• Food Science and Biotechnology
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• 제17권6호
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• pp.1171-1177
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• 2008

This study was undertaken to classify Enterobacter sakazakii isolates from 13 powdered infant formula products, 25 powdered weaning diet products, and 33 weaning diet ingredients on polymerse chain reaction (PCR) methods. The numbers of the isolates from 1 powdered infant formula product, 7 powdered weaning diet products, and 6 weaning diet ingredients were 1, 14, and 8, respectively. The contaminated ingredients were 1 rice powder, 2 millet powders, 2 vegetable powders, and 1 fruit and vegetable premix. PCR with the primer of repetitive extragenic palindromic element (REP-PCR) and random amplification of polymorphic DNA(RAPD) were effective in discriminating among the isolates, but tRNA-PCR and PCR with the primer of l6S-23S internal transcribed spacer (ITS-PCR) were not. Some of E. sakazakii isolates from vegetable powders, fruit and vegetable premix, and millets powders were classified into the clonal groups based on the DNA patterns in the REP-PCR and RAPD analysis. A close genetic relationship among the isolates from some of the powdered weaning diet products and the rice powder was also detected in the cluster analysis based on the DNA patterns in RAPD.

• 한국축산식품학회지
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• 제29권5호
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• pp.647-652
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• 2009

유제품에 들어 있는 우유와 산양유를 동정하기 위해 미토콘드리아의 12S rRNA 유전자를 목표로 하는 primer을 이용하는 duplex PCR 분석을 적용하였다. 소와 산양의 특이성 primer을 이용한 duplex PCR 분석은 우유와 산양유 DNA에 대해 각각 233 bp와 326 bp의 특이성 단편을 나타냈다. Duplex PCR 분석이 라벨에 표시된 성분을 확인하기위하여 시중마트에서 구입한 15개 유제품에 적용하였다. Duplex PCR 분석 결과 4개 시유, 3개 요구르트, 1개 전지분유는 표시된 성분과 완전히 일치하였다. 그러나 7개의 조제분유 중 5개만 표시성분과 일치하고 2개 조제분유제품은 산양유와 우유가 각각 오염되어 있는 것으로 나타났다. 제안된 duplex PCR 분석은 산양유에 들어있는 우유를 0.1%까지 측정할 수 있는 민감하고 신속한 방법이다. Duplex PCR 분석은 유제품 속에 들어있는 우유와 산양유를 one-step 방법으로 동시에 탐지할 수 있다.

• The Plant Pathology Journal
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• 제31권1호
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• pp.25-32
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• 2015

Pseudomonas coronafaciens causes halo blight on oats and is a plant quarantine bacterium in many countries, including the Republic of Korea. Using of the certificated seed is important for control of the disease. Since effective detection method of P. coronafaciens is not available yet, PCR and TaqMan PCR assays for specific detection of P. coronafaciens were developed in this study. PCR primers were designed from the draft genome sequence of P. coronafaciens LMG 5060 which was obtained by the next-generation sequencing in this study. The PCR primer set Pc-12-F/Pc-12-R specifically amplified 498 bp from the 13 strains of P. coronafaciens isolated in the seven different countries (Canada, Japan, United Kingdom, Zimbabwe, Kenya, Germany, and New Zealand) and the nested primer set Pc-12-ne-F/Pc-12-ne-R specifically amplified 298 bp from those strains. The target-size PCR product was not amplified from the non-target bacteria with the PCR and nested primer sets. TaqMan PCR with Pc-12-ne-F/Pc-12-ne-R and a TaqMan probe, Pc-taqman, which were designed inside of the nested PCR amplicon, generated Ct values which in a dose-dependent manner to the amount of the target DNA and the Ct values of all the P. coronafaciens strains were above the threshold Ct value for positive detection. The TaqMan PCR generated positive Ct values from the seed extracts of the artificially inoculated oat seeds above 10 cfu/ml inoculation level. PCR and TaqMan PCR assays developed in this study will be useful tools to detect and identify the plant quarantine pathogen, P. coronafaciens.

