• 제목/요약/키워드: Species-specific PCR

검색결과 649건 처리시간 0.027초

URP-PCR 다형성에 의한 국내 느타리버섯 품종의 유전적 특성 분석 (Genetic Analysis of Cultivars in Pleurotus spp. of Korea by URP-PCR Polymorphism)

  • 김종군;임선화;이대성;지정현;서건식;주영철;강희완
    • 한국균학회지
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    • 제35권2호
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    • pp.61-67
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    • 2007
  • 일반 느타리(P. ostreatus) 59품종, 사철느타리(P. florida) 2품종, 여름느타리(P. sajor-caju) 1품종, 전복느타리(P. abalonus) 1품종, 큰 느타리(P. eryngii) 2품종을 포함 하는 국내 등록된 총 65느타리버섯 품종이 URP-PCR다형성 분석에 적용되었다. 12종류의 URP primer 중 6종류의 URP primer가 품종간의 PCR 다형성 분석에 유효하였으며, URP2F primer는 높은 PCR 다형성 밴드를 형성하면서 품종간 PCR 다형성을 15 type으로 분류할 수 있었다. URP2F, URP6R, URP4R, URP2R에 의해 생성된 느타리 품종의 PCR다형성 밴드가 유전적 유사도 산출에 이용되어 UPGMA cluster분석을 적용 dendrogram을 작성하였다. P. ostreatus의 품종군은 group 1에서 group 5까지를 포함하고 있었으며, 그룹간에 70% 이상의 유전적 유연관계를 보였으며 기 장려품종으로 보급된 원형느타리 1, 2, 3호와 춘추 1, 2호 농기2-1, 농기201, 농기202 등 8품종은 group 1에서 4에 포함되어 있었다. group 5는 수한 및 신농 품종군이 밀접한 유전적 유사도를 보여 특징적인 품종군을 이루고 있었다. outside group으로서는 전복느타리, 큰 느타리, 여름느타리, 백송이가 group 6과 group 7에 포함되었다.

Detection of Campylobacter jejuni in food and poultry visors using immunomagnetic separation and microtitre hybridization

  • Simard, Ronald-E.
    • 한국어업기술학회:학술대회논문집
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    • 한국어업기술학회 2000년도 춘계수산관련학회 공동학술대회발표요지집
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    • pp.71-73
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    • 2000
  • Campylobacter jejuni is most frequently identified cause of cause of acute diarrhoeal infections in developeed countries, exceeding rates of illness caused by both salmonella and shigilla(Skirrow, 1990 ; Lior 1994). Previous studies on campylobacter jejuni contamination of commercial broiler carcasses in u.s.(Stern, 1992). Most cases of the disease result from indirect transmission of Campylobactor from animals via milk, water and meat. In addition to Campylobactor jejuni. the closely relates species Campylobactor coli and Campylobactor lari have also been implicated as agents of gastroenteritis in humans. Campylobactor coli represented only approximately 3% of the Campylobactor isolates from patients with Campylobactor enteritis(Griffiths and Park, 1990) whereas Campylobactor coli is mainly isolated from pork(Lmmerding et al., 1988). Campylobactor jejuni has also been isolated from cases of bacteremia, appendicitis and, recently, has been associated with Guillai-Barre syndrome(Allos and Blaser, 1994; von Wulffen et al., 1994; Phillips, 1995). Studies in volunteers indicated that the infectious dose for Campylobactor jejuni is low(about 500 organisms)(Robinson, 1981). The methods traditionally used to detect Campylobactor ssp. in food require at least two days of incubation in an enrichment broth followed by plating and two days of incubation on complex culture media containing many antibiotics(Goossens and Butzler, 1992). Finnaly, several biochemical tests must be done to confirm the indentification at the species level. Therfore, sensitive and specific methods for the detection of small numbers of Campylobactor cells in food are needed. Polymerase chain reaction(PCR) assays targeting specific DNA sequences have been developed for the detection of Campylobactor(Giesendorf and Quint, 1995; Hemandex et al., 1995; Winter and Slavidk, 1995). In most cases, a short enrichment step is needed to enhance the sensitivity of the assay prior to detection by PCR as the number of bacteria in the food products is low in comparison with those found in dinical samples, and because the complex composition of food matrices can hinder the PCR and lower its sensitivity. However, these PCR systems are technically demanding to carry out and cumbersome when processing a large number of samples simutaneously. In this paper, an immunomagnetic method to concentrate Campylobactor cells present in food or clinical samples after an enrichment step is described. To detect specifically the thermophilic Campylobactor. a monoclonal antibody was adsorbed on the surface of the magnetic beads which react against a major porin of 45kDa present on the surface of the cells(Huyer et al., 1986). After this partial purification and concentration step, detection of bound cells was achieved using a simple, inexpensive microtitre plate-based hybridization system. We examined two alternative detection systems, one specific for thermophilic Campylobactor based on the detection of 23S rRNA using an immobilized DNA probe. The second system is less specific but more sensitive because of the high copy number of the rRNA present in bacterial cell($10^3-10^4$). By using specific immunomagnetic beads against thermophilic Campylobactor, it was possible to concentrate these cells from a heterogeneous media and obtain highly specific hybridization reactions with good sensitivity. There are several advantages in using microtitre plates instead of filter membranes or other matrices for hybridization techniques. Microtitre plates are much easier to handle than filter membranes during the adsorption, washing, hybridization and detection steps, and their use faciilitates the simultanuous analysis of multiple sample. Here we report on the use of a very simple detection procedure based on a monoclonal anti-RNA-DNA hybrid antibody(Fliss et al., 1999) for detection of the RNA-DNA hybrids formed in the wells.

