• Journal of Dairy Science and Biotechnology
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• v.33 no.4
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• pp.253-262
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• 2015

The authentication and implications of misleading labeling in milk and dairy products is important to protect against cheating consumers from adulteration and to alert sensitive consumers to any undeclared potential allergens. This need to support milk and dairy products labeling has led to the development of specific analytical techniques for the analysis of milk and dairy products ingredients. Recently, several methods based on polymerase chain reaction (PCR), including restriction fragment length polymorphism (PCR-RFLP), multiplex PCR, species-specific PCR, and real-time PCR, have been proposed as useful means for identifying species of origin in milk and dairy products, as well as quantifying and detecting any adulteration. These methods have particular advantages owing to their high specificity and sensitivity, as well as rapid processing time. In this review, we provide an updated and extensive overview of the PCR-based methods used for milk and dairy products authentication with a particular focus on the application of PCR methods to detect adulteration.

• Journal of Microbiology
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• v.45 no.4
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• pp.297-304
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• 2007

The detection of virulence factors of Aeromonas is a key component in determining potential pathogenicity because these factors act multifunctionally and multifactorially. In this study water samples were collected from a trout farm on a seasonal basis, and diseased fish and Aeromonas species were isolated and identified. For rapid detection of six virulence factors of isolated Aeromonas, a hexaplex-polymerase chain reaction (hexaplex-PCR) assay was used. The detected virulence factors include aerolysin (aer), GCAT (gcat), serine protease (ser), nuclease (nuc) lipase (lip) and lateral flagella (laf). The dominant strain found in our isolates was Aeromonas sobria, and the dominant virulence factors were aer and nuc for all seasons. We confirmed that A. sobria and two of the virulence genes (aer and nuc) are related. We proposed a method by which one can identify the major strains of Aeromonas: A. hydrophila, A. sobria, A. caviae, and A. veronii, using hexaplex-PCR.

• Korean Journal of Microbiology
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• v.48 no.4
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• pp.275-283
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• 2012

Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Asteropus simplex collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and MA media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 94% similarities compared with known bacterial species, and the isolates belonged to five phyla, Alphaproteobacteria, Gammaproteobacteria Actinobacteria, Bacteroidetes, and Firmicutes, of which Gammaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge-derived total gDNA showed 12 DGGE bands, and their sequences showed more than 90% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven phyla, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteira, Chloroflexi, and Nitrospira. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with A. simplex by both RFLP and DGGE methods, however, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture-independent method than in culture-dependent method.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.322-327
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• 2009

A total of 23 isolates of Botrytis cinerea causing the grey mold were collected from infected ginseng in several fields of Korea. The sensitivity to carbendazim and the mixture of carbendazim plus diethofencarb was determined through a mycelial inhibition test on PDA amended with or without fungicides. B. cinerea isolates were classified as 3 phenotypes, which were the first phenotype resistant to both of carbendazim and the mixture ($Car^RMix^R$), the second one resistant to carbendazim and sensitive to the mixture ($Car^RMix^S$), and the last one sensitive to both of them ($Car^RMix^S$). Carbendazim resistance correlated with a single mutation $\beta$-tubulin gene of B. cinerea amplified with primer pair tubkjhL and tubkjhR causing a change of glutamate to alanine at amino acid position 198. Furthermore, the substitution of valine for glutamate led the resistance to carbendazim and the mixture at the same position of amino acid. PCR-restriction fragment length polymorphism (PCR-RFLP) analysis using the restriction endonuclease, Tsp451 and BstUI allowed differentiation of the PCR fragment of $\beta$-tubulin gene of $Car^SMix^S$ isolates from that of $Car^RMix^R$ and $Car^RMix^S$ isolates. This method will aid in a fast detection of resistance of carbendazim and the mixture of carbendazim plus diethofencarb in B. cinerea in ginseng field.

• The Plant Pathology Journal
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• v.38 no.6
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• pp.629-636
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• 2022

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 ㎍/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

• Journal of Korean Society on Water Environment
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• v.22 no.4
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• pp.590-598
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• 2006

In-situ Air sparging (IAS) is a groundwater remediation technique, in which organic contaminants volatilize into air form the saturated to vadose zone. This study was carried out to evaluate the effect of sludge and soil microbial community structure on air sparging of Total Petroleum Hydrocarbons (TPH) contaminated groundwater soils. In the laboratory, diesel (10,000 mg TPH/kg) contaminated saturated soil. The Air was injected in intermittent (Q=1500 mL/min, 10 minute injection and 10 minute idle) modes. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for experiment with sludge soil samples that were closely related to Agrococcus, Flavobacterium, Thermoanaerobacter, Flexibacter and Shewanella, etc, in the clone library. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil the fate of microorganisms in natural microbial community.

