenic and time required analysis and, due to their sensitivity and non-aflatoxigenic fungal strains were stored as slant cultures specificity, provide precise estimations of species with- on potato dextrose agar (PDA) medium at 4 °C. To obtain out the need for any complex cultivation and additional fresh cultures of fungi, all the fungal isolates were streaked on confirmation steps (Ahmad et al. 2010). As a result, the PDA plates under aspetic conditions and transferred to incu- detection of specific microorganisms can be done in bators already set at 28±2 °C for proper growth. After 3– hours instead of the days required with traditional sys- 4 days of incubation, the plates were stored at 4 °C. tems (Rompre et al. 2002). Through a simple PCR- based method, Shapira et al. (1996) detected Screening of fungal cultures versicolorin A dehydrogenase gene (ver-1) and sterigmatocystin-o-methyltransfersase 1 gene (omt-1) Extraction of aflatoxins from fungal agents followed the from A. flavus and A. parasiticus only, and achieved a method of Ahmad et al. (2013). Filtered M1 medium sensitivity of 102 spores g−1 from A. parasiticus. (5 mL) (De Jesus et al. 1988) was placed in a freshly Similarly, Geisen (1996) and Farber et al. (1997) autoclaved tube. An equal amount of chloroform was targeted the norsolorinic acid reductase gene (nor-1) added to it. After vortexing vigorously, the mixture was together with ver-1 and omt-1 genes, respectively. On left for 30 min at ambient temperature for separation of the other hand, Manonmani et al. (2005)usedanaflR two (organic and aqueous) phases. The lower phase gene specific primer to evaluate the presence of pure- as (organic part) was transferred to a fresh tube. The same well as mixed-cultures of fungi in foodstuffs. Specific procedure was repeated 2–3 times. The organic phase detection of aflatoxigenic molds was carried out in was then transferred to a Petri dish and left overnight at wheat flour using primers aimed at internal transcribed room temperature to evaporate. After evaporation, region (ITS1-5.8S-ITS2) (González-Salgado et al. 2008; 300 μL methanol was added and finally the sample Sardinãs et al. 2010). was collected in an Eppendorf tube and kept at 4 °C In recent years, restriction fragment length polymor- for further use. phism of PCR products (PCR-RFLP) has been used The samples and standard (different concentrations; multi- widely for distinguishing mycotoxigenic species after level) dissolved in methanol were spotted onto the absorbent detection. El Khoury et al. (2011) digested the PCR layer using a sample applicator (Linomat5, CAMAG, products of the aflR-aflJ intergenic region from both Muttenz, Switzerland) at a distance of 8 mm from the bottom pure and aflatoxin-contaminated grapes, while edge of the width band and spaced at a distance of 6 mm Somashekar et al. (2004) restricted the aflRamplicons between spots. Chromatographic development was carried out with the enzyme PvuII to differentiate A. flavus from in the dark in an unsaturated TLC chamber (CAMAG) using A. parasiticus. PCR-RFLP has shown the difference toluene:isoamyl alcohol:methanol (in the ratio 90:32:30) as between A. niger, A. tubingensis, A. carbonarius,and the mobile phase. The developed plate was dried and finally A. aculeatus isolates (Medina et al. 2005; Bau et al. scanned under UV illumination at single wavelength of 2006; Zanzotto et al. 2006; Martinez-Culebras and 366 nm using a scanner (Linomat 5, CAMAG) attached to a Ramon 2007). Similar results have shown distinction CAMAG system for HPTLC. Analysis was carried out using at species level using species-specific primers. Perrone the winCATS software provided along with the CAMAG et al. (2004)andSuscaetal.(2007) differentiated HPTLC system. A. carbonarius from A. niger by amplifying the calmod- ulin region, while, targeting ITS regions, Haugland and Artificial spiking of peanut samples Vesper (2002) detected A. carbonarius and A. niger and