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PCR을 이용한 육류 내 Salmonella sp. 및 Salmonella Typhimurium 분리 검출 (Selective Detection of Salmonella sp. and Salmonella Typhimurium in Meat by Polymerase Chain Reaction)

  • 주종원;홍경표;김용휘;조상범
    • 동아시아식생활학회지
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    • 제19권2호
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    • pp.295-300
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    • 2009
  • 본 연구에서는 육류 식품 시료에서 단시간 내에 살모넬라를 검출하기 위하여 PCR을 이용한 검출용 프라이머들의 특이성과 민감성을 평가하였다. 실험에 사용된 프라이머들은 Salmonella Typhimurium의 mdh와 invA 유전자의 염기서열에 기초하여 제작되었다. 각각의 primer들의 검출 감도를 평가하여 최종적으로 broad spectrum primer SLM1과 S. Typhimurium specific primer SLT4를 선발하였다. 또한, 시료에 오염된 병원균의 최소 검출량이 어느 정도인지를 확인하기 위하여 살모넬라 균수를 reaction tube당 $10^0{\sim}10^3$ cell까지 다양하게 하여 검출 감도를 측정한 결과, 최소 1 cell에서도 PCR 산물을 나타내어 검출 감도가 매우 우수한 것으로 나타났다. 살모넬라 균주를 혼합한 소고기와 돼지고기 시료에서 각각 프라이머들의 검출 감도를 평가한 결과, 증균하지 않은 시료 자체와 세균학적 방법으로 증균 배양한 시료 그리고 증균 배양된 시료로부터 세균의 DNA를 추출한 시료 간에 큰 차이를 나타내지 않았다. 이에 육류 식품에서 살모넬라 혹은 S. Typhimurium의 검출을 위한 프라이머로서 효율적으로 사용이 가능할 것으로 판단된다.

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Molecular Identification and Sequence Analysis of Coat Protein Gene of Ornithogalum mosaic virus Isolated from Iris Plant

  • Yoon, Hye-In;Ryu, Ki-Hyun
    • The Plant Pathology Journal
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    • 제18권5호
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    • pp.251-258
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    • 2002
  • A potyvirus was isolated from cultivated Iris plants showing leaf streak mosaic symptom. Reverse transcription and polymerase chain reaction (RT-PCR) product of 1 kb long which encoded partial nuclear inclusion B and N-terminal region of viral coat protein (CP) genes for potyviruses was successfully amplified with a set of potyvirus-specific degenerate primers with viral RNA samples from the infected leaves: The RT-PCR product was cloned into the plasmid vector and its nucleotide sequences were determined. The nucleotide sequence of a CDNA clone revealed that the virus was an isolate of Ornithogalum moseic virus (OrMV) based on BLAST search analysis and was denoted as OrMV Korean isolate (OrMV-Ky). To further characterize the CP gene of the virus, a pair of OrMV-specific primers was designed and used for amplification of the entire CP gene of OrMV-Kr, The virus was easily and reliably detected from virus-infected Iris leaves by using the RT-PCR with the set of virus-specific primers. The RT-PCR product of the CP gene of the virus was cloned and its sequences were determined from selected recombinant CDNA clones. Sequence analysis revealed that the CP of OrMV-Kr consisted of 762 nucleotides, which encoded 253 amino acid residues. The CP of OrMV-Ky has 94.1-98.0% amino acid sequence identities (20 amino acid alterations) with that of other three isolates of OrMV, Two NT rich potential N-glycosylation motif sequences, NCTS and NWTM, and a DAC triple box responsible for aphid transmission were conserved in CPs of all the strains of OrMV. The virus has 58.5-86.2% amino acid sequence identities with that of other 16 potyviruses, indicating OrMV to be a distinct species of the genus. OrMV-Ky was the most related with Pterostylia virus Yin the phylogenetic tree analysis of CP at the amino acid level. This is the first report on the occurrence of OrMV in Iris plants in Korea. Data in this study indicate that OrMV is found in cultivated Iris plants, and may have mixed infection of OrMV and Iris severe mosaic virus in Korea.

