Duck-Hwa Chung

Development and validation of a gold nanoparticle immunochromatographic assay (ICG) for the detection of zearalenone.

A monoclonal antibody (mAb)-based gold nanoparticle immunochromatographic assay (ICG) for zearalenone detection was developed, optimized, and validated. The detection limits of ICG optimized with appropriate amounts of zearalenone-bovine serum albumin and gold nanoparticle-mAb to zearalenone were 2.5 ng/mL and 30 μg/kg for the standard solution and spike sample, respectively, and a weak cross-reaction for α-zearalenol and β-zearalenol was observed. The assay required only 15 min to obtain results and one step to perform the assay. In validation, the results obtained from spiked corn (10, 20, 30, 50, and 100 μg/kg) and naturally contaminated corn samples by the ICG were in good agreement with those obtained by direct competitive enzyme-linked immunosorbent assay (DC-ELISA) and high-performance liquid chromatography (HPLC). Therefore, the results obtained in this study could be used as basic research for the development of zearalenone-ICG, and the ICG developed could be a useful on-site screening tool for the rapid detection of zearalenone in corn without special instrumentation.

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 ± 0.02 µg l−1 and an IC50 value of 1.13 ± 0.16 µg l−1. Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0–909.8 µg kg−1. Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant® zearalenone kit (r 2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.

Natural Occurrence of Aflatoxin B1 in Marketed Foods and Risk Estimates of Dietary Exposure in Koreans

Aflatoxin B1 (AFB1) is an unavoidable food contaminant. To evaluate the potential health risk of AFB1 to Koreans posed by food consumption, we determined the natural occurrence of AFB1 in food and estimated the excess risk for liver cancer through dietary exposure to AFB1. A total of 694 food samples collected from six different regions of South Korea were analyzed for their AFB, content. One hundred four of the 694 samples were found to give positive enzyme-linked immunosorbent assay (ELISA) readings for AFB1 and were further investigated with high-performance liquid chromatography. Thirty-two samples, including 2 maize samples, 3 soybean products, 20 peanut samples, nut samples, and their products, and 7 spices, were found to be contaminated with AFB1 (4.6% incidence), up to 48.6 microg kg(-1). The level of AFB1 contamination in 28 of the 32 food products was below 10 microg kg(-1), which is the legal tolerance limit in Korea. From data on daily food consumption, the exposure dose of AFB1 was estimated to be 6.42 x 10(-7) mg kg(-1) body weight (bw) day(-1). The major contributors to the dietary intake of AFB1 were soybean paste and soy sauce, which composed 91% of the total exposure to AFB1. The excess risk of liver cancer for those exposed to AFB1 through food intake was estimated to be 5.78 x 10(-6) for hepatitis B-negative individuals and 1.48 x 10(-4) for hepatitis B-positive individuals. These results suggest that special consideration is required to reduce the intake of AFB1 in hepatitis B-positive individuals.

Enhanced Rapidity for Qualitative Detection of Listeria monocytogenes Using an Enzyme-Linked Immunosorbent Assay and Immunochromatography Strip Test Combined with Immunomagnetic Bead Separation

An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.

Detection of aflatoxin-producing molds in Korean fermented foods and grains by multiplex PCR.

An assay based on multiplex PCR was applied for the detection of potential aflatoxin-producing molds in Korean fermented foods and grains. Three genes, avfA, omtA, and ver-1, coding for key enzymes in aflatoxin biosynthesis, were used as aflatoxin-detecting target genes in multiplex PCR. DNA extracted from Aspergillus flavus, Aspergillus parasiticus, Aspergillus oryzae, Aspergillus niger, Aspergillus terreus, Penicillium expansum, and Fusarium verticillioides was used as PCR template to test specificity of the multiplex PCR assay. Positive results were achieved only with DNA that was extracted from the aflatoxigenic molds A. flavus and A. parasiticus in all three primer pairs. This result was supported by aflatoxin detection with direct competitive enzyme-linked immunosorbent assay (DC-ELISA). The PCR assay required just a few hours, enabling rapid and simultaneous detection of many samples at a low cost. A total of 22 Meju samples, 24 Doenjang samples, and 10 barley samples commercially obtained in Korea were analyzed. The DC-ELISA assay for aflatoxin detection gave negative results for all samples, whereas the PCR-based method gave positive results for 1 of 22 Meju samples and 2 of 10 barley samples. After incubation of the positive samples with malt extract agar, DC-ELISA also gave positive results for aflatoxin detection. All Doenjang samples were negative by multiplex PCR and DC-ELISA assay, suggesting that aflatoxin contamination and the presence of aflatoxin-producing molds in Doenjang are probably low.

Monitoring of Pesticide Residues and Risk Assessment for Fruit Vegetables and Root Vegetables of Environment-friendly Certified and General Agricultural Products

BACKGROUND: This study was conducted to monitor the residue of pesticides and to assess their risk in domestic agricultural products, such as fruit vegetables, tomatoes, oriental melons and root vegetables, garlic, potatoes and onions. METHODS AND RESULTS: 250 samples containing both general and environment-friendly certified agricultural products were collected from traditional markets and supermarkets in 6 cities. 132 pesticides except for herbicides were analysed using the multi-residue methods by GC/ECD, GC/NPD and HPLC/UVD. 17 kinds of pesticides were detected from 42 samples, which were 32 general, 1 organic, 4 pesticide-free and 5 low pesticide agricultural products. Among those, myclobutanil detected in 1 potato and procymidone detected in 10 oriental melons were unregistered pesticides for using in Korea. Fenbuconazole detected in 1 potato and phorate detected in 1 tomato were exceeded over the MRLs established by Korea Food and Drug Administration. CONCLUSION: Based on these results, a risk assesment was conducted using a percentage of acceptable daily intake(%ADI). %ADI ranged from 0.0064% to 4.6035%, and showed these values have no effect on human health.