Gonzalez-Salgado et al. (2005) were successful in Sterilized (0.1 % aqueous solutions of HgCl2 for 2 min) species-specific identification of a number of black peanut seed samples were artificially contaminated with aspergilli. aflatoxin-producing fungi by plating 20 grains (6 per The aim of this study was to determine the specificity and plate). The spore suspension was prepared by mixing threshold limit of A. flavus and A. parasiticus in pure cultures spores at various concentrations (102,104,106,108 per and contaminated peanuts followed by restriction analysis of mL) in autoclaved MilliQ water with 0.05 % Tween 20. amplified products of the aflPgene. Spore suspension (1 mL) was sprayed on separate plates, Ann Microbiol (2014) 64:1597–1605 1599

Table 1 Aflatoxigenic and non-aflatoxigenic fungal strains analyzed in this study, indicating species, source and the occurrence of a PCR amplification product

Genus Species Strain/isolate Isolation source Aflatoxin production PCR signal

Aspergillus parasiticus MTCC 2796a Arachis hypogea rhizosphere, + h + flavus MTCC 277 NId ++ flavus JH 11b Jamia Hamdard, New Delhi + + flavus JH 03 Jamia Hamdard, New Delhi -h - flavus JH 02 Jamia Hamdard, New Delhi - - flavus JH 01 Jamia Hamdard, New Delhi - - flavus ITCC 5192c Arachis hypogea,Hyderabad - - flavus ITCC 6284 Arachis hypogea rhizosphere, Junagarh, Gujarat - - versicolor MTCC 280 Phoenix dactylifera, California, USA - - awamori ITCC 1418 Zea mays rhizosphere, New Delhi - - carbonarius ITCC 1415 Zea mays rhizosphere, IARI, New Delhi - - clavatus ITCC 454 NRRL 8, USAe -- ochraceus ITCC 1740 Phaseolus vulgaris seeds, New Delhi - - ochraceus ITCC 3470 Glycine max seeds, New Delhi - - oryzae ITCC 4712 NRRL 458, USA - - tamarii ITCC 6285 Arachis hypogea rhizosphere, Junagarh, Gujarat - - niger ITCC 545 γ-ray induced mutant, ATCC 76557, USAf -- nidulans ITCC 145 CBS, Netherlandsg -- Emericella sp. JH 05 Jamia Hamdard, New Delhi - - Fusarium oxysporum f. pisi MTCC 2480 Arachis hypogea,India - - equiseti JH 04 Jamia Hamdard, New Delhi - - tenuissima JH 08 Jamia Hamdard, New Delhi - - brassicae JH 07 Jamia Hamdard, New Delhi - - brassicae JH 15 Jamia Hamdard, New Delhi - - Trichoderma viride MTCC 3114 Soil, India - - a Microbial Type Culture Collection, Chandigarh, Punjab b Jamia Hamdard, New Delhi c Indian Type Culture Collection, IARI, New Delhi d No Information e Northern Regional Research Laboratory, USA f American Type Culture Collection, USA g Centraalbureau voor Schimmelcultures, Netherlands h +Aflatoxin production and PCR product observed, - aflatoxin production and PCR was not observed respectively, containing uniformly wounded seeds. The DNA isolation plates were then shaken to allow uniform distribution of inoculum over the seeds (Babu et al. 2005). Inoculated Powdered mycelia (0.5 g) was mixed with lysis buffer (CTAB samples were incubated at 28±2 °C for different time intervals, 2 %) and 1 % polyvinyl pyrrolidone (PVP). The mixture