• Research in Plant Disease
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• v.21 no.4
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• pp.326-329
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• 2015

Fusaric acid is a mycotoxin produced by species of the fungus Fusarium and can act synergistically with other Fusarium toxins. In order to develop a specific detection method for fusaric acid-producing fungus, PCR primers were designed to amplify FUB10, a transcription factor gene in fusaric acid biosynthetic gene cluster. When PCR with Fub10-f and Fub10-r was performed, a single band (~550 bp) was amplified from F. oxysporum, F. proliferatum, F. verticillioides, F. anthophilum, F. bulbicola, F. circinatum, F. fujikuroi, F. redolens, F. sacchari, F. subglutinans, and F. thapsinum, all of which were known for fusaric acid production. Whereas the FUB10 specific band was not amplified from Fusarium species known to be trichothecene producer. Because production of fusaric acid can co-occur in species that also produce fumonisin mycotoxins, we developed a multiplex PCR assay using the FUB10 primers as well as primers for the fumonisin biosynthetic gene FUM1. The assay yielded amplicons from fumonisin producers such as F. proliferatum and F. verticillioides, allowing for the simultaneous detection of species with the genetic potential to produce both types of mycotoxins.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.10
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• pp.1522-1527
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• 2010

This study was conducted to find out the minimal time needed for detection of Salmonella spp. which exist at very low concentration in foods by using real-time PCR. The sal-F and sal-R sequences were used as primers and sal-P was used as a probe. The detection limit of Salmonella spp. was $3.77{\times}10^2\;cfu/mL$ in buffered peptone water (BPW). Microbial growth was monitored after artificially inoculated Salmonella spp. into BPW. The obtained growth curve was well fitted with the equation, y＝$0.0127x^2$+0.5927x-0.4317 ($R^2$=0.99), if assuming that 1 cell exists in 25 g sample (0.04 cfu/mL). The microbial concentration will be reduced to 10 fold by adding BPW during sample treatment, so actual initial concentration at the starting point of enrichment is 0.004 cfu/mL. At this condition, real-time PCR detection would be possible only when microbial concentration increase occurs to exceed the detection limit (377 cfu/mL). The time needed for microbial increase was calculated from the growth curve equation as 7 hours and 20 minutes. Therefore the total time required for detection was less than 10 hours including the PCR operating time.

• Journal of Microbiology and Biotechnology
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• v.31 no.12
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• pp.1709-1715
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• 2021

Outbreaks of food poisoning due to the consumption of norovirus-contaminated shellfish continue to occur. Male-specific (F+) coliphage has been suggested as an indicator of viral species due to the association with animal and human wastes. Here, we compared two methods, the double agar overlay and the quantitative real-time PCR (RT-PCR)-based method, for evaluating the applicability of F+ coliphage-based detection technique in microbial contamination tracking of shellfish samples. The RT-PCR-based method showed 1.6-39 times higher coliphage PFU values from spiked shellfish samples, in relation to the double agar overlay method. These differences indicated that the RT-PCR-based technique can detect both intact viruses and non-particle-protected viral DNA/RNA, suggesting that the RT-PCR based method could be a more efficient tool for tracking microbial contamination in shellfish. However, the virome information on F+ coliphage-contaminated oyster samples revealed that the high specificity of the RT-PCR- based method has a limitation in microbial contamination tracking due to the genomic diversity of F+ coliphages. Further research on the development of appropriate primer sets for microbial contamination tracking is therefore necessary. This study provides preliminary insight that should be examined in the search for suitable microbial contamination tracking methods to control the sanitation of shellfish and related seawater.

