• The Plant Pathology Journal
• /
• 제35권6호
• /
• pp.654-661
• /
• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

• 한국균학회지
• /
• 제36권2호
• /
• pp.138-143
• /
• 2008

토마토 잿빛곰팡이병균(B. cinerea)은 비닐하우스에서 재배할 때 토마토의 꽃과 줄기의 감염을 통해 매우 심각한 피해를 입힌다. 이 연구에서 토마토에 발병하는 잿빛 곰팡이병균의 검출 및 종 동정을 위해 새로운 종 특이적 primer set가 개발되었다. 종 특이적 primer(BTF1/BTR1)는 B. cinerea와 유전적으로 매우 유사한 진균들의 pyruvate carboxylase(pyc) 유전자 내부의 변이영역으로부터 설계되었다. 10개의 다른 기주식물에서 분리된 13균주의 모든 B. cinerea에서 112 bp 크기의 PCR 산물들이 만들어졌다. 그러나 6종의 다른 Botrytis 속균, 4종의 Botryotinia 속균, 5종의 Sclerotinia 속균 및 그 이외 16속의 다른 식물병원균들에 대해서는 PCR 반응이 나타나지 않았다. 종 특이적 primer의 반응민감도 한계는 대략 2 pg이었다. 자연상태에서 B. cinerea에 감염된 토마토 식물체와 인공적으로 접종된 식물체로부터 종 특이적 primer를 활용한 병원균의 PCR 검출이 이루어졌다. 이 연구결과로 미루어 새롭게 개발된 primer는 높은 반응민감도와 종 특이성을 나타내 추후 토마토 잿빛곰팡이병의 빠른 진단 및 병원균의 정확한 동정에 활용될 수 있을 것으로 판단된다.

• Journal of Ginseng Research
• /
• 제43권1호
• /
• pp.1-9
• /
• 2019

Background: Korean ginseng is an important cash crop in Asian countries. However, plant yield is reduced by pathogens. Among the Ilyonectria radicicola-species complex, I. mors-panacis is responsible for root-rot and replant failure of ginseng in Asia. The development of new methods to reveal the existence of the pathogen before cultivation is started is essential. Therefore, a quantitative real-time polymerase chain reaction method was developed to detect and quantify the pathogen in ginseng soils. Methods: In this study, a species-specific histone H3 primer set was developed for the quantification of I. mors-panacis. The primer set was used on DNA from other microbes to evaluate its sensitivity and selectivity for I. mors-panacis DNA. Sterilized soil samples artificially infected with the pathogen at different concentrations were used to evaluate the ability of the primer set to detect the pathogen population in the soil DNA. Finally, the pathogen was quantified in many natural soil samples. Results: The designed primer set was found to be sensitive and selective for I. mors-panacis DNA. In artificially infected sterilized soil samples, using quantitative real-time polymerase chain reaction the estimated amount of template was positively correlated with the pathogen concentration in soil samples ($R^2=0.95$), disease severity index ($R^2=0.99$), and colony-forming units ($R^2=0.87$). In natural soils, the pathogen was recorded in most fields producing bad yields at a range of $5.82{\pm}2.35pg/g$ to $892.34{\pm}103.70pg/g$ of soil. Conclusion: According to these results, the proposed primer set is applicable for estimating soil quality before ginseng cultivation. This will contribute to disease management and crop protection in the future.

• Journal of Microbiology
• /
• 제44권1호
• /
• pp.92-97
• /
• 2006

We developed an mPCR assay for the simultaneous detection, in one tube, of Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Listeria monocytogenes using species-specific primers. The mPCR employed the E. coli O157:H7 specific primer Stx2A, Salmonella spp. specific primer Its, S. aureus specific primer Cap8A-B and L. monocytogenes specific primer Hly. Amplification with these primers produced products of 553, 312, 405 and 210 bp, respectively. All PCR products were easily detected by agarose gel electrophoresis, and the sequences of the specific amplicons assessed. Potential pathogenic bacteria, in laboratory-prepared and four commercially available kimchi products, were using this mPCR assay, and the amplicons cloned and sequenced. The results correlated exactly with sequences derived for amplicons obtained during preliminry tests with known organisms. The sensitivity of the assay was determined for the purified pathogen DNAs from four strains. The mPCR detected pathogen DNA at concentrations ranging from approximately 0.45 to $0.05\;pM/{\mu}l$. Thus, this mPCR assay may allow for the rapid, reliable and cost-effective identification of four potentially pathogens present in the mixed bacterial communities of commercially available kimchi.

• Proceedings of the National Institute of Ecology of the Republic of Korea
• /
• 제4권4호
• /
• pp.154-158
• /
• 2023

The Asiatic black bear, Ursus thibetanus, is among the most threatened or endangered species in Asia. For its conservation and management, sex identification of U. thibetanus using non-invasive samples (e.g., hair and/or feces) is potentially valuable. In this study, a non-invasive molecular method for sex identification of U. thibetanus samples collected from various countries was first utilized, and it was based on polymerase chain reaction (PCR) amplification of the amelogenin gene via PCRs. Thirty-three bear DNA samples, extracted not only from blood (n=9) but also from hair (n=18) and feces (n=6), were used. We performed sex-specific PCR amplifications of the amelogenin gene using a primer set, SE47 and SE48. The primer set could successfully amplify a single X-specific band for females and both X- and Y-specific bands for males from all blood (100%) and hair (100%) samples. In addition, the primer set could distinguish the sex of bears in four out of a total of six fecal samples (approximately 67%). This study's findings suggest that this molecular method can be applied to sex identification of Asiatic black bears from various Asian regions using non-invasive samples, such as hair and feces.

