Developing a PCR assay to detect Aspergillus parasiticus

Aspergillus parasiticus is one of the producers of aflatoxins in food. They may produce aflatoxin B and G which can lead to serious diseases in human. Aflatoxin is known as a type of carcinogens. People ingesting infected food even in a very low level for a long time can get cancer because of aflatoxin accumulation. Detection of aflatoxin or aflatoxin producers in foods is necessary to improve the life quality by decreasing the risk of getting diseases. Recently, the contemporary method to detect A. parasiticus is a morphological method but it still retains many limitations. In this study, a PCR based method was developed to provide a basic for develop a new method to detect A. parasiticus in food that overcame the disadvantages of the conventional morphological method. A specific set of primers was designed based on norB-cypA genes and successfully optimized for best amplification results at 62  ̊C. The sensitivity of the test was identified to be 0.005 ng/μL of target fungi DNA. A DNA isolation protocol was also optimized to ensure the success of the PCR assay, using SDS lysis, sand and thermal shock. The protocol from DNA isolation to PCR was successfully developed and provided a useful tool to improve the diagnosis of aflatoxins at an early stage and control all stages of food production. The success of this study is designing a pair of primers and a PCR assay which is specific for detection of A. parasiticus among other aspergilus species. This PCR assay can be used in the future for further development a PCR method for detection of A. parasiticus in food.


INTRODUCTION
Aspergillus species are among the most ubiquitously found mold fungi throughout the world which are of high importance in medicine, agriculture and biotechnology. Besides inducing direct pathogenesis, they also produce various types of toxic secondary metabolites, mycotoxins and cause non-contagious mycotoxicoses [1]. Out of all different types of mycotoxins, aflatoxins are the most potent natural carcinogens known, possessing hepatoxic and immunosuppressive properties which can cause acute liver damage, liver cirrhosis, tumor induction and teratogenesis [2]. They have been recognized as a possible human carcinogen by International Agency of Research on Cancer [3]. These mycotoxins are produced primarily by Aspergillus parasiticus and A. flavus which may invade agricultural products during plant growth, during harvest and finally in storage, resulting in significant economic losses [4].
A. parasiticus, unlike A. flavus which only produces aflatoxin B, may produce both aflatoxins B and G. Discrimination between these two species and distinguishing them from closely related species is difficult when using conventional methods, which are mainly based on morphological or immunological features including culturing the fungus in suitable inducing media, extracting aflatoxins with other solvents, and monitoring their presence by chromatographic and ELISA techniques [5,6]. These methods are time-consuming and require considerable performing expertise, which are their major drawbacks. Furthermore, the complexity in the presence of aflatoxinogenic and non-aflatoxinogenic Aspergillus species can cause false negative results in detection [7,8].
The application of DNA-based techniques, particularly polymerase chain reaction (PCR), permits rapid, sensitive and specific detection that overcomes the disadvantages of conventional method, necessary to devise strategies to control and reduce fungal mass and toxin production at early and critical stages of the food chain. Up to date, there have been few studies on detection of A. parasiticus using molecular methods. RT-PCR with target genes aflD, aflO, aflP [9], PCR-RFLP with target gene aflR, multiplex-PCR with target genes nor-1, ver-1, omt-1 and apa-2 [10], realtime PCR with primers and probe designed on multicopy ITS2 rDNA target sequence have been used [11]. However, these methods were established to detect both A. parasiticus and A. flavus with the aim of monitoring aflatoxinproducing Aspergillus contamination of food and feedstuffs. A recent study on detection of only A. parasiticus using real-time PCR assay was performed by Sardinas N. in 2010 [11,12] with primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). This study was successful in accurately detecting and quantifying A. parasiticus at spore concentrations equal or higher than 10 6 spores/g. Nevertheless, this method needs to be performed in expensively equipped laboratories, which may increase the detection cost when applied in practice.
In this study, a cheap, accurate, sensitive and specific PCR assay is developed to detect A. parasiticus. The specificity of the assay was considerably improved when norB-cypA genes were used. These genes, which belong to the aflatoxin gene cluster, were shown to involve in biosynthesis of aflatoxins G [13][14][15]. Furthermore, they are highly variable among closely related species in Aspergillus genus, allowing successful detection of A. parasiticus. The possibility of this method will provide further information to develop a new method to predict mycotoxins profiles as well as to detect aflatoxin-producing species in food quality assurance labs in the future.

Fungal isolates and culture conditions
Two different genera of filamentous fungi were used in this study (Table 1), including five Aspergillus species and one Penicillium species. Fungal strains were obtained from different Culture Collections in Vietnam or isolated from food. Morphological and genetic variations of A. parasiticus among intraspecies and extraspecies would be the important points to identify this species. Among these, both A. parasiticus and A. flavus belong to an important group of foodborne fungi which can produce aflatoxins. A. oryzae and A. niger are two fermented fungi which are "generally regarded as safe" (GRAS) by the FDA [16]; while A. candidus acts as a human pathogen causing invasive infection [17]. Finally, P. aethiopicum is an Aspergillus-related species. [18].
The isolates were maintained by regular subculturing on slant tubes containing Potato Dextrose Agar (PDA) (Merck, Germany) at 25 ˚C in the dark for 48 to 72 hours and then stored as spore suspension in sterile paraffin oil at 4˚C. Fungal strains were cultured for DNA extraction in Erlenmeyer flasks containing 50 mL of Malt Extract (ME) broth (Himedia, India) and incubated at 25 ˚C in an orbital shaker (120 rpm) for 48 to 72 hours. Mycelial mass was filtered through a filter paper, washed 3 times with NaCl 0.8 M and kept at -20 ˚C until DNA extraction.

