Center for Agricultural Biotechnology PERDO CHE

Bangkok, Thailand

Center for Agricultural Biotechnology PERDO CHE

Bangkok, Thailand
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Khapanya W.,Kasetsart University | Hongprayoon R.,Kasetsart University | Hongprayoon R.,Center for Agricultural Biotechnology PERDO CHE | Chinaphuti A.,Postharvest and Processing Research and Development Office
Journal of the International Society for Southeast Asian Agricultural Sciences | Year: 2017

Aflatoxins are cancer-causing chemicals produced primarily by Aspergillus flavus and A. parasiticus. Aflatoxin B1 (AFB1) is the most commonly found aflatoxin in improperly stored staple commodities such grain and feed. Its presence in the food supply, can be carried over to animal products such as meat, liver, kidney, pig blood and milk. A specific and sensitive detection method is required for preliminary screening of these samples. This research sought to develop a detection kit for total aflatoxin by immunochromatographic technique using monoclonal antibody (MAb) from the hybridoma cell line 4G6. The experiments were conducted at the Serology and Diagnostic Laboratory, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom province during 2014-2016. The MAb is composed of IgG2b isotype and lambda light chain. Its specificity recognized four aflatoxins including AFB1, AFB2, AFG1 and AFG2 with cross reactivity at 100%, 89.2%, 82.6%, and 72.7%, respectively by direct competitive enzyme-linked immunosorbent assay (dcELISA). In vitro propagation of the hybridoma was carried out using an Integra CELLine Culture System and the antibody was purified by affinity column chromatography. The conjugate probe was prepared by comparing two sizes of colloidal gold particles at 20 and 40 nm in diameter for the conjugation with the MAb. The MAb conjugate with 40 nm colloidal gold was selected and sprayed onto the conjugate release pad (CRP). The target cut-off value for the developed immunochromatographic strip (ICS) was 20 ng/mL according to a regulation limit in Thailand. The study on the appropriate conditions for this strip showed that aflatoxin B1 conjugated to bovine serum albumin (AFB1-BSA) and goat anti-mouse immunoglobulin (GAM) should be immobilized at the test line and control line at the same concentrations of 0.25 mg/mL. The testing sample was extracted with 70% methanol and further diluted 1:4 with Tris buffer saline with 0.05% Tween-20 (TBST) before application on the sample application pad (SAP) and the reaction could be visualized within 15 min. The analysis of 5 naturally contaminated corn samples (n=7) indicated that 2 samples contained ≥ 20 µg/kg and 3 samples contained < 20 µg/kg. Five samples, analyzed by dcELISA, showed contamination levels at <4, 9.6, 19.9, 10.5 and 39.7 µg/kg which delivered a good correlation to the results from ICS analysis. © 2017, International Society for Southeast Asian Agricultural Sciences. All rights reserved.

Phatsaman J.,Kasetsart University | Phatsaman J.,Center for Agricultural Biotechnology PERDO CHE | Hongprayoon R.,Kasetsart University | Hongprayoon R.,Center for Agricultural Biotechnology PERDO CHE | Mahakarnjanagul W.,Kasetsart University
Journal of the International Society for Southeast Asian Agricultural Sciences | Year: 2017

Aflatoxins are metabolites produced by Aspergillus spp. and can be found as contaminants in various food and agricultural products. A specific antibody is needed for the development of serological method, such as Enzyme-linked immunosorbent assay (ELISA), as a screening process to determine toxin contamination. In this study, nine clones of specific single chain variable fragments (scFv) were selected from naïve mouse phage display scFv library and their reactivity with aflatoxins were determined. The experiments were conducted in the Serology and Diagnostic Laboratory, Center of Agricultural Biotechnology, Kasetsart University from 2012-2013. The scFv gene from the recombinant phagemid clone 22A12 (scFv-22A12 gene), which gave the strongest reaction, was selected for further investigation on aflatoxin analysis. The recombinant protein product was 30 kDa. Concurrently, an alkaline phosphatase (AP) gene was amplified from Escherichia coli strain HB2151. The scFv-22A12 and the AP genes were ligated into pCANTAB-5E phagemid and transformed into E. coli TG1 and HB2151 to produce phage scFv-AP and soluble scFv-AP, respectively. Comparison on the efficiency of Phage scFv, Phage scFv-AP and soluble scFv-AP to whole molecule antibody for detecting AFB1 was performed by ELISA. The resulst showed that the soluble scFv-AP gave highest reactivity and in accordance with those obtained from the whole molecule antibody. Cross reactivity with other aflatoxins (B2, G1 and G2) was reported to be 38.63%, 21.24 % and 9.64%, respectively. When using soluble scFv-AP to analyze ground samples of corn and groundnut spiked with 100 µg/kg of AFB1, acceptable results were obtained with 87.02 and 94.41% recovery, respectively. Analysis of the certified reference material (TMAF No.2 and TMAF No.3) showed comparable results with those analyzed through high performance liquid chromatography. © 2017, International Society for Southeast Asian Agricultural Sciences. All rights reserved.

