• Korean Journal Plant Pathology
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• v.14 no.6
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• pp.589-593
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• 1998

DNA amplification by polymerase chain reaction (PCR) was used to specifically detect Plasmodiophora brassicae, causing clubroot of crucifers. On the basis of DNA sequence informations, an oligonucleotide primer set specific for the pathogen was designed form small subunit gene (18S-like) and internal transcribed spacer (ITS) region of ribosomal DNA. Primer ITS 5/PB-C produced an amplification product of approximately 520 bp in length with DNA from P. brassicae. However, no amplification product was produced with DNAs from several soil-borne fungi, Didymella bryoniae and Rhizopus stolonifer. Using these primers, the clubroot pathogen was readily detected from infected roots of crucifers, but not from healthy roots. Southern hybridization analysis further confirmed that the amplification product was originated from P. brassicae.

• Research in Plant Disease
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• v.14 no.3
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• pp.219-222
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• 2008

Real time PCR was used to discriminate Colletotrichum acutatum and C. gloeosporioides for analysis of population density. Two primers, caInt2 and cgint, used for conventional PCR to discriminate two species were modified with fluorescent dye to make probe for real time PCR. Fluorescence signals were successfully detected by fCaInt2 and vCgint probe coupled with primer pair Unicon and Unicor1 resulting in discrimination of C. acutatum and C. gloeosporioides by comparison of delta Rn value.

• Research in Plant Disease
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• v.14 no.3
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• pp.229-232
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• 2008

An isolate of Cucumber mosaic virus (CMV), designated as Cas-CMV, was isolated from Chinese aster (Callistephus chinensis) showing severe mosaic symptom, and its properties was compared to the well-characterized Fny-CMV (subgroup IA) and As-CMV (subgroup IB) by host reaction in several indicator plants, dsRNA analysis, RT-PCR analysis, and restriction enzyme profile of the PCR products. Cas-CMV differed markedly in their host reaction to Fny-CMV or As-CMV in Cucurbita pepo cv. Black beauty. In the zucchini squash, all strains induced chlorotic spot on inoculated leaves and mosaic symptoms on upper leaves. However, symptoms induced by Cas-CMV were developed lethal necrosis on the young plants 15 to 20 days after inoculation. In experiments of dsRNA analysis and RT-PCR analysis, properties of Cas-CMV was come within subgroup I CMV. Moreover, restriction enzyme analysis using HindIII of the RT-PCR products showed that Cas-CMV belong to a member of CMV subgroup IA.

• Parasites, Hosts and Diseases
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• v.55 no.1
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• pp.95-98
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• 2017

Fasciola hepatica is a trematode that causes zoonosis, mainly in cattle and sheep, and occasionally in humans. Few recent studies have determined the infection status of this fluke in Korea. In August 2015, we collected 402 samples of freshwater snails at Hoenggye-ri (upper stream) and Suha-ri (lower stream) of Song-cheon (stream) in Daegwalnyeong-myeon, Pyeongchang-gun in Gangwon-do (Province) near many large cattle or sheep farms. F. hepatica infection was determined using PCR on the nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 402 samples, F. hepatica 1TS-2 marker was detected in 6 freshwater snails; thus, the overall prevalence in freshwater snails was 1.5%. The prevalence varied between collection areas, ranging from 0.0% at Hoenggye-ri to 2.9% at Suha-ri. However, F. gigantica ITS-2 was not detected in the 6 F. hepatica-positive samples by PCR. The nucleotide sequences of the 6 F. hepatica ITS-2 PCR-positive samples were 99.4% identical to the F. hepatica ITS-2 sequences in GenBank, whereas they were 98.4% similar to F. gigantica ITS-2 sequences. These results indicated that the prevalence of F. hepatica in snail intermediate hosts was 1.5% in Gangwon-do, Korea; however the prevalence varied between collection areas. These results may help us to understand F. hepatica infection status in natural environments.

• Parasites, Hosts and Diseases
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• v.52 no.3
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• pp.257-261
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• 2014

Toxoplasma 3 main clonal lineages are designated as type I, II, and III; however, atypical and mixed genotypes were also reported. This study was conducted for detection of Toxoplasma gondii genotypes in rats (Rattus rattus) in Riyadh region, Saudi Arabia. PCR test on T. gondii B1 gene was conducted on ELISA IgM positive samples for confirmation of the infection. However, genetic analysis of the SAG2 locus was performed to determine T. gondii genotypes using PCR-RFLP technique. PCR test on T. gondii B1gene showed that 22 (81.5%) out of the 27 ELISA IgM positive samples have T. gondii DNA. Genotypic analysis shows that, of the total 22 PCR positive samples, only 13 (59.1%) were of type II, 7 (31.8%) were of type III, and 2 (9.1%) were of an unknown genotype. It is obvious that the prevalence of both type II and III is high in rats. No reports have been available on T. gondii genotypes among rats in Riyadh region, and only little is known about its seroprevalence in rats. Future studies on T. gondii genotypes in rats using multi-locus markers is needed in Riyadh region, Saudi Arabia for better understanding of T. gondii pathogenesis and treatment in humans and animals.