was viz., 0, 12, 24, 36 and 48 h. then transferred to a centrifuge tube containing 50 μL β- mercaptoethanol, and incubated at 65 °C for 1 h with intermit- Aflatoxin estimation of artificially spiked peanut samples tent vortexing. After incubation, the mixture was cooled to room temperature. An equal volume of phenol:chloroform:isoamyl Aflatoxin was extracted from each -contaminated pea- alcohol (PCIA; 25:24:1 v/v) was added to each tube and the nut sample as follows: 10 g unground peanut sample was tubes were vortexed gently for 15 min. The tubes were then crushed by mixing the samples with 100 mL sterile MilliQ centrifuged (Beckman, Avanti™ 30) at 18,000 rpm for 20 min water in a beaker. The slurry was then used for aflatoxin at 4 °C. The supernatant was transferred to an autoclaved tube extraction using the method described above. and two-thirds (v/v) chilled iso-propanol was added to 1600 Ann Microbiol (2014) 64:1597–1605 precipitate the DNA. The mixture was mixed gently and kept at Primer design −20 °C for 4–5 h followed by centrifugation at 15,000 rpm to recover the DNA pellet. Finally, the pellet was washed with For primer design, the AF biosynthetic gene aflP was used as a absolute ethanol, air-dried and dissolved in 100 μL TE (pH 8.0). target for amplification and restriction analysis of the ampli- Spore DNA was isolated by dissolving aflatoxigenic fungal fied products. A primer pair aflP-F, 5′-CATGCTCCATCATG spores at different concentrations (102,104,106,108 per mL) with GTGACT-3′; aflP-R, 5′-CCGCCGCTTTGATCTAGG-3′ was 0.05 % Tween 20. Spore DNA was collected using the same designed from the consensus sequences of four Aspergillus protocol, replacing the mycelia with variable spore concentrations. isolates retrieved from NCBI GenBank (A. flavus isolate Extraction of DNA from each fungus-contaminated peanut AF13, Acc. AY510451; A. flavus isolate AF36, Acc. sample, however, was performed as follows: 10 g unground AY510455; A. flavus isolate AF70, Acc. AY510453; peanut sample was washed by mixing the samples with 10 mL A. parasiticus, Acc. AY371490) (Fig. 1). The primers were sterile distilled water containing 0.05 % Tween 20 in sterile synthesized by Sigma-Aldrich (Bangalore, India). 50-mL tubes. After draining the washing fluid into a fresh tube, the sample was spun at 6,000 g for 5 min. The superna- Polymerase chain reaction tant was discarded and the remaining pellet was washed three times with 5 mL sterile ultra pure water with intermediate PCR was performed in duplicate with Taq recombinant polymer- spinning under the conditions previously described. After that, ase (Fermentas, Mumbai, India). Amplification was carried out genomic DNA was isolated from peanuts coated with in a 25-μL reaction mixture containing: 10× Taq DNA polymer- aflatoxin-contaminated spores using the CTAB method as ase buffer, 100 nM each primer, 0.5 μM dNTPs and 100 ng described above. template DNA with 1 unit Taq DNA polymerase. The reaction