• 미생물학회지
• /
• 제39권1호
• /
• pp.36-39
• /
• 2003

본 연구에서는 16S rDNA복합중합효소연쇄반응을 이용하여 중이 삼출액에서 미생물병인원에 대한 특성을 알아보았다. 중이염환자의 중이 삼출액에서의 미생물 병인원은 주로 streptococcus pneumoniae, Haemophilus influenzae와 Moraxella catarrhalis이다. 26명의 환자로부터 39개의 중이염의 삼출액을 얻었고, 중이 삼출액에서 DNA를 추출하였다. PCR은 16S rDNA의 C4 region에서 21 base pair의 common primer와 각각 bacterium specific primers [(i) Haemophilus-specific primer (ii) Moraxella-specific primer and (iii) Streptococcus-specific primer]를 이용하여 수행하였다. 39 개의 중이염의 삼출액 시료 중에서, H. influenzae가 24 개(61.5%) 검출되었고, M. catarrhalis는 10 개(25.6%), S.pneumoniae는 3개 (7.7%)가 검출되었다. 16s rDNA 복합중합효소연쇄 반응 진단 결과,11 개(28%)의 중이삼출액 시료에서 음성을 나타내었다. 중이염의 중복감염은 9개의 중이 삼출액시료에서 관찰되었고, 이들은 모두 H.influenzae 와 M. catarrhalis 에 의한 중복감염이었다. 본 연구에서 저자 등은 165 rDNA 복합중합효소연쇄반응이 병원성 미생물을 빠르게 진단하고, 중이삼출액의 미생물 병인론을 추적할 수 있는 좋은 방법으로 제시하는 바이다.

• BMB Reports
• /
• 제37권2호
• /
• pp.213-222
• /
• 2004

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

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

• 미생물학회지
• /
• 제48권4호
• /
• pp.309-313
• /
• 2012

본 연구는 multiplex PCR 방법을 적용하여 질 내부에 서식하는 유산균을 동시에 그리고 신속하게 검출하는 방법을 개발하는 것을 목적으로 수행되었다. 세균의 감염에 의한 질염 증상이 없는 건강한 20대 여성 166명으로부터 질 분비물을 채취하였다. 건강한 여성의 질 내부에 서식하는 것으로 확인된 Lactobacillus 속에 속하는 6종의 유산균을 종 특이 multiplex PCR primer를 제작하기 위해 선정하였다. Multiplex primer I은 L. iners, L. crispatus, L. gasseri를 선택적으로 검출하기 위해 특성화하였고 multiplex primer II는 L. acidophilus, L. jensenii, L. fermentum를 선택적으로 검출하기 위해 특성화하였다. Multiplex PCR 기술을 이용하여 분석한 결과 L. crispatus (77%)가 가장 높은 빈도로 검출되었고 L. acidophilus (57%)과 L. jensenii (57%)는 상대적으로 높은 빈도로 검출되었다. L. iners (59%)는 L. acidophilus (57%)와 L. jensenii (57%) 보다 높은 빈도로 검출되었지만 항생제 치료 과정 후 또는 세균성 질염의 경우에도 발견되는 균종으로 건강한 여성에게서 주로 서식하는 종이라고 결론 내리기는 어렵다. 결론적으로, 특정 유산균의 특이적인 primer를 이용한 multiplex PCR 기술은 질의 건강상태의 변화 또는 질의 질병으로부터 회복 과정을 예측할 수 있는 도구로서 유용할 것으로 생각된다.

• Journal of Microbiology and Biotechnology
• /
• 제27권5호
• /
• pp.909-915
• /
• 2017

Bifidobacterium bifidum BGN4 (BGN4) has many proven beneficial effects, including antiallergy and anticancer properties. It has been commercialized and used in several probiotic products, and thus strain-specific identification of this strain is very valuable for further strain-dependent physiological study. For this purpose, we developed novel multiplex polymerase chain reaction (PCR) primer sets for strain-specific detection of BGN4 in commercial products and fecal samples of animal models. The primer set was tested on seven strains of B. bifidum and 75 strains of the other Bifidobacterium species. The BGN4-specific regions were derived using megaBLAST against genome sequences of various B. bifidum databases and four sets of primers were designed. As a result, only BGN4 produced four PCR products simultaneously whereas the other strains did not. The PCR detection limit using BGN4-specific primer sets was $2.8{\times}10^1CFU/ml$ of BGN4. Those primer sets also detected and identified BGN4 in the probiotic products containing BNG4 and fecal samples from a BGN4-fed animal model with high specificity. Our results indicate that the PCR assay from this study is an efficient tool for the simple, rapid, and reliable identification of BGN4, for which probiotic strains are known.