• 식물병연구
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• 제11권1호
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• pp.48-55
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• 2005

Cylindrocarpon destructans는 인삼 및 수목에 뿌리썩음병을 일으키는 토양 전염병 식물병원균이다. 신속 정확한 검출 가능성을 알아보기 위하여 종 특이적인 primer와 nested PCR 기법을 활용하여 인삼 유묘로부터 뿌리썩음 병균 C. destructans로 2차 PCR증폭을 실시한 결과 병원성이 확인된 C.destructans에서만 400bp의 종특이적 증폭산물을 얻을 수 있었다. 종 특이성 primer 와 nested PCR 기법을 이용한 인삼뿌리썩음병균 DNA에 대한 반응 민감도는 최저 약 1fg으로 나타나 단 몇 개의 포자만 존재해도 검출이 가능하였다. 또한, nested PCR 기법은 실제 이병토양에 심었을 경우에도 C.destructans 에 감염된 인삼 유묘로부터만 정확하게 병원균을 검출해 내었다. 종특이적 primer 와 nested PCR 기법을 이용한 본 연구 결과는 실제 재배농가에서 인삼 경작시 뿌리썩음병 진단에 매우 유용하게 활용될 수 있을 것으로 판단된다.

• 한국미생물·생명공학회지
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• 제45권4호
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• pp.358-364
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• 2017

Asymmetric PCR preferentially amplifies one DNA strand for use in DNA hybridization studies. Linear-After-The-Exponential-PCR (LATE-PCR) is an advanced asymmetric PCR method which uses innovatively designed primers at different concentrations. This study aimed to optimise LATE-PCR parameters to produce single-stranded DNA of Candida spp. and Aspergillus spp. for detection via probe hybridisation. The internal transcribed spacer (ITS) region was used to design limiting primer and excess primer for LATE-PCR. Primer annealing and melting temperature, difference of melting temperature between limiting and excess primer and concentration of primers were optimized. In order to confirm the presence of single-stranded DNA, the LATE-PCR product was hybridised with digoxigenin labeled complementary oligonucleotide probe specific for each fungal genus and detected using anti-digoxigenin antibody by dot blotting. Important parameters that determine the production of single-stranded DNA in a LATE-PCR reaction are difference of melting temperature between the limiting and excess primer of at least $5^{\circ}C$ and primer concentration ratio of excess primer to limiting primer at 20:1. LATE-PCR products of Candida albicans, Candida parapsilosis, Candida tropicalis and Aspergillus terreus at up to 1:100 dilution and after 1 h hybridization time, successfully hybridised to respective oligonucleotide probes with no cross reactivity observed between each fungal genus probe and non-target products. For Aspergillus fumigatus, LATE-PCR products were detected at 1:10 dilution and after overnight hybridisation. These results indicate high detection sensitivity for single-stranded DNA produced by LATE-PCR. In conclusion, this advancement of PCR may be utilised to detect fungal pathogens which can aid the diagnosis of invasive fungal disease.

• 미생물학회지
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• 제43권3호
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• pp.179-185
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• 2007

본 연구는 polymerase chanin reaction(PCR)기법과 DNA enzyme-linked immunosorbent assay(DNA ELISA) 기법을 이용하여 토양내 식물병원균인 Ralstonia solanacearum를 검출하고자 하였다. 토양 시료로부터 분석에 사용될 R. solanacearum DNA를 추출하기 위하여 몇 가지 방법을 비교 평가한 결과 기존의 DNA 추출 방법에 비하여 Guanidin isothiocyanate와 Chelex-100 resin을 사용하는 방법 이 토양 내에 존재하는 다양한 중류의 반응 저해 물질과 R. solanacearum만의 고유한 PCR반응 저해물질들을 제거하는 데에 효과적이었다. R. solanacearum만을 특이적으로 검출하기 위해 fliC유전자 부위에 특이적인 몇 종의 primer들을 제작하였다. 이들 중 높은 민감도와 특이도를 나타내는 두 set의 primer RsolfliC(forward; 5-GAACGCCAACGGTGCGAACT-3 and reverse; 5-GGCGGCCTTCAGGGAGGTC-3, designed by J. $Sch\ddot{o}nfeld$ et al.)와 RS_247 (forward; 5-GGCGGTCTGTCGGCRG-3 and reverse; 5-CGGTCGCGTTGGCAAC-3, designed by this study)를 선정하여 nested PCR을 수행할 수 있도록 고안하였다. Nested PCR primer에 biotin을 표지하였고 nested PCR산물의 내부 서열과 특이적으로 교잡반응을 할 수 있는 probe를 제작하여 PCR 결과를 DNA-EIA반응으로 확인 분석할 수 있도록 하였다. Primary PCR과 nested PCR의 산물을 전기영동 상에서 확인한 결과, nested PCR이 약 $10^2$정도의 높은 민감도를 나타내었고 DNA-EIA의 경우 $10^2P{\sim}10^3$정도의 민감도를 상승시켜주는 것으로 확인되었다.