우리나라 일부 해안 지역 야생화들로부터 분리한 효모들의 분자 생물학적 동정 (Identification of Yeasts Isolated from Wild Flowers Collected in Coast Areas of Korea Based on the 26S rDNA Sequences)

  • 민진홍;이향범;이종수;김하근
    • 한국균학회지
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    • 제41권3호
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    • pp.185-191
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    • 2013
  • 국내 자연환경으로부터 다양한 효모들을 분리, 동정하고 나아가 이들로부터 유용물질을 생산하는 효모자원을 확보하기 위한 연구의 일환으로 우리나라 동해안, 서해안, 남해안에 서식하는 야생화들을 채집하여 이들로부터 효모들을 분리한 후 분자생물학적 방법으로 동정하였다. 동해시에서 수집한 야생화로부터는 Candida silvae 등을 포함한 15종에 속하는 27균주의 효모들을 분리하였다. 서해안의 대천시 해수욕장 주위 야생화에서는 Bulleromyces albus를 비롯한 17종 34균주가 분리, 동정되었다. 또한 남해안의 완도군 대문리 주위의 야생화들로부터는 Cryptococcus flavus를 포함하여 13종에 속하는 효모 22 균주들이 분리 동정되었다. 전체적으로 우리나라 동해안, 서해안, 남해안의 야생화로부터 모두 45종에 속하는 효모들 83균주를 분리, 동정하였다.

PCR-RAPD 기법에 의한 무지개송어 Genome DNA 의 다형현상 (Genomic Polymorphisms of Genome DNA by Polymerase Chain Reaction-RAPD Analysis Using Arbitrary Primers in Rainbow Trout)

  • Yoon, J.M.
    • 한국가축번식학회지
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    • 제23권4호
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    • pp.303-311
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    • 1999
  • 본 연구는 정자세포로부터 분리된 genome 내 DNA를 PCR 기법으로 증폭시킨 후 random amplified polymorphic DNA(RAPD) 분석을 통해 무지개송어의 품종 내 유전적 특성과 변이성을 해석하고 품종의 특이 유전적 표지를 개발하기 위해서 수행되었다. 20 종류의 primer를 사용하여 RAPD 양상을 검색한 후 다형현상의 출현빈도와 band 수에 기초하여 이들 중 6개의 primer를 선정하여 이용하였다. 그 중에 4개의 primer는 17개의 RAPD marker를 나타내었고, 그중 primer 당 8개인 48개 (28%)의 band 가 다형성을 보여주었다. 6개의 primer 중 4개는 개체들 사이에 다형성을 나타내는 band를 나타내었다. Bandsharing 의 경우 연어와 비교될 만큼 무지개송어는 3개의 특이적인 DNA marker를 가지고 있었다 (2.3. 2.0 및 1.3kb). 같은 무작위 primer를 이용해서 나타난 개별적인 band는 단일 primer 가 무지개송어의 정자핵 DNA의 경우 적어도 3개의 독립적인 genome 내 다형성을 탐지해 낼 수 있다는 것을 제시하고 있다. 이러한 primer에 의해서 나타난 RAPD 다형성은 개체식별을 위한 유전적 표지인자로서 사용될 수 있는 가능성을 제시하였으며, RAPD-PCR은 많은 어종에서 다형현상을 밝혀내는 기술이라 할 수 있다. 본 연구는 RAPD marker가 풍부하고 재현성이 있으며 RAPD 다형성을 지닌 marker를 사용하여 이러한 중요한 양식대상어종에서 미래의 gene mapping과 MAS 를 위한 기초를 제공해 줄 수 있다. RAPD 다형성은 어류의 품종 분화를 위한 유전적 표지로서 유용한 것으로 결론지을 수 있을 것이다.

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Discovery of a new primer set for detection and quantification of Ilyonectria mors-panacis in soils for ginseng cultivation

  • Farh, Mohamed El-Agamy;Han, Jeong A.;Kim, Yeon-Ju;Kim, Jae Chun;Singh, Priyanka;Yang, Deok-Chun
    • Journal of Ginseng Research
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    • 제43권1호
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    • pp.1-9
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    • 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.