Srinives P.,Kasetsart University | Kitsanachandee R.,Kasetsart University | Kitsanachandee R.,Center for Agricultural Biotechnology PERDO CHE | Chalee T.,Kasetsart University | And 3 more authors.
Plant and Soil | Year: 2010

Iron deficiency chlorosis (IDC) is a major problem reducing yield of mungbean in many countries. In this study, we crossed "KPS1", the most popular Thai mungbean cultivar susceptible to IDC with "NM10-12", a mungbean line from Pakistan resistant to IDC. Segregation analysis of the F2 population revealed that the resistance is controlled by a major gene (IR) with dominant effect. Two AFLP markers, E-ACT/M-CTA and E-ACC/M-CTG were identified closely linking with the IR gene. The frequencies of these markers were assessed in 241 mungbean accessions from several countries. The accessions could be divided, in relative to total chlorophyll content of the resistant check (NM10-12) and the susceptible check (KPS1), into the resistant group with 125 accessions and the susceptible group with 116 accessions. Among 125 resistant accessions, E-ACT/M-CTA and E-ACC/M-CTG were present in 119 (95%) and 109 (87%) accessions, respectively. Both markers can identify all resistant accessions from England, Indonesia and Pakistan, but only E-ACT/M-CTA linked to all resistant accessions from Australia, India, Iraq, Taiwan and Thailand. Understanding the inheritance and identifying molecular markers linking to the IR gene can help plant breeders to improve this crop for growing in iron-deficient soils. © 2010 Springer Science+Business Media B.V.

Kositcharoenkul N.,Kasetsart University | Kositcharoenkul N.,Center for Agricultural Biotechnology PERDO CHE | Chatchawankanphanich O.,BIOTEC Central Research Unit | Bhunchoth A.,BIOTEC Central Research Unit | And 2 more authors.
Plant Pathology | Year: 2011

A single-tube nested PCR was developed for detection of Xanthomonas citri subsp. citri (Xcc), the causal agent of citrus canker disease. The assay targets the pthA gene of Xcc and utilizes different annealing temperatures for the two primer pairs. It reliably detected as few as 1·0×102Xcc cells, and was unaffected by the presence of PCR inhibitors. It was 10-fold and 8500-fold more sensitive than standard PCR and ELISA, respectively. Increased sensitivity was also achieved via the use of a washing method for DNA extraction, as opposed to direct extraction from leaf tissue. When evaluated for Xcc detection in 90 samples collected from affected pomelo orchards, the single-tube nested PCR was superior to standard PCR, detecting the pathogen in 67 vs. 54 samples. It was also able to detect Xcc from samples with and without symptoms. This assay can be used as a rapid and sensitive technique for routine Xcc detection in field samples for surveillance of citrus canker. © 2010 The Authors. Plant Pathology © 2010 BSPP.

