Determination of Aflatoxins in Feed by HPLC and PCR

: The aim of this study is to identify aflatoxin secretion isolates in animal feeds by using HPLC and PCR methods . In this study we collected fourty three samples of animal feed from different sites in Iraq (maize ,soybean ,sunflower grain ,barley grain, wheat). we isolated fungi on potato dextrose agar, Aspergillus flavus fungi was isolated from this samples and identified the enzyme activities were tested for this isolate. The detection and determination for aflatoxin secretion of the isolates were done by using High Performance Liquid Chromatography (HPLC) technique. Twelve isolates shown Aflatoxin B1 secretion . Polymrease chain reaction ( PCR) technique is an alternative method to detect for Aspergillus spp. strains that secret aflatoxin by using specific primers( ITS1) endogenous gene for Aspergillus flavus and (ord , nor) genes for aflatoxin B1 secreation, the PCR technique considered to be an important role for safety and quality in industrial food and feed .


INTRODUCTION
Aspergillus flavus and Aspergillus parasiticus fungi produced the Aflatoxins (AF) are secondary metabolites. At least 17 different forms have been identified and among them Aflatoxins G1, G2, B1 and B2 are considered the most dangerous, showing properties of genotoxicity, carcinogenicity and immune toxicity [1][2][3]. Aflatoxins are responsible for contamination of various plant-based foods including cereals, oil seeds, fruit ashell, dried fruit, baby food and feed [4]. AFs belong to category B of Annex 1 of (Directive 23/1996/EC) and, as required by [5], for these substances have set a maximum level value allowed in food for direct human consumption of (2 µg/kg for aflatoxin B1 and 4 µg/kg) for Total aflatoxins (G1,G2, B1and B2) For animal feed [6], in accordance with [7], establishes for AFB1 a maximum feedstock content of 0.02 mg/kg and variable limits in the range 0.005-0.02 mg/kg for products intended for animal feed. Among the numerous methods reported in the literature for the determination of AF in products food (enzyme immunoassays, chromatographic methods with pre-post-column chemical derivatization or online electrochemical derivatization), combined reverse phase separationto the fluorimetric detection with post-column photochemical derivatization.
It is economic and easy to use and ensures high reproducibility of analysis, without the need for control of the derivatization reaction. Furthermore, this analytical method is rapid and highly sensitive and allows to quantify separately the different Aflatoxins, requirement indispensable when it is necessary to ascertain the level of AFB1 only, as in the case of food for zoo technical use.
The validation procedure of the analytical method (HPLC) High Performance Liquid Chromatography with derivatization post-column photochemistry for determination of AFB1, AFB2, AFG1 and AFG2 in products food and feed materials [8]. The requirements of the analytical method have been verified through a validation procedure, performed in accordance with the provisions of [9], which allows to integrate the [10] with the European protocols regarding validation, as reported in [11]. The aim of this study is to identify aflatoxin secretion isolates in animal feeds by using HPLC and PCR methods and determinate the concentration of aflatoxin and study the ability of this isolates to enzyme activity [12].

Equipment
The equipment used was an Agilent Technologies SL 1200 lc Series (Germany) equipped with a fluorimetric detector (model G1321A) and UVETM online photochemical derivatization device (LC Tech GmbH, Dorfen, Germany).
The chromatographic column chosen was (150 × 4.6) mm in size with (5) μm particles. The conditions for chromatography are shown in Table 1 .

Collection of samples
The samples are collected and prepared to testing for aflatoxin and other mycotoxins. Forty three Samples of feed animal collected from different places in Iraq. The samples include (maize, soya bean, sunflower grain, barley grain, wheat) were prepared to analyzed.

Aflatoxin extraction
Aflatoxin were extracted according to [12]

RESULT AND DISCUSSION
Animal feed samples were examined for quantification of fungal screening using a potato dextrose agar media, 12 isolates were identified as Aspergillus flavus by microscope according to [18], as seen Figure 1. The isolate were examined on Aspergillus differentiation medium base agar to determination aflatoxin production by production orange zone around colony which are positive for aflatoxin production the zone were measured as mentioned in Table 2. The petridishes were examined under U.V. light and the colonies were fluoresces as aflatoxin production (Fig.2). Thirty one isolates were positive for some enzyme activity as mentioned below in Table 3 and   HPLC with fluorescence detection can be used as a quantitative method ofconfirmation for the determination of Aflatoxins, provided that the method requirements meet as indicated in [10].
This legislation identifies the criteria of precision and accuracy to be satisfied and defines, with [9], the performance parameters of the method to be evaluated.The proposed method of analysis has been validated for both feed materials that for food products. The performance criteria, assessed over 2 analytical sessions for the first and on 3 sessions for the second, at 0.5, 1 and 1.5 the maximum allowed limit were results complying with what is defined in [10].
The standard injected with a concentration 0.05 mg/g. The retention time appeared at 5.048 min with a recovery area 23600 Mv.s. The injected sample (aflatoxin) appeared at 5.14 min and the area peak was 2176 Mv.s. The concentration of sample by using B1 aflatoxin standard solution the result is 0.01 ppm (Fig. 4) [21]. The selectivity of the method, assessed by analyzing 10 samples per species (wheat, almonds,pistachios, corn and oats), allowed, in combination with the robustness tests,the application of the analysis method to the matrices indicated above.
That the relative uncertainty values% for AFB1 (10.1%) and for Total aflatoxins (13.0%), guarantee an adequate level of precision for the analysis of confirmation.
The proposed analytical method has been successfully applied for the determination simultaneous Aflatoxins, Ochratoxin A and Fusarium toxins in corn, wheat and barley.The experimental approach developed, based on a double extraction with phosphate buffer and methanol/water (70:30) and purification of the extracts by immune affinity columns multi-antibody, allows the co-extraction of all 11 mycotoxins considered with recoveries acceptable. The characteristics of the method in terms of recoveries and repeatability conform tocriteria established by [10]. The method is also sufficiently sensitive to be applied to the analysis of naturally  it has been possible to optimize in any way the PCR conditions, it is conceivable that the low amplification efficiency is due to the highly specificity of the primers, which amplify too long a sequence. Therefore it is necessary to revise the design of the ITS primer sequence.
The efficiency values obtained for NOR and ORD are always within acceptable limits. This means that all the reaction conditions have been optimised: the quantity and the quality of the extracted DNA, the concentration of the reagents, the specificity of the primers, the thermal protocol together with the fact that PCR operates with good efficiency, confirms that the assay is well designed and can be used for measurements of type quantity of DNA present in Figure 6.

CONCLUSION
PCR are: sampling efficiency, associated efficiency to DNA extraction, the number of genomic copies in the target sample, the inhibition of the PCR (primer dimmers, etc.) and the internal variables of the PCR tool (especially a low initial sample concentration). The results presented in the work show that the molecular method, developed in the biology laboratory Ministry of Science and Technology, including the DNA extraction and its amplification, applied to fungal spores, has good performance characteristics.
If you want to apply this method in the future for the characterization and quantification of the mycotoxins , it is certainly necessary to continue the experimentation on Aspergillus spp. but also on other types of conids/spores, in order to obtain a method of DNA extraction applicable to a mixed sample, including many fungal genera.