Immuno‐ultrafiltration as a new strategy in sample clean‐up of aflatoxins

The present paper describes the development of a new clean-up strategy for the analysis of aflatoxins (AFs) in food. The sample preparation method is based on immuno-ultrafiltration (IUF) which, in contrast to immunoaffinity chromatography, makes use of antibodies in free form. After selecting an appropriate ultrafiltration (UF) device and optimizing different operation conditions the IUF method was applied to the clean-up of maize and rice. Quantification of AFs was carried out by HPLC and fluorescence detection, after postcolumn derivatization in a Kobracell. The IUF method was shown to be as selective as sample clean-up using commercial immunoaffinity columns. Recovery rates and RSD for the AFs G(2), G(1), B(2) and B(1) in spiked rice were found to be 76 +/- 3, 76 +/- 2, 83 +/- 5 and 99 +/- 14%, respectively. The analysis of a FAPAS (food analysis performance assessment scheme) maize material resulted in AFs concentrations which were in the range assigned by the producer of the reference material.

cDNA sequence and gene expression of storage protein-2 — a juvenile hormone-suppressible hexamerin from the fall webworm, Hyphantria cunea Drury

We isolated and sequenced a cDNA clone corresponding to storage protein-2 (SP-2) from the fall webworm, Hyphantria cunea. The cDNA for SP-2 (2572 bp) codes for a 747-residue protein with a predicted molecular mass of 88.5 kDa. The calculated isoelectric point is 7.6. Multiple alignment analysis of amino acid sequence revealed that SP-2 is most similar to BJHSP2 (74.3% identity). According to both the phylogenetic analyses and criteria for amino acid composition, SP-2 belongs to the subfamily of moderately methionine-rich storage proteins (3.2% methionine, 11.8% aromatic amino acid). Topical application of the JH analog, methoprene, after head ligation of larvae, suppressed transcription of the SP-2 gene, indicating hormonal effects at the transcriptional level. The SP-2 transcript was detected by Northern blot analysis in Malpighian tubules, in addition to the fat body where it was most abundant. The local expression of SP-2 in Malpighian tubules suggests that it may have some function in that organ.

Monitoring of Pesticide Residues and Risk Assessment for Cereals and Leafy Vegetables of Certificated and General Agricultural Products

BACKGROUND: This study was conducted to monitor the current status of pesticide residues and to assess their risk in domestic agricultural products. The samples were rice, barley, lettuce and perilla leaf. These four types of agricultural products were those with GAP(Good Agricultural Practice) certification, organic agricultural products, pesticide-free agricultural products or general agricultural products. METHODS AND RESULTS: They were purchased from traditional markets and supermarkets of 12 regions in Korea from July to August 2010. The total number of samples was 259 for agricultural products and these were analyzed by GC/ECD, GC/NPD and GC/MSD. We used multiresidue methods to analyze for 110 different pesticides except for herbicides. CONCLUSION: In this study, residual pesticides were detected in 18 samples. Among these general agricultural products, organic agricultural products and products with GAP-certification were detected in 12, 4 and 2 samples, respectively. Detection rates of general agricultural products, organic agricultural products and products with GAP- certification were 4.6%, 1.5% and 0.8% respectively. Pesticides were not detected in pesticide-free agricultural products. Their detection levels were less than their maximum residue levels. Their estimated daily intakes ranged from 0.0003% to 0.04302% of their acceptable daily intakes, of which the values have no effect on human health.

Rapid detection of enterotoxigenic Clostridium perfringens in meat samples using immunomagnetic separation polymerase chain reaction (IMS-PCR).

Rapid detection of enterotoxigenic Clostridium perfringens in meat samples was accomplished with an immunomagnetic separation polymerase chain reaction (IMS-PCR). First, a monoclonal antibody (mAb) specific to C. perfringens was generated. The antibody showed strong binding to C. perfringens and no binding to non- Clostridia bacteria, except a weak cross-reaction to Staphylococcus aureus based on the enzyme-linked immunosorbent assay (ELISA). Then, magnetic beads were coated with the mAb, and the IMS-PCR system was developed. With the optimized conditions, the IMS-PCR assay was capable of detecting as few as 10 colony forming units (CFU)/g of C. perfringens cells in the meat sample within 10 h. Of the 116 collected samples (26 chicken samples, 20 beef samples, 30 pork samples, 20 fish samples, and 20 processed meat samples) examined with IMS-PCR, 36 (31%) were C. perfringens -positive samples and 2 (1.7%) were enterotoxigenic C. perfringens -positive samples. The IMS-PCR results gave a good agreement with the results obtained by conventional culture methods. In comparison to conventional culture methods, the IMS-PCR is a rapid and specific method and has potential use as a screening tool for enterotoxigenic C. perfringens in food samples.