Fig. 1 Alignment of three Aspergillus flavus isolates [AF13 (GenBank accession number AY510451), AF36 (AY510455), AF70 (AY510453)] and one Aspergillus parasiticus (GenBank accession number AY371490) exonic region of the aflPgene. Red Amplified region; mutations are shown in white. Horizontal arrows Primer positions. Restriction site for BanIand NlaIV endonucleases enzyme are indicated by open and filled downward pointing arrows, respectively Ann Microbiol (2014) 64:1597–1605 1601 was performed in a thermal cycler (GStorm, Ramsey, MN) with Rf value. A. parasiticus MTCC 2796 proved to be a strong apreheatingstepat94°Cfor1min;35cyclesof94°Cfor1min, producer of AFB1 when compared with A. flavus MTCC 277 62 °C for 1 min, 72 °C for 1 min with final incubation at 72 °C and A. flavus JH 11. Other strains of the same species/genus or for 3 min. A negative control contained all the PCR reaction of different genera showed no AFB1 signals (data not shown). mixture components except the template DNA. Each PCR- Similar results were observed in aflatoxin production when amplified sample (5 μL)wasexaminedona1.2%w/vagarose peanut samples were contaminated with variable dilutions of (HiMedia, Mumbai, India) gel in separate lanes. spores of three positive aflatoxigenic molds and incubated at different time intervals to observe the presence of AFB1 Restriction site analysis of PCR products content secreted in the samples. A. parasiticus MTCC 2796 showed maximum production followed by A. flavusJH 11 and The PCR products were subjected to restriction enzyme digestion A. flavus MTCC 277 (Table 2). using BanIandNlaIV (NEB, Hitchin, UK). The reactions were performedinatotalvolumeof30μL containing 2 U restriction PCR specificity with aflP-F/aflP-R enzyme, 2 μL10×buffer,10μL PCR product, and ultrapure water up to 18 μL. The reaction mixture was incubated at 37 °C The specificity of primers aflP-F/aflP-R was studied by con- for2hfollowedbyenzymedeactivationat65°Cfor20min.The ventional PCR in different Aspergillus species as well as in resulting amplified DNA fragments were finally detected by closely related genera that frequently arise in the same food electrophoresis on a 2 % w/v agarose (HiMedia) gel. items. A single fragment of ~236 bp was amplified from genomic DNA of A. flavus MTCC 277, A. flavus JH 11 and A. parasiticus MTCC 2796 only. No product was observed Results with genomic DNA from strains of the same species/genus or from different genera (Fig. 2). HPTLC analysis PCR detection with spore samples To determine the kinetics of aflatoxin production by the studied fungal isolates, HPTLC was performed and results After determining primer specificity, amplification from dilut- were compared with standard aflatoxin B1. Only three fungal ed spore samples at variable concentrations was evaluated. agents tested positive and showed a positive peak at the same Detection of target DNA was achieved with a minimum spore