DNA extraction
DNA was extracted from mycelium following SDS method which was modified from Plaza's method [19]. The yield of the method was independently evaluated in all species shown in table 2. DNA concentrations were determined using a NanoDrop® 2000c spectrophotometer (Nanodrop Technologies, Wilmington, USA). The purity of the extractions was between 1.8 and 2.0. The DNA of the samples was diluted to 100 ng/μL.

PCR amplification
The assays were performed in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, PCR assay was performed using the thermal cycle of 5 minutes at 95 °C, then 35 cycles of 30 seconds at 95 °C, 30 second at annealing temperature, 30 seconds at 72 °C and finally 3 min at 72 °C. The annealing temperature was checked in the range of 58 -66 o C to get the best annealing temperature for further analysis. PCR product was then analyzed in 2 % agarose ethidium bromide gel at 150 V in TBE 0.5 X buffer for 30 minutes and observed under an ECX-20. M transilluminator (Vilber Lourmat, Germany).

Primer design
Primers were designed on the basis of sequence alignments of the norB-cypA genes, the farthest upstream portion of the aflatoxin biosynthesis gene cluster. This design, evaluated by Ehrlich [20,21] Figure 1). To avoid extra-products at low temperature and lost of product at high temperature, 62 o C is selected at the best temperature for further experiments. Hence, the optimal temperature for the designed primer set was 62 o C.

Specificity testing
The specificity of the primer pairs APA1/APA2 for A. parasiticus was tested in PCR assays by using genomic DNA extracted from closely related species and genera that usually contaminate the same foods. The samples were prepared independently with DNA from individual strain at the concentration of 100 ng/µL. An additional sample containing DNA mixture of these molds with the same ratio in a concentration of 100 ng/µL was also used to test the capacity of the assay in detection of

A. parasiticus contaminated with other species
The results shown in Figure 3 illustrated that the test could be used to detect the presence of A. parasiticusonly in the tested samples (lane 2, 3, 4), while samples containing DNA solution of other species gave negative results (lane 5, 6,7,8,9,10,11,12,13). The specificity of the test was also demonstrated when A. parasiticus was detected in the mixture of the DNA solution of all tested species (lane 1, Figure 3). This indicated that the test was specific for detecting A. Parasiticus in the presence of other species.
The test was performed using the samples containing both DNA of Aspergillus parasiticus (VTCC-F-1132) only ( Figure 4) and DNA mixture of all selected species (Table 1) at similar amount ratio ( Figure 5). Figure 4 and Figure 5 showed a decrease in the intensity of bands from lane 1 to lane 11, corresponding to 100 ng to 0.001 ng in each microliter of PCR reaction, respectively. However, bands expressed in Figure 5 appeared significantly smeared when great amount of DNA was used (lane 1, 2, 3 and 4). This phenomenon might be caused by high productivity of amplified products. The bands in lane 11 of both Figure 4 and Figure 5 were considerably faint and difficult to be observed by naked eyes under UV luminescence. Thus, the previous band (lane 10), corresponding to 0.005 ng of DNA in each microliter, was selected as the limit of detection (LOD), i.e. LOD = 0.005 ng/µL.

DISCUSSION
In this work, a highly specific method was developed to allow the detection of Aspergillus parasiticus, which would permit the prediction of whether aflatoxins type Gis present besides aflatoxins type B.
The sensitivity of PCR assay is dependent on the concentration of the target DNA input. The lowest detection limit of target DNA was estimated to be 0.005 ng/µL in comparison with 0.002 ng/µL in a similar study using real-time PCR method done by Sardinas [11]. However, with real-time PCR, the laboratories need to be equipped with expensive machine and costly chemicals. The validity of this assay was also confirmed when a mixture of target DNA and those of other related species with similar ratio were employed. A similar result to the one of pure target DNA was obtained.
The specificity of the assay was examined among a relatively diverse selection of strains and confirmed using a sample containing a DNA mixture of these strains. The use of the target sequence based on norB-cypA genes, which have shown to be an important factor in biosynthesis of aflatoxin type G in aflatoxin gene cluster, might enhance the specificity in the detection of A. parasiticus in comparison with other gene regions. Therefore, regarding its sensitivity, the PCR method would provide a better tool with high accuracy, rapidity and inexpensiveness for A. parasiticus as well as aflatoxins detection in comparison with the morphological methods.
However, to use this PCR assay for detection A.paraciticus in food it is necessary to perform more experiments to test the sensitivity of the test. Adding more controls such as internal control is necessary to avoid the fault results such as fault negative.

CONCLUSION
In this work, a PCR method using novel primers was successfully designed to detect the target genes norB-cypA, which is a part of the aflatoxin gene cluster. This PCR assay worked well at the annealing temperature of 62 ˚C, providing a specific, accurate and sensitive tool to detect Aspergillus parasiticus among others Aspergillus species. The limit of detection in this PCR assay was 0.005 ng/µL of pure genomic DNA. This assay and the designed primer pair can be used for continue developing a PCR method which can apply for detection of A.paraciticus in food in the future.