• The Plant Pathology Journal
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• 제26권4호
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• pp.409-416
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• 2010

Fusarium species, a large group of plant pathogens, potentially pose quarantine concerns worldwide. Here, we focus on the development of a method for detecting four Fusarium species in quarantined plants in Korea: F. solani f. sp. cucurbitae, F. stilboides, F. redolens, and F. semitectum var. majus. Species-specific primers were designed from the nucleotide sequences of either the translation elongation factor-1 alpha (TEF1) gene or RNA polymerase II subunit (RPB2) gene. Two different primer sets derived from TEF1, all specific to F. solani f. sp. cucurbitae, were able to differentiate the two races (1 and 2) of this species. A set of nested primers for each race was designed to confirm the PCR results. Similarly, two primer sets derived from RPB2 successfully amplified specific fragments from five F. stilboides isolates grouped within a single phylogenetic clade. A specific TEF1 primer set amplified a DNA fragment from only four of the 12 F. redolens strains examined, which were grouped within a single phylogenetic clade. All of the F. semitectum var. majus isolates could be specifically detected with a single RPB2 primer set. The specificity of the primer sets developed here was confirmed using a total of 130 Fusarium isolates.

• International Journal of Oral Biology
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• 제38권4호
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• pp.181-188
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• 2013

The presence of distinct bacterial species is found to be dependent on age, diet, and disease. We compared the detection rate of several oral bacterial strains in a cohort of 36 subjects including healthy volunteers, periodontal patients, and oral cancer patients. Gargling samples were obtained from these subjects from which DNA was then extracted. Specific primers for 29 bacterial species were used for PCR detection. In the oral cancer patients, Capnocytophaga ochracea, Gemella morbillorum, and Streptococcus salivarius were detected more frequently compared with the healthy volunteers and periodontitis patients. Fusobacterium nucleatum/ polymorphym and Prevotella nigrescens were significantly less prevalent in oral cancer patients than the other groups. In periodontitis patients, Porphyromonas gingivalis and Treponema denticola were more frequently found compared with the healthy volunteers. In the healthy volunteer group, Peptostreptococcus anaerobius was more frequently found than the other groups. The detection rate of several oral bacterial species was thus found to differ between healthy volunteers, periodontitis patients and oral cancer patients.

• Fisheries and Aquatic Sciences
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• 제7권3호
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• pp.122-129
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• 2004

Harmful Cochlodinium polykrikoides is a notorious harmful algal bloom (HAB) species that is causing mass mortality of farmed fish along the Korean coast with increasing frequency. We analyzed the sequence of the large subunit (LSD) rDNA D1-D3 region of C. polykrikoides and conducted phylogenetic analyses using Bayesian inference of phylogeny and the maximum likelihood method. The molecular phylogeny showed that C. polykrikoides had the genetic relationship to Amphidinium and Gymnodinium species supported only by the relatively high posterior probabilities of Bayesian inference. Based on the LSU rDNA sequence data of diverse dinoflagellate taxa, we designed the C. polykrikoides-specific PCR primer set, CPOLY01 and CPOLY02 and developed PCR detection assays for its sensitive, accurate HAB monitoring. CPOLY01 and CPOLY02 specifically amplified C. polykrikoides and did not cross-react with any dinoflagellates tested in this study or environmental water samples. The effective annealing temperature $(T_{p})$ of CPOLY01 and CPOLY02 was $67^{\circ}C$. At this temperature, the conventional and nested PCR assays were sensitive over a wide range of C. polykrikoides cell numbers with detection limits of 0.05 and 0.0001 cells/reaction, respectively.

• Journal of Microbiology and Biotechnology
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• 제13권5호
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• pp.794-799
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• 2003

Enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Fusarium species, known as harmful fungi in food. One of the hybridoma cell lines (lB8) which produced a monoclonal antibody (Mab) specific to Fusarium extracellular polysaccharide (EPS) was screened and the Mab was produced and purified. A detection limit of the sandwich ELISA against F. moniliforme EPS was $0.001\;\mu\textrm{g}/ml$ in the standard curve. Among the 59 strains tested, most Fusarium species showed hight reactivity with Mab lB8, even when the culture broths were diluted 100,000 times. On the other hand, the other genera, except A. versicolor and Trichoderma viride, had no reactivity. When 1 to $100\;\mu\textrm{g}$ of F. moniliforme EPS was spiked into rice, potato, and mandarine orange, the average recoveries were 151%, 84%, and 94%, respectively, determined by sandwich ELISA. The correlation coefficients between the EPS levels determined by sandwich ELISA and the dry mycelial weight of the liquid culture of F. moniliforme, as well as between the EPS and colony forming unit in solid culture of potato, were 0.97 and 0.91, respectively.