Table 2 Estimation of aflatoxin content and PCR assay in peanuts with variable spore counts

Aspergillus sp. Time interval (h) Aflatoxin content (μgkg−1) PCR signala

Spore count Spore count

102 104 106 108 102 104 106 108

A. flavus MTCC 277 0 NDb ND 0.12 1.49 −−−+ 12 2.023.674.976.10++++ 24 4.336.528.2111.54++++ 36 5.147.0711.8313.28++++ 48 6.788.2212.8914.61++++ A. parasiticus MTCC 2796 0 ND ND 0.86 1.97 −−−+ 12 2.833.315.116.30++++ 24 4.936.718.9812.34++++ 36 5.367.6112.0313.80++++ 48 7.118.5713.4915.21++++ A. flavus JH 11 0 ND ND 0.26 1.62 −−−+ 12 2.313.915.176.10++++ 24 4.436.728.5911.81++++ 36 5.347.3611.8313.51++++ 48 7.038.2813.1514.97++++ a − Band not observed in PCR, + band observed in PCR b Not Determined 1602 Ann Microbiol (2014) 64:1597–1605

M 1 2 3 4 5 6 7 8 9 10 11 12 13 C comparison of restriction maps of the PCR product of the aflP gene fragment (A. flavus isolate AF13 Acc. AY510451; A. flavus isolate AF36 Acc. AY510455; A. flavus isolate AF70 Acc. AY510453; A. parasiticus Acc. AY371490) allowed the identification of two type II restriction endonu- (a) cleases (BanIandNlaIV) that could be used to differentiate the M 14 15 16 17 18 19 20 21 22 23 24 25 C two species (Table 3;Fig.1). Both restriction enzymes BanI and NlaIV were able to cut PCR product of A. flavus at single site producing two band fragments of 206 and 31; and 208 and 31 bp, respectively. On the other hand, there was no restriction (b) site found in the sequence of A. parasiticus,resultingina single band of undigested PCR product (Fig. 5). Fig. 2 a,b Specificity of PCR primers for aflatoxigenic and non- aflatoxigenic molds. a Lanes: M Molecular marker (100 bp), 1 A. parasiticus MTCC 2796, 2 A. flavus MTCC 277, 3 A. flavus JH 11, 4 A. flavus JH 03, 5 A. flavus JH 02, 6 A. flavus JH 01, 7A.flavusITCC Discussion 5192, 8 A. flavus ITCC 6284, 9 A. versicolor MTCC 280, 10 A. awamori ITCC 1418, 11 A. carbonarius ITCC 1415, 12 A. clavatus ITCC 454, 13 A. ochraceus ITCC 1740, C negative control, b Lanes: M Molecular We have developed a precise PCR protocol for detecting marker (100 bp), 14 A. ochraceus ITCC 3470, 15 A. oryzae ITCC 4712, aflatoxigenic molds and discriminating A. flavus from the 16 A. tamari ITCC 6285, 17 A. niger ITCC 545, 18 A. nidulans ITCC closely related species A. parasiticus in food/feed matrices 145, 19 Emericella sp. JH 05, 20 Fusarium oxysporum f. pisi MTCC that are contaminated frequently with aflatoxins. This pro- 2480, 21 Fusarium equiseti JH 04, 22 Alternaria tenuissima JH 08, 23 Alternaria brassicae JH 07, 24 Alternaria brassicae JH 15, 25 vides significant knowledge with which to devise strategies Trichoderma viride MTCC 3114, C negative control to minimize fungal infection and the risk of aflatoxin poison- ing. Exonic regions of the aflP gene of aflatoxin-producing A. flavus and A. parasiticus isolates obtained from the concentration of 104 spore mL−1 without any incubation in all GenBank database were aligned and a consensus sequence three aflatoxin-producing fungal species (Fig. 3). of 361 bp was identified (Fig. 1). Selection from the coding regions gives more consensus sequences because exons are Detection of fungal spores in contaminated peanut samples more conserved than introns (Li et al. 2010). The specificity of the designed primer pairs was analyzed by conventional PCR To test the efficacy of the method under practical conditions, on aflatoxigenic as well as non-aflatoxin producing surface sterilized peanut samples were artificially inoculated Aspergillus species and other common fungal phytopatho- with variable dilutions of spores (102–108). Amplification of gens. Contrary to the amplification bands obtained with aflP target was obtained with the highest spore concentration tested primers from aflatoxigenic species (A. flavus, A. parasiticus), (108 spores) at 0 h of incubation. However, upon increasing no amplification signal was obtained with these primers in the the incubation time, detection at low spore concentration was case of other mycotoxin producers. This shows the specificity further improved (12 and 24 h with a spore concentration of of these primers for amplifying the aflPgenefragmentonly; 102). No amplification products were obtained when DNA the amplicon size corresponds to the expected product with no was extracted from un-inoculated peanut samples (control) subsidiary bands. The aflP-F/aflP-R primers appeared to be (Fig. 4). more efficient in amplifying exclusively aflatoxin-producing fungi than the primer pairs FLA1/FLA2 and PAR1/PAR2 Restriction site analysis of amplified products from ITS regions (Gonzalez-Salgado et al. 2008; Sardinãs et al. 2010) and primers from aflR (Somashekar et al. 2004). In order to differentiate A. flavus from A. parasiticus,restric- Previous studies have detected specifically single aspergillus tion digestion of the target product was carried out. A detailed fungus rather than aflatoxigenic mold. We, however, were

2 4 6 8 2 4 6 8 2 4 6 8 Fig. 3 a–c Electrophoretic M 10 10 10 10 C M 10 10 10 10 C M 10 10 10 10 C analysis of aflPamplified products from serially diluted spores. a A. parasiticus MTCC 2796, b A. flavus MTCC 277, c A. flavus JH 11. Lanes: M Molecular marker (100 bp), C negative control (a) (b) (c) Ann Microbiol (2014) 64:1597–1605 1603

Fig. 4 a–c PCR amplification of aflP gene of serially diluted spores. a A. parasiticus MTCC 2796, b A. flavus MTCC 277, c A. flavus JH 11. Lanes: M Molecular marker (100 bp), C negative control