• Journal of Applied Biological Chemistry
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• 제49권4호
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• pp.143-147
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• 2006

A rapid PCR-based assay for detecting hepatitis B viral DNA(HBV DNA) in serum and plasma was developed using a new PCR instrument named GenSpector(TMC-1000, Samsung electronics). PCR was carried out using a chip-based platform, which enabled 50 PCR cycles with internal controls, and melting-curve analysis in 30 minutes. Verification of the amplified HBV DNA product and the internal control was based on specific melting temperatures(Tm) analysis, executed by the GenSpector software. Primers were designed within the region conserved through HBV genotypes A to F. The lower limit of detection was 840 copies/ml serum, conducted with serial dilutions of a HBV DNA positive control(ACCURUN 325 series 700, Boston Biomedica Inc.). The assay was also compared to another assay for HBV DNA(Versant HBV DNA 3.0 assay, Bayer HealthCare) for 200 samples(each 100 clinical negative and positive samples). The sensitivity and specificity were 100% matched. This rapid PCR-based assay is specific, reproducible, and enables qualitative detection of HBV DNA.

• 한국버섯학회지
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• 제2권3호
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• pp.145-148
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• 2004

이 연구는 약용버섯 번데기 동충하초로부터 특이적 자실체 형성 유전자를 선별하기 위하여 수행하였다. cDNA는 버섯의 분화 4단계 균사체, 원기, 미성숙 자실체, 성숙한 자실체로부터 분리한 각각의 total RNA를 이용하여 합성하였다. 특이적으로 발현된 유전자의 선별은 cDNA와 랜덤한 primer set을 이용한 DD-PCR에 의해 수행되었고, pGEM 클로닝 벡터를 이용하여 6개의 partial 유전자 서열을 확인하였다. 6개의 DD-PCR product는 보고된 유전자와 유사도를 확인하기 위해 NCBI BLAST search를 사용하여 GenBank에서 비교하였고, 6개의 유전자는 보고되지 않은 유전자임을 확인하였다.

• 대한미생물학회지
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• 제35권4호
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• pp.283-288
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• 2000

The PCR primer set JW21-JW22 of Weiss et al. (19), which was reported to amplify a 139-bp fragment of the l6S rRNA gene of Helicobacter pylori, has been recently used for the detection of H. pylori in clinical specimens. However, when we applied JW21-JW22 PCR to other members of the genus Helicobacter and unrelated microorganisms, all of these bacteria produced a 139-bp PCR product. Therefore, we designed a novel primer set, HPU185-HPL826, which produced a 642-bp amplicon of the l6S rRNA gene of H. pylori. Then we further examined the specificity of the novel PCR assay using Southern blot hybridization with an internal probe, HPP225. The PCR assay described in this study was shown to be highly sensitive and specific only to the H. pylori 16S rRNA gene sequences.

• 대한수의학회지
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• 제45권3호
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• pp.335-339
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• 2005

The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo, we performed a modified PCR-RFLP analysis of MC1R gene using single nucleotide polymorphism (SNP) within MC1R as a target. A size of 538 bp (537 bp for Hanwoo) was amplified by PCR, digested with Hpa II, and electrophoresed on a 1.5% agarose gel. A PCR product from Hanwoo showed a single band of 537 bp, whereas two fragments of 328 bp and 210 bp were detected in both Holstein and Black angus. The current result suggests that the PCR-RFLP using our primers and enzyme digestion system would be very accurate, easy and reproducible method to discriminate between Hanwoo and Holstein/Black angus meat.