Poolsawat O.,Suranaree University of Technology | Poolsawat O.,Center for Agricultural Biotechnology PERDO CHE | Tharapreuksapong A.,Suranaree University of Technology | Wongkaew S.,Suranaree University of Technology | And 2 more authors.
Journal of Phytopathology | Year: 2010

Anthracnose is one of the major diseases affecting grape (Vitis vinifera L.) cultivars in Thailand. Isolates of Sphaceloma ampelinum, the anamorph stage of Elsinoe ampelina, were collected from various regions of Thailand. Nineteen single-conidial isolates were evaluated for differences in conidial morphology, DNA patterns and pathogenicity. These isolates could not be unambiguously distinguished based on conidial morphology; however, they were genetically differentiated using random amplified polymorphic DNA markers. Cluster analysis by the unweighted paired grouped mean arithmetic average classified these isolates into four groups. Pathogenicity analysis using nine grape genotypes and five S. ampelinum isolates showed that 'Wilcox321' and 'Illinois547-1 were highly resistant to all isolates, suggesting their usefulness as resistant sources in future breeding programmes. © 2010 Blackwell Verlag GmbH.

Narit T.,Prince of Songkla University | Narit T.,Center for Agricultural Biotechnology PERDO CHE | Anuchit C.,National Biological Control Research Center
Philippine Agricultural Scientist | Year: 2011

Factors affecting the attraction of two fruit fly species [Bactrocera cucurbitae (Coquillett) and B. papayae Drew and Hancock] to the odor of the bacterium Enterobacter cloacae were studied. The experimental factors analyzed were sex (S), mating experience (M), feeding status (F) and host fruit provision (H). For B. cucurbitae, all main factors and their interaction did not affect net attractancy to bacterial odor except for the interaction between M and F where protein-deprived virgin flies gave significantly the lowest percentage net attractancy. In B. papayae, the main effects of S, M and H and the interaction effect of (S×M×F) and (M×F×H) significantly influenced net attractancy. Male flies which have mated and those not given host fruit showed high response to bacterial odor. For the interactive factors, mated male and protein-fed flies had higher net attractancy to bacterial odor compared with the virgin female and protein-fed flies. On the other hand, flies which had mated which were protein-deprived and were not provided with host fruit, gave higher net attractancy compared with virgin flies which were fed with protein and host fruit. B. papayae was more attracted to the bacterial odor than B. cucurbitae.

Panlawan P.,Mahidol University | Panlawan P.,Center for Agricultural Biotechnology PERDO CHE | Luangthongkam P.,Mahidol University | Wiemann L.O.,Fraunhofer Institute for Interfacial Engineering and Biotechnology | And 7 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2013

The synthetic activity of lipases in biphasic o/w systems was investigated with respect to their use in the synthesis of polyester chains via transesterification reactions. Lipase-catalyzed ring-opening polymerization (ROP) of pentadecalactone (ω-PDL) dispersed in water was used as a model reaction to understand the synthetic activity of lipases in biphasic o/w system. We conducted a systematic investigation of the influence of reaction conditions on the macromolecular characteristics of oligo(ω-PDL) encompassing chemical, thermophysical and colloidal properties of the reaction medium. A model was proposed assuming Michaelis-Menten interfacial kinetics followed by chain extension via lipase-catalyzed linear polycondensation. The solidification of oligo(ω-PDL) chains with a degree of polymerization of approximately three was identified as a major factor limiting the molecular weight of obtained oligomers to ∼870 g mol-1, despite the fast reaction rate and complete conversion of ω-PDL. The addition of toluene into the dispersed phase at a volumetric ratio of 0.3-0.5 of toluene to ω-PDL allowed us to circumvent this problem and increase the molecular weight of obtained oligomers up to 1460 g mol-1. The molecular weight of polymer product according to this model was thus inversely related to the weight ratio percentage of interfacial lipase PS to ω-PDL per droplet and correspondingly correlated with the activity of lipase. Taking into account all these parameters allowed increasing the molar mass of oligo(ω-PDL) from 870 g mol-1 to 3507 g mol-1. © 2012 Elsevier B.V.

