• The Plant Pathology Journal
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• v.34 no.6
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• pp.490-498
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• 2018

Panicle blight and seed rot disease caused mainly by Burkholderia glumae and Burkholderia gladioli is threatening rice cultivation worldwide. The bacteria have been reported as seed-borne pathogens from rice. Accurate detection of both pathogens on the seeds is very important for limiting the disease dissemination. Novel primer pairs targeting specific molecular markers were developed for the robust detection of B. glumae and B. gladioli. The designed primers were specific in detecting the target species with no apparent cross-reactions with other related Burkholderia species at the expected product size. Both primer pairs displayed a high degree of sensitivity for detection of B. glumae and B. gladioli separately in monoplex PCR or simultaneously in duplex PCR from both extracted gDNA and directly preheated bacterial cell suspensions. Limit of detection was as low as 0.1 ng of gDNA of both species and $3.86{\times}10^2cells$ for B. glumae and $5.85{\times}10^2cells$ for B. gladioli. On inoculated rice seeds, the designed primers could separately or simultaneously detect B. glumae and B. gladioli with a detection limit as low as $1.86{\times}10^3cells$ per rice seed for B. glumae and $1.04{\times}10^4cells$ per rice seed of B. gladioli. The novel primers maybe valuable as a more sensitive, specific, and robust tool for the efficient simultaneous detection of B. glumae and B. gladioli on rice seeds, which is important in combating rice panicle blight and seed rot by early detection and confirmation of the dissemination of pathogen-free rice seeds.

• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.555-559
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• 2013

Vibrio cholerae O1 and O139 are the major serotypes associated with illness, and some V. cholera non-O1 and non-O139 isolates produce cholera toxin. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the species-specific detection and quantitation of V. cholera using a primer pair based on an outer membrane lipoprotein lolB gene for the amplification of a 195 bp DNA fragment. The qPCR primer set for the accurate diagnosis of V. cholera was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.

• Food Science of Animal Resources
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• v.31 no.2
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• pp.158-165
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• 2011

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at $65^{\circ}C$ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

• Journal of Practical Agriculture & Fisheries Research
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• v.24 no.3
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• pp.5-14
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• 2022

Trichoderma is known as pathogen caused serious green mold disease on commercial production. T. pleuroti and T. pleuroticola were common species in various mushroom media. Many strains of T. pleuroti, known as aggressive species causing major economic losses in Korea, showed benomyl resistance. Accurate identification and detection of Trichoderma species associated with oyster mushrooms is very important for disease control. We developed species-specific primers for T. pleuroticola, T. pleuroti, T. harzianum, and T. atroviride based on species-specific fragments isolated from amplified fragment length polymorphism analysis. PCR products corresponding to the predicted fragment of 500bp, 230bp, 180bp, and 410bp were amplified from T. pleuroticola, T. pleuroti, T. harzianum, and T. atroviride, respectively. Multiplex PCR assay using species-specific primers quickly and accurately identified and detected T. pleuroti from mushroom media in which various species co-exist. Our results can be useful for the effective control of mushroom disease.

• Mycobiology
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• v.31 no.3
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• pp.133-138
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• 2003

Analysis of phylogenetic relationship was performed among Phellinus species based on 18S ribosomal subunit sequence data. Twenty-five strains of 19 Phellinus species including P. linteus were examined in this study. Regions of 18S ribosomal subunit were very conserved, but some variable regions between Phellinus species were observed. The species-specific detection primers, modified by 2 or 3 nucleotides in sense primer were designed based on 18S ribosomal DNA(rDNA) sequence data. The 210 by PCR bands were detected with annealing temperature $48^{\circ}C$. The 18S 2F-18S 4R detection primer set distinguished P. linteus from various Phellinus species but some species like P. baumii, P. weirianius, P. rhabarberinus and P. pomaceus also had weak reactivity on this primer set. The 18S 3F-18S 4R primer set distinguished only P. linteus from various Phellinus species, although sensitivity with this primer set was lower than that of 18S 2F-18 4R primer set. These primer sets would be useful for the detection of only P. linteus among unknown Phellinus species rapidly.

• Food Science of Animal Resources
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• v.29 no.5
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• pp.647-652
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• 2009

A duplex PCR technique was applied for specific identification of cow and goat milk in milk products by using primers targeting the mitochondrial 12S rRNA gene. Duplex PCR using primers specific for cow and goat generated specific fragments of 223bp and 326bp from cow and goat milk DNA, respectively. Duplex PCR was applied to 15 milk products purchased from the market to verify label statements. The labeling statements of four market milk products, three yoghurt products, and one whole milk powder product were confirmed in the duplex PCR. The labeling statements of five of seven infant milk powder products were also confirmed by duplex PCR but the other two products were shown to be contaminated with either cow or goat milk. The proposed duplex PCR provides a rapid and sensitive approach to detection of as little as 0.1% cow milk in goat milk and one-step detection of cow or goat milk in milk products.