• Journal of Food Hygiene and Safety
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• v.36 no.1
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• pp.93-99
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• 2021

This study was undertaken to compare the efficacy of different sample preparation (stomaching, pulsifying, and sonication) and DNA extraction methods (boiling and commercial kit) for detection of enterotoxin-producing Clostridium perfringens from produce by polymerase chain reaction (PCR). Each produce type was inoculated at concentrations of 102, 103, 104, 105, 106, and 107 spores/g. Produce inoculated with spores was treated with three sample preparation methods, and DNA was extracted by boiling method and a commercial kit, followed by PCR. The detection limit of stomached samples was lower than that of pummeled and sonicated samples by 10-100 times. Moreover, the DNA extraction efficiency of the commercial kit was found to be superior to that of boiling. In particular, the PCR efficiency of cherry tomato and perilla leaf samples was greatly affected by sample preparation and DNA extraction method. These data suggest that DNA extraction with a commercial kit after pulsification is an optimum sample preparation method for detection of C. perfringens by PCR.

• Journal of Ecology and Environment
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• v.48 no.1
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• pp.17-23
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• 2024

Background: Honey bees play a crucial role in pollination and ecological balance. Apis mellifera L. colonies, especially those located in specific geographic regions, such as the palm garden in Eastern Thailand, are susceptible to potential threats from microbial contaminants. Understanding and detecting microbial organisms in these beehives is essential for the preservation of bee health, honey production, and the broader ecosystem. However, the problem of microbial infection and antibiotic-resistant bacteria is more severe and continuously increasing, resulting in a health, economic, and social crisis. The purpose of this study is to determine the prevalence of microorganisms in A. mellifera beehives in palm gardens in Rayong province, Eastern Thailand. Results: Ten swabs in transport media were swabbed and obtained from different parts of each beehive (1 swab per beehive), for a total of 10 hives. Traditional microbial culture-based methods, biochemical tests, and antimicrobial susceptibility (disc-diffusion) tests were used to detect microbial organisms and antibiotic resistance in bacteria. The swab tests from nine beehives resulted in the detection of Gram-positive bacteria (63.64%), Gram-negative bacteria (27.27%), and fungi/yeast (9.09%). These microorganisms are classified as a group of coagulase-negative Staphylococcus spp. and made up 40.91% of the bacteria discovered. Other bacteria found were Coryneform bacteria (13.64%), Pantoea spp. (13.64%), Bacillus spp. (9.09%), yeast (9.09%), glucose non-fermentative Gram-negative bacilli (9.09%), and Pseudomonas spp. (4.55%). However, due to the traditional culture-based and 0biochemical tests usually used to identify the microbial organisms in clinical specimens and the limitation of identifying some environmental microbial species, the results of the antimicrobial susceptibility test cannot reveal if the organism is resistant or susceptible to the drug. Nevertheless, drug-sensitive inhibition zones were formed with each antibiotic agent. Conclusions: Overall, the study supports prevention, healthcare, and public health systems. The contamination of microorganisms in the beehives may affect the quality of honey and other bee products or even the health of the beekeeper. To avoid this kind of contamination, it is therefore necessary to wear personal protective equipment while harvesting honey and other bee products.

• Korean Journal of Microbiology
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• v.43 no.4
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• pp.317-320
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• 2007

In this paper, we have developed base-calling error detection program and algorithm which show the list of the genes or sequences that are suspected to contain base-calling errors. Those programs detect dubious bases in a few aspects in the process of microbial genome project. The first module detects base-calling error from the Phrap file by using contig assembly information. The second module analyzes frame shift mutation if it is originated from real mutation or artifact. Finally, in the case that there is control microbial genome annotation information, the third module extracts and shows the candidate base-calling error list by comparative genome analysis method.

• Environmental Engineering Research
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• v.12 no.4
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• pp.129-135
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• 2007