able to detect aflatoxin-producing molds first followed by spiked samples and pure cultures might be due to food com- species-specific differentiation using RFLP. ponents, which could interfere with Taq polymerase giving Specific detection from uncontaminated cultures is much false negative results (Rossen et al. 1992). Moreover, the easier compared to the analysis of products containing several accuracy of the detection method to quantify fungus in artifi- mycobiota together with other compounds of food or feed cially contaminated peanuts was established by detecting the items that may affect the efficiency and sensitivity of the assay target after longer incubation periods. The possibility to quan- (Rossen et al. 1992; Färber et al. 1997). Additionally, in food tify contamination levels in food matrices is essential because samples enrichment techniques are required for several days previous studies have demonstrated that levels of (Shapira et al. 1996;Chenetal.2002; Gonzalez Salgado et al. mycotoxigenic fungi can be related with mycotoxin concen- 2008), as molds are present as asexual spores or dried mycelia trations that exceed legal limits (Lund and Frisvad 2003). that contain very little DNA. In our case, we could detect the The protocol developed here can be used for accurate presence of aflatoxigenic molds at the highest spore concen- discrimination of the most relevant aflatoxigenic species tration (104 spores mL−1), even without incubation, while (Wilson et al. 2002). A. flavus and A. parasiticus discrimina- fungus-infected peanut samples were detectable at 108 spores tion is imperative because they produce different secondary at 0 h incubation. No incubation time was needed to detect extrolites. A. flavus strains are known to produce aflatoxins, molds in contaminated samples. The aflatoxin content evalu- cyclopiazonic acid, versicolorin and sterigmatocystin, while ated through the HPTLC method and the positive amplifica- A. parasiticus specifically produces aflatoxins (Wilson et al. tion of the aflP gene also showed that detection at 108 spores is 2002). Additionally, A. flavus is a more ubiquitous species in within the maximum limit (2 μgkg−1) of aflatoxin B1 in food contamination than A. parasiticus (Bankole et al. 2004; peanuts allowed by the European Commission (EU No. 165/ Melki Ben Fredj et al. 2007). Most of the work in the literature 2010; http://eur-lex.europa.eu/LexUriServ/LexUriServ.do? cited involves monomeric or multiplex PCR, which detect uri=CELEX:32010R0165:EN:NOT). This suggests the aflatoxigenic strains of A. flavus and A. parasiticus, but does applicability of our method for commercial use. not always permit differentiation between them and between PCR amplification from pure fungal cultures is much sim- non-aflatoxigenic strains (Criseo et al. 2001). RFLPs result pler compared to detection in food samples. The variation in from a particular variation of DNA sequences, by which the

Table 3 Restriction band patterns of four aflatoxin-producing Aspergillus isolates selected from NCBI GenBank M AF FB FN JH HB HN AP PB PN C

Fungi name Accession no. Restricted fragments (bp)

BanI A. flavus AF13 AY510451 31, 206 A. flavus AF36 AY510455 31, 206 A. flavus AF70 AY510453 31, 206 Fig. 5 PCR-RFLP analysis of the aflP gene product of aflatoxin-produc- ing molds. Lanes: M Molecular marker (100 bp), AF A. flavus MTCC A. parasiticus AY371490 0, 236 277, FB restriction digestion of A. flavus MTCC 277 PCR product with NlaIV BanI, FN restriction digestion of A. flavus MTCC 277 PCR product with A. flavus AF13 AY510451 31, 208 NlaIV, JH A. flavus JH 11, HB restriction digestion of A. flavus JH 11 PCR product with BanI, HN restriction digestion of A. flavus JH 11 PCR A. flavus AF36 AY510455 31, 208 product with NlaIV, AP A. parasiticus MTCC 2796, PB restriction A. flavus AF70 AY510453 31, 208 digestion of A. parasiticus MTCC 2796 PCR product with BanI, PN A. parasiticus AY371490 0, 236 restriction digestion of A. parasiticus MTCC 2796 PCR product with NlaIV, C negative control 1604 Ann Microbiol (2014) 64:1597–1605 generated fragments become altered after digestion with type Chen RS, Tsay JG, Huang YF, Chiou RYY (2002) Polymerase chain II restriction endonucleases. For this reason, bands with sizes reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A. parasiticus in peanut of 206 and 31, and 208 and 31 bp were observed when the kernels by multiplex polymerase chain reaction. J Food Prot 65: amplified products from A. flavus and A. parasiticus are 840–844 digested with BanIandNlaIV, respectively. Consequently, this Criseo G, Bagnara A, Bisignano G (2001) Differentiation of aflatoxin method can be employed for the detection of aflatoxigenic producing and non-producing strains of Aspergillus flavus group. Lett Appl Microbiol 33:291–295 molds and for differentiating them at species level. De Jesus AE, Gorst-Allman CP, Horak RM, Vleggaar R (1988) Large- In the food industry, it is essential to develop specific and scale purification of the mycotoxins aflatoxin B1, B2 and G1. 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