• Journal of Dairy Science and Biotechnology
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• v.31 no.2
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• pp.127-131
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• 2013

The aim of this study was to investigate adulteration of caprine milk products by bovine milk using biomolecular techniques with bovine-specific primers for the mitochondrial cytochrome b gene. Polymerase chain reaction (PCR) and real-time PCR assays were applied to caprine milk products including infant formula, city milk, and fermented milk. The results indicated that six out of the eight caprine infant formula products tested contained bovine milk components. In addition, two of the three tested caprine city milk products and two caprine fermented milk products were shown to be adulterated with bovine milk. Conventional PCR results corroborated with results obtained by real-time PCR. This study demonstrates that DNA-based species identification procedures would be useful and applicable in routine examinations of the dairy industry to ensure the quality and safety of dairy foods.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.110-115
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• 2004

This study was undertaken to develop PCR primers for the simultaneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans, using two species-specific reverse primers in combination with a single conserved forward primer. These primers target the variable and conserved regions of the 16S rDNA. The primer specificity was tested against (i) four F. nucleatum and three A. actinomycetemcomitans strains and (ii) seven representatives of the different species of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of F. nucleatum and A. actinomycetemcomitans. The data indicate that species-specific amplicons could be obtained for all the F. nucleatum and A. actinomycetemcomitans strains tested, which were not found in the seven other species. The multiplex PCR could detect as little as 4 fg of chromosomal DNA of F. nucleatum and A. actinomycetemcomitans simultaneously. These findings suggest that these PCR primers are highly sensitive and are suitable for applications in epidemiological studies, diagnosis, and monitoring F. nucleatum and A. actinomycetemcomitans after the treatment of periodontitis.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.173-180
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• 2000

The intern1 spacer regions (ISR) between the 16s and 23s $1_RNA$ genes of Aeronzonus iwonii blogroupsobria and A. caviae were investigated by PCR fragment length typing and DNA sequencing. A. iwonii bv.sobria has a speciIic 16s-23s pattern of 2-4 fiagments ranging Goin 479-539 bp, with the exception of thespecies Aeron7onns cmiae, which has 3 fragments ranglog from 470-602 bp. In all of the.4 vei*onii bv. sobr,iaand A, caviae strains examined in this study, the 470-481bp Tragnent, designated TSR-1, invariably contained $tDNA^{uc(GAT)$ and $tDNA^{Ala(TGC)$ in contrast to ISR-2 (513-525 bp). ISR-3 (537-539 bp) and ISR-4 (568-602 bp)containing TEX>$tDNA^{Olu(ITC)$ A stretch of 20 nucleotides (178-197 bp) in the ISR-4 was conserved only wit11mA.caiiue, from which the A. caiiae specific primer, named prAC-F, was designed and used for PCR with aAcaviae coimnon reverse primer A PCR product of 450 bp was apparent alnong I , caiizne strains, but not ii1.4.ijeronii bv. sob~ia strains. The PCR product was oot detected t"-om strains belonging to A. hjili-o~~hila, Ebrio,aud the family Ef\ulcornertei,obncteriaceae. This study provides the first molecular tool for mdentifying the species 8.caviae.ing the species 8. caviae.

• Journal of Animal Science and Technology
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• v.48 no.5
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• pp.637-650
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• 2006

Genomic DNA isolated from two crucian carp species obtained from Yesan (Carassius auratus) and Dangjin (Carassius cuvieri) in Korea were amplified at several times by polymerase chain reaction (PCR). The amplified products were separated by agarose gel electrophoresis (AGE) with oligonucleotides decamer primer and stained with ethidium bromide. The seven arbitrarily selected primers OPC-11, OPC-14, OPC-18, OPD- 02, OPD-11, OPD-15 and OPD-20 generated the shared loci by each species, the polymorphic and specific loci. The seven primers generated the total 458 loci that can be scored from the crucian carp obtained in C. auratus species. 358 fragments were generated from the species obtained in C. cuvieri species. The size of DNA fragments varies from 150 to 1,600bp. The complexity of the banding patterns varies dramatically between the primers and two locations. In this study, 458 loci were identified in the crucian carp species from Yesan and 358 in the crucian carp species from Dangjin: 84 polymorphic loci (18.3%) in the C. auratus species and 48 (13.4%) in the C. cuvieri species. 154 shared loci by each species, the average 22 per primer, were observed in the C. auratus species and 187 loci, the average 26.7 per primer, in the Dangjin species. Based on the average bandsharing (BS) values of all samples, the similarity matrix ranged from 0.434 to 0.868 in the C. auratus species and from 0.449 to 0.924 in the C. cuvieri species. The average BS value was 0.641±0.013 within the C. auratus species and 0.684±0.013 within the C. cuvieri species. The average BS value between two crucian carp species 0.484 ± 0.007, ranged from 0.307 to 0.682. The BS value between the individual No. 09 and No. 16 was 0.682, which was the highest between two crucian carp species. Compared separately, the BS value of individuals within the C. cuvieri species was higher than the C. auratus species. The dendrogram obtained by the seven primers, indicates three genetic clusters: cluster 1 (AURATUS No. 01, 02, 03, 04, 05, 06, 07, 08, 09, 10 and 11), cluster 2 (CUVIERI No. 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21) and cluster 3 (CUVIERI no. 22). The shortest genetic distance displaying significant molecular difference was between the individual AURATUS No. 09 and AURATUS No. 08 from Yesan (genetic distance=0.064). The longest genetic distance displaying significant molecular differences was between the individual CUVIERI No. 17 and AURATUS No. 11 between two crucian carp species (0.477). RAPD-PCR analysis has revealed the significant genetic distance between two crucian carp species pairs.(Key words: Carassius auratus, Carassius cuvieri, Crucian Carp, DNA Polymorphism, Genetic Distance)