Saktaweewong S.,Mahidol University | Saktaweewong S.,Center for Agricultural Biotechnology PERDO CHE | Phinyocheep P.,Mahidol University | Ulmer C.,Helmholtz Center for Infection Research | And 3 more authors.
Journal of Molecular Catalysis B: Enzymatic | Year: 2011

This work focused on lipase-catalyzed triglyceride hydrolysis in biphasic media. The effect of specific interfacial area of oil-in-water emulsions on the hydrolysis activity of lipase was particularly investigated following a rigorous methodology and using two different oils, tributyrin and olive oil. The specific interfacial area was varied over several orders of magnitude by changing either the amount of emulsified oil or the average diameter of oil droplets. This work particularly focused on the effect of changing droplet size (at given amount of oil) on lipase activity. When the specific interfacial area was varied over several orders of magnitude, the specific activity of the enzyme exhibited a non-monotonic variation with a pronounced maximum. At low specific interfacial area, initial velocity increased with specific interfacial area. Inhibition of enzyme activity at a high interfacial concentration of triglyceride was observed. Experimental results were interpreted on the basis of a theoretical mechanism assuming Michaelis-Menten mechanism for enzyme catalysis, Langmuir-type adsorption isotherm for enzyme and limitation of enzyme-substrate formation by enzyme adsorption process. © 2011 Elsevier B.V.

Thaochan N.,Walailak University | Thaochan N.,Center for Agricultural Biotechnology PERDO CHE | Drew R.A.I.,Griffith University | Hughes J.M.,Griffith University | And 2 more authors.
Journal of Insect Science | Year: 2010

Bacteria were isolated from the crop and midgut of field collected Bactrocera cacuminata (Hering) and Bactrocera tryoni (Froggatt) (Diptera: Tephritidae). Two methods were used, firstly isolation onto two types of bacteriological culture media (PYEA and TSA) and identification using the API-20E diagnostic kit, and secondly, analysis of samples using the 16S rRNA gene molecular diagnostic method. Using the API-20E method, 10 genera and 17 species of bacteria in the family Enterobacteriaceae were identified from cultures growing on the nutrient agar. The dominant species in both the crop and midgut were Citrobacter freundii, Enterobacter cloacae and Klebsiella oxytoca. Providencia rettgeri, Klebsiella pneumoniae ssp ozaenae and Serratia marcescens were isolated from B. tryoni only. Using the molecular cloning technique that is based on 16S rRNA gene sequences, five bacteria classes were dignosed Alpha-, Beta-, Gamma- and Delta- Proteobacteria and Firmicutes including five families, Leuconostocaceae, Enterococcaceae, Acetobacteriaceae, Comamonadaceae and Enterobacteriaceae. The bacteria affiliated with Firmicutes were found mainly in the crop while the Gammaproteobacteria, especially the family Enterobacteriaceae, was dominant in the midgut. This paper presents results from the first known application of molecular cloning techniques to study bacteria within tephritid species and the first record of Firmicutes bacteria in these flies.

Wichaphon J.,Mahidol University | Wichaphon J.,Center for Agricultural Biotechnology PERDO CHE | Thongthai C.,Mahidol University | Assavanig A.,Mahidol University | Lertsiri S.,Mahidol University
Flavour and Fragrance Journal | Year: 2012

Numerous investigations on aroma characteristics of fish sauce in particular samples have been conducted extensively; however, the relation of those volatile aroma profiles and aroma characteristics to quality and categorization of the product have never been reported. This study explored the contribution of volatile aroma components on product quality categorization of 52 Thai fish sauce samples. First, odour-active compounds were investigated by dynamic headspace gas chromatography-olfactometry (GC-O) from eight selected samples with four different qualities, including mature-unblended samples, premium-grade samples, first-grade samples and second-grade samples. Eleven odour-active compounds, i.e. acetic acid, propanoic acid, 2-methylpropanoic acid, butanoic acid, 3-methylbutanoic acid, dimethyl trisulfide, 3-(methylthio)propanal, 1-octen-3-ol, 2-butanol, trimethylamine and n-propanol, contributed to the aroma characteristics of Thai fish sauce. A combination score was assigned to express the integration of flavour dilution on dynamic headspace dilution analysis and intensity of odour perceived on GC-O. The combination scores were analysed with principal component analysis to categorize these eight selected samples. Furthermore, these odour-active compounds detected as released volatile compounds from 52 fish sauce samples were applied for categorization of 52 samples. As a result, both combination scores and relative concentrations of these odour-active compounds could discriminate the fish sauce products according to their conventional grading which is based on total nitrogen of the sample. © 2011 John Wiley & Sons, Ltd.

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