• 식물병연구
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• 제20권1호
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• pp.21-24
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• 2014

Plasmodiophora brassicae는 십자화과 작물에 뿌리혹병을 일으키는 주요 병원균이다. 본 연구에서는 뿌리혹병균의 신속 정확한 검출을 위해서 뿌리혹병균에 대한 새로운 종 특이적 프라이머를 개발하고자 하였다. 새롭게 개발된 프라이머들은 10종의 주요 토양병원균을 비롯하여 기주인 배추 DNA와는 반응하지 않고 P. brassicae와만 반응하는 특이성을 갖고 있었다. 그 가운데 Primer ITS1-1/1-2는 민감도 검정 결과, 10 spores/ml의 DNA까지 검출이 가능함으로써, first round PCR용임에도 불구하고 이전의 검출법 보다 감도가 높고 정확한 결과를 얻었다. Quantitative real-time PCR로 분석할 경우에는 더 적은 수의 포자까지 안정적으로 검출해 낼 수 있어 새로운 P. brassicae 종 특이적 프라이머로서의 유용성을 확인할 수 있었다.

• 한국약용작물학회지
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• 제26권1호
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• pp.26-31
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• 2018

Background: Angelica gigas, A. sinensis, and A. acutiloba are commercially important in the herbal medicine market, and among them, A. gigas has the highest economic value and price. However, their similar morphological traits are often used for fraud. Despite their importance in herbal medicine, recognition of the differences between Angelica species is currently inadequate. Methods and Results: A multiplex polymerase chain reaction (PCR) method was developed for direct detection and identification of A. gigas, A. sinensis, and A. acutiloba. The gene for the distinction of species was targeted at ITS in the nucleus and trnC-petN gene in chloroplasts. The optimized multiplex PCR in the present study utilized each Angelica species-specific primer pairs. Each primer pair yielded products of 229 base pairs (bp) for A. gigas, 53 bp for A. sinensis, 170 bp for A. acutiloba. Additionally non-specific PCR products were not detected in similar species by species-specific primers. Conclusions: In the present study, a multiplex-PCR assay, successfully assessed the authenticity of Angelica species (A. gigas, A. sinensis, and A. acutiloba). and whole genome amplification (WGA) was performed after DNA extraction to identify, the species in the product. The detection method of raw materials developed in the present study could be applied to herbal medicine and health functional food management.

• 현장농수산연구지
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• 제24권3호
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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.

• 동아시아식생활학회지
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• 제18권5호
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• pp.753-759
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• 2008

본 연구는 식품 샘플에서 단시간 내에 간단한 방법으로 Campylobacter jejuni를 검출하기 위하여 10가지의 Campylobacter genus-specific primer와 C. jejuni species-specific oligonucleotide를 제작하였고, amplification efficiency test를 통하여 4종으로 축소한 후 다시 specificity, sensitivity analysis를 통하여 최종적으로 CB4, CJ1 2종의 oligonucleotide primer를 선별하였다. 선별된 oligonucleotide primer는 각각 Campylobacter genus specific, Campylobacter jejuni에 대한 species specific한 특성을 지닌다. 또한, sensitivity analysis를 통하여 isolated colony에서 reaction tube당 $10^0{\sim}10^1$까지의 detection limit을 확보하였다. 육류 시료에서는 Sensitivity가 $10^1{\sim}10^2$으로 떨어지는 양상을 보였으며, 이는 쇠고기나 돼지고기에 존재하는 hemoglobin이나 immunoglobulin 등의 PCR inhibitor의 영향에 의한 것으로 추정된다.

• Journal of Microbiology and Biotechnology
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• 제33권7호
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• pp.964-972
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• 2023

Bacteriophage endolysins are peptidoglycan hydrolases composed of cell binding domain (CBD) and an enzymatically active domain. A phage endolysin CBD can be used for detecting bacteria owing to its high specificity and sensitivity toward the bacterial cell wall. We aimed to develop a method for detection of Enterococcus faecalis using an endolysin CBD. The gene encoding the CBD of ECP3 phage endolysin was cloned into the Escherichia coli expression vector pET21a. A recombinant protein with a C-terminal 6-His-tag (CBD) was expressed and purified using a His-trap column. CBD was adsorbed onto epoxy magnetic beads (eMBs). The bacterial species specificity and sensitivity of bacterial binding to CBD-eMB complexes were determined using the bacterial colony counting from the magnetic separations after the binding reaction between bacteria and CBD-eMB complexes. E. faecalis could bind to CBD-eMB complexes, but other bacteria (such as Enterococcus faecium, Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, Streptococcus mutans, and Porphyromonas gingivalis) could not. E. faecalis cells were fixed onto CBD-eMB complexes within 1 h, and >78% of viable E. faecalis cells were recovered. The E. faecalis recovery ratio was not affected by the other bacterial species. The detection limit of the CBD-eMB complex for E. faecalis was >17 CFU/ml. We developed a simple method for the specific detection of E. faecalis using bacteriophage endolysin CBD and MBs. This is the first study to determine that the C-terminal region of ECP3 phage endolysin is a highly specific binding site for E. faecalis among other bacterial species.

• Journal of Microbiology and Biotechnology
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• 제19권3호
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• pp.265-270
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• 2009

An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCR-RFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.