• Journal of Korean Society on Water Environment
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• v.24 no.1
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• pp.19-29
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• 2008

Hot air sparging is a groundwater remediation technique, in which organic contaminants volatilized into hot air from the saturated to vadose zone. In the laboratory diesel (10,000 mg TPH/kg) was spiked in contaminated saturated aquifer soil. The hot air ($34.9{\pm}2.7^{\circ}C$) was injected in intermittent (Q=1,500 mL/min, 10 minute injection and 10 minute idle) modes. We performed microcosm tests using the groundwater samples to assess TPH reductive remediation activity. For Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis of eubacterial communities in sludge of wastewater treatment plants and soil of experiment site, the 16S rDNA was amplified by Polymerase Chain Reaction (PCR) from the sludge and the soil. The obtained 16S rDNA fragments were digested with Msp I and separated by electrophoresis gel. We found various sequence types for hot air sparging experiment with sludge soil samples that were closely related to Bacillus (149 bp, Firmicutes), Methlobacterium (149 bp, Euryarchaeotes), Pseudomonas (492 bp, ${\gamma}$-Proteobacteria), etc., in the clone library. In this study we find that TPH-water was reduced to 78.9% of the initial value in this experiment aquifer. The results of the present study suggests that T-RFLP method may be applied as a useful tool for the monitoring in the TPH contaminated soil fate of microorganisms in natural microbial community.

• Environmental Analysis Health and Toxicology
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• v.21 no.1 s.52
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• pp.27-34
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• 2006

Numerous studies reported the significant association between genetic polymorphisms in the vitamin D receptor (VDR) gene and various bone phenotypes such as bone mineral density (BMD) and bone quality, although conflicting results were produced. The objective of this study was to investigate the association between a TaqI RFLP in the VDR gene and calcaneal BMD in Korean vegetarian men, and its interaction with nutrition status as an environmental factor. BUA (broadband ultrasound attenuation), SOS (sound of speed) and stiffness index of the calcaneus were measured using an ultrasound bone densitometer in 266 Korean men (age: $mean{\pm}SD;\;50.9{\pm}12.0$ year), and a TaqI RFLP in the VDR gene analysed by PCR-RFLP method. In total subjects, the distribution of TT, Tt and tt genotypes occurred with frequencies of 90.8%, 8.8% and 0.4%, respectively. There were no significant associations between this polymorphism and osteopenia-osteoporosis or several bone phenotypes in our subjects irrespectively of nutrition status (P>0.05). Therefore, our results suggest that a TaqI RFLP in the vitamin D receptor gene does not contribute to the susceptibility to the calcaneal BMD in Korean men.

• Korean Journal of Microbiology
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• v.37 no.1
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• pp.72-79
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• 2001

The effect of phenol on the change of bacterial community in the effluent water from a wastewater treatment plant was analyzed by PCR and terminal restriction fragment length polymorphism (T-RFLP). The fragments of 16S rDNA were amplified by PCR with bacterial primers, where one of the primers was biotinylated at the 5'-end. After digestion with restriction enzymes, HaeIII and AluI, the biotinylated terminal restriction tragments (T-RFs) of the digested products were selectively isolated by using streptavidin paramagnetic particles. The single-stranded DNA of T-RFs was separated by electrophoresis on a polyacrylamide gel and detected by silver staining technique. When 10 standard strains were analyzed by our method, each strain had a unique T-RF which corresponded to the calculated size from the known sequences of RDP database. The T-RFLP fingerprint generated from the effluent water was very complex, and the predominant T-RFs corresponded to members of the genus Acinetobacter, Bacillus and Pseudomonas. In addition, the perturbation of bacterial community was observed when phenol was added to the sample at the final concentration of 250 $l^{-1}$. The number of T-RFs increased and the major bacterial population could be assigned to the genus Acinetobacter, Comamonas, Cytophaga and Pseudomonas. A intense band assigned to the putative genera of Acinetobacter and Cytophaga was eluted, amplified, and sequenced. The nucleotide sequence of the T-RF showed close relationship with the sequence of Acinetobacter junii.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.7
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• pp.942-948
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• 2005

New polymorphism of major histocompatibility complex B-G genes was investigated by amplification and digestion of a 401bp fragment including intron 1 and exon 2 using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique with two restriction enzymes of Msp I and Tas I in eight breeds of Chinese indigenous chickens and one exotic breed. In the fragment region of the gene, three novel single nucleotide polymorphisms (SNPs) were detected at the two restriction sites. We found the transition of two nucleotides of A294G and T295C occurred at Tas I restriction site, and consequently led to a non-synonymous substitution of asparagine into serine at position 54 within the deduced amino acid sequence of immunoglobulin variable-region-like domain encoded by the exon 2 of B-G gene. It was observed at rare frequency that a single mutation of A294G occurring at the site, also caused an identical substitution of amino acid, asparagine 54-to-serine, to that we described previously. And the transversion of G319C at Msp I site led to a non-synonymous substitution, glutamine 62-to-histidine. The new alleles and allele frequencies identified by the PCR-RFLP method with the two enzymes were characterized, of which the allele A and B frequencies at Msp I and Tas I loci were given disequilibrium distribution either in the eight Chinese local breeds or in the exotic breed. By comparison, allele A at Msp I locus tended to be dominant, while, the allele B at Tas I locus tended to be dominant in all of the breeds analyzed. In Tibetan chickens, the preliminary association analysis revealed that no significant difference was observed between the different genotypes identified at the Msp I and Tas I loci and the laying performance traits, respectively.