This study evaluated microbial risk that could develop within soil microbial communities after amended with organic fertilizers from stabilized swine manure waste. For this purpose, we assessed the occurrences and competitiveness of antibiotic resistance and pathogenicity in soil microbial communities that were amended with swine manure wastes stabilized by a traditional lagoon fermentation process and an autothermal thermophilic aerobic digestion process, respectively. According to laboratory cultivation detection analysis, soil applications of the stabilized organic fertilizers resulted in increases in absolute abundances of antibiotic resistant bacteria and of two tested pathogenic bacteria indicators. The increase in occurrences might be due to the overall growth of microbial communities by the supplement of nutrients from the fertilizers. Meanwhile, the soil applications were found to reduce competitiveness for various types of antibiotic resistant bacteria in the soil microbial communities, as indicated by the decrease in relative abundances (of total viable heterotrophic bacteria). However, competitiveness of pathogens in response to the fertilization was pathogens-specific, since the relative abundance of Staphylococcus was decreased by the soil applications, while the relative abundance of Salmonella was increased. Further testes revealed that no MAR (multiple antibiotic resistance) occurrence was detected among cultivated pathogen colonies. These findings suggest that microbial risk in the soil amended with the fertilizers may not be critical to public health. However, because of the increased occurrences of antibiotic resistance and pathogenicity resulted from the overall microbial growth by the nutrient supply from the fertilizers, potential microbial risk could not be completely ruled out in the organic-fertilized soil samples.

• Journal of Food Hygiene and Safety
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• v.35 no.1
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• pp.13-22
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• 2020

A total of 244 cereal samples (oat, sorghum, adlay, and proso millet) were collected from fields to examine the contamination of Fusarium mycotoxins in cereals during harvest season in 2017 and 2018. The contamination levels of deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEA) were analyzed individually by using the immunoaffinity column clean-up method with ultra performance liquid chromatography, and fumonisins (FUM) were analyzed by using the QuEChERS method with liquid chromatography-mass spectrometry. Highest level of NIV contamination (120.0-3277.0 mg/kg) was observed in oat samples among the analyzed cereals. In the adlay samples, DON contamination was the highest (maximum level 730.0 ㎍/kg). The proso millet samples had a high frequency of detection of NIV and ZEA (61.5% and 57.9%, respectively), but the levels were low (average detection level of NIV, 75.6 ㎍/kg, for ZEA, 21.5 ㎍/kg). Among the cereal samples, sorghum had the highest contamination frequency of DON, ZEA, and FUM, and the co-occurrence of Fusarium mycotoxin was 70.0%, which was higher than the average of 29.9%. In order to safely manage Fusarium mycotoxin levels in cereals, continuous research on the development of contamination prevention technologies together with monitoring of mycotoxin contamination is needed.

• Journal of Life Science
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• v.21 no.1
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• pp.159-164
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• 2011

Microbial whole-cell biosensors can be excellent analytical tools for monitoring environmental pollutants. They are constructed by fusing reporter genes (e.g., lux, gfp or lacZ) to inducible regulatory genes which are responsive to the relevant pollutants, such as aromatic hydrocarbons and heavy metals. A large spectrum of microbial biosensors has been developed using recombinant DNA technology and applied in fields as diverse as environmental monitoring, medicine, food processing, agriculture, and defense. Furthermore, their sensitivity and target range could be improved by modification of regulatory genes. Recently, microbial biosensor cells have been immobilized on chips, optic fibers, and other platforms of high-throughput cell arrays. This paper reviews recent advances and future trends of genetically modified microbial biosensors used for monitoring of specific environmental pollutants.

• Mycobiology
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• v.32 no.3
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• pp.123-127
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• 2004

Sweet persimmons have been increasingly cultivated in the southern part of Korea. However, anthracnose disease caused by Colletotrichum species is one of the major hindrances in cultivation and productions. In this study, we used polymerase chain reaction(PCR) to detect Colletotrichum species with the AFLP(amplified fragment length polymorphism) method. In AFLP, we used E3(5'-GACTGCGTACCAATTCTA-3') and M1(5'-GATGAGTCCTGAGTAACAG-3') primer combination and, as a result, 262 bp segment was observed in Colletotrichum species only. Specific PCR primers were designed from the sequence data and used to detect the presence of the fungus in genomic DNA isolated from symptomless sweet persimmon plants. Based on sequence data for specific segments, Co.B1(5'-GAGAGAGTAGAATTGCGCTG-3') and Co.B2(5'-CTACCATTCTTCTA GGTGGG-3') were designed to detect Colletotrichum species. The 220 bp segment was observed in Colletotrichum species only, but not in other fungal and bacterial isolates.