• Horticultural Science & Technology
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• v.33 no.2
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• pp.242-250
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• 2015

Insertional mutagenesis induced by T-DNA or transposon tagging offers possibilities for analysis of gene function. However, its potential remains limited unless good methods for detecting the target locus are developed. We describe a PCR technique for efficient identification of DNA sequences adjacent to the inserted T-DNA in a higher plant, Chinese cabbage (Brassica rapa ssp. pekinensis). This strategy, which we named variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR), was designed by modifying a single-step annealing-extension PCR by including a touch-up PCR protocol and using long gene-specific primers. Amplification efficiency of this PCR program was significantly increased by employing an autosegment extension method and linked sequence strategy in nested long gene-specific primers. For this technique, arbitrary degenerate (AD) primers specific to B. rapa were designed by analyzing the Integr8 proteome database. These primers showed higher accuracy and utility in the identification of flanking DNA sequences from individual transgenic Chinese cabbages in a large T-DNA inserted population. The VA-TAIL PCR method described in this study allows the identification of DNA regions flanking known DNA fragments. This method has potential biotechnological applications, being highly suitable for identification of target genomic loci in insertional mutagenesis screens.

• Food Science of Animal Resources
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• v.26 no.3
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• pp.394-401
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• 2006

Forty Clostridium perfringens isolates were obtained from twelve animal products, following the examination of eighty six beef, pork, broiler chicken and salami meat products, and eleven milk powder products. There were 21 isolates from salami stored at $25^{\circ}C$, 3 isolates from pork, 4 isolates from beef, 9 isolates from broiler chicken, and 3 isolates from milk powder. Only the cpa gene encoding a toxin among the 5 toxin genes tested (cpa, cpb, etx, iap, and cpe) was detected in all forty isolates, suggesting contamination with C. perfringens type A. DNA fingerprinting analysis using PCR of the tRNA intergenic spacer (tDNA-PCR) and the 16S-23S internal transcribed spacer (ITS-PCR), and randomly amplified polymorphic DNA (RAPD) analysis were attempted to differentiate the isolates. RAPD analysis was the most discriminating method among the three PCR analyses. Isolates from the same products tended to show similar RAPD patterns. Antimicrobial susceptibility tests showed that some isolates from broiler chickens had the same antibiogram with multiple resistance to streptomycin, colistin, and ciprofloxacin. Antibiograms were similar between isolates from the same livestock products, but differed considerably between the products.

• The Korean Journal of Mycology
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• v.40 no.1
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• pp.11-18
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• 2012

This study was carried out to identify the genetic diversity of Penicillium isolates that were isolated from pears with postharvest decay in storage. URP-PCR was used to detect DNA diversity of 84 Penicillium isolates. Based on URP-PCR profiles, 18 Penicillium isolates were selected and their PCR polymorphic bands were produced by additional primers URP1F, URP2R, URP2F, and URP4R. UPGMA cluster analysis using the polymorphic bands showed four clustered groups and futhermore cultural and morphological features characterized the 18 Penicillium isolates. Group 1 was dominant, which occupies 70% in the four clustered groups and identified as P. expansum based on ITS sequence and morphological features.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1146-1153
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• 2024

The increasing economic losses associated with growth retardation caused by Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid shrimp, require effective monitoring. The internal transcribed spacer (ITS)-1 region, the non-coding region of ribosomal clusters between 18S and 5.8S rRNA genes, is widely used in phylogenetic studies due to its high variability. In this study, the ITS-1 region sequence (~600-bp) of EHP was first identified, and primers for a polymerase chain reaction (PCR) assay targeting that sequence were designed. A newly developed nested-PCR method successfully detected the EHP in various shrimp (Penaeus vannamei and P. monodon) and related samples, including water and feces collected from Indonesia, Thailand, South Korea, India, and Malaysia. The primers did not cross-react with other hosts and pathogens, and this PCR assay is more sensitive than existing PCR detection methods targeting the small subunit ribosomal RNA (SSU rRNA) and spore wall protein (SWP) genes. Phylogenetic analysis based on the ITS-1 sequences indicated that the Indonesian strain was distinct (86.2% nucleotide sequence identity) from other strains collected from Thailand and South Korea, and also showed the internal diversity among Thailand (N = 7, divided into four branches) and South Korean (N = 5, divided into two branches) samples. The results revealed the ability of the ITS-1 region to determine the genetic diversity of EHP from different geographical origins.

• Biomedical Science Letters
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• v.26 no.4
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• pp.275-287
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• 2020

Since its introduction in the forensic field, quantitative PCR (qPCR) has played an essential role in DNA analysis. Quality of DNA should be evaluated before short tandem repeat (STR) profiling to obtain reliable results and reduce unnecessary costs. To this end, various human DNA quantification kits have been developed. Among these kits, the PowerQunat® System was designed not only to determine the total amount of human DNA and human male DNA from a forensic evidence item, but also to offer data about degradation of DNA samples. However, a crucial limitation of the PowerQunat® System is its high cost. Therefore, to minimize the cost of DNA quantification, we evaluated kit performance using a reduced volume of reagents (1/2-volume) using DNA samples of varying types and concentrations. Our results demonstrated that the low-volume method has almost comparable performance to the manufacturer's method for human DNA quantification, human male DNA quantification, and DNA degradation index. Furthermore, using a reduced volume of regents, it is possible to run 2 times more reactions per kit. We expect the proposed low-volume method to cut costs in half for laboratories dealing with large numbers of DNA samples.