Distribution of Rickettsia spp. in Ticks from Northwestern and Southwestern Provinces, Republic of Korea

  • Jiang, Ju;Choi, Yeon-Joo;Kim, Jeoungyeon;Kim, Heung-Chul;Klein, Terry A;Chong, Sung-Tae;Richards, Allen L.;Park, Hye-Jin;Shin, Sun-Hye;Song, Dayoung;Park, Kyung-Hee;Jang, Won-Jong
    • Parasites, Hosts and Diseases
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    • 제57권2호
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    • pp.161-166
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    • 2019
  • This study was done to characterize distribution of Rickettsia spp. in ticks in the northwestern and southwestern provinces in the Republic of Korea. A total of 2,814 ticks were collected between May and September 2009. After pooling, 284 tick DNA samples were screened for a gene of Rickettsia-specific 17-kDa protein using nested PCR (nPCR), and produced 88 nPCR positive samples. Of these positives, 75% contained 190-kDa outer membrane protein gene (ompA), 50% 120-kDa outer membrane protein gene (ompB), and 64.7% gene D (sca4). The nPCR products of ompA, ompB, and sca4 genes revealed close relatedness to Rickettsia japonica, R. heilongjiangensis, and R. monacensis. Most Rickettsia species were detected in Haemaphysalis longicornis. This tick was found a dominant vector of rickettsiae in the study regions in the Republic of Korea.

미토콘드리아 DNA 염기서열 변이를 이용한 인삼 종 판별 연구 (Analysis of Mitochondrial DNA Sequence and Molecular Marker Development for Identification of Panax Species)

  • 조익현;방경환;김영창;김장욱;신미란;문지영;노봉수;현동윤;김동휘;차선우;김홍식
    • 한국약용작물학회지
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    • 제21권2호
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    • pp.91-96
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    • 2013
  • This study describes the identification of Panax species using mitochondrial consensus primers. Initially, a total of thirty primers were tested in ten Korean ginseng cultivars and two foreign Panax species, P. quinquefolius and P. notoginseng. In the polymerase chain reaction (PCR) amplification results, three primers (cox1, nad1/2-3 and nad2/1-2) generated co-dominant polymorphic banding patterns discriminating Korean ginseng cultivars from P. quinquefolius and P. notoginseng. However, these primers could not generated polymorphisms among the Korean ginseng cultivars, and simply represented species-specific polymorphisms for P. quinquefolius and P. notoginseng. Primers PQ91 and PN418 were designed from the consensus sequence of nad1/2-3 region. Two banding patterns (A or B) were detected in PQ91. Korean ginseng cultivars and P. notoginseng shared the same banding pattern (A type) and P. quinquefolius was identified another banding pattern (B type). In the case of PN418, two banding patterns (A or B) were detected in the Korean ginseng cultivars and two foreign Panax species. Korean ginseng cultivars and P. quinquefolius shared the same banding pattern (A type) and P. notoginseng was identified another banding pattern (B type). The combination banding patterns of three Panax species, Korean ginseng cultivars (Panax ginseng C. A. Mey.), P. quinquefolius and P. notoginseng, was identified as 'AA', 'BA' and 'AB', respectively. Consequently, PQ91 and PN418 primer sets can be used to distinguish among Panax species.

Multiplex-PCR에 의한 먹는샘물 및 야채류로부터의 병원성 Yersinia enterocolitica의 신속검출 (Detection of Pathogenic Yersinia Enterocolitica in Drinking Water and Vegetables by Mutiplex-PCR)

  • 이택수;박부길;오덕환
    • 한국식품영양과학회지
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    • 제32권1호
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    • pp.35-41
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    • 2003
  • 본 연구는 식품에 존재하는 Y. enterocolitica균의 신속한 검출방법을 조사하기 위하여 이균에 특이적인 특이적인 ail, yst 및 virF 유전자와 Yersinia 속균을 구별하는 subgenus-specific Y16S primer를 도입하여 multiplex PCR을 수행하였으며 Y. enterocolitica균의 검출 민감도와 특이도 및 먹는 샘물과 야채류에서의 적용실험을 각각 조사하였다. PCR 특이성 실험에서는 Y enterorolitica ATCC 27729균은 355 bp(ail), 134 bp(yst) 및 200 bp(Y16S) 3종의 유전자에 대한 DNA 증폭밴드를 나타내었으며, Y. enteroculitica ATCC 9610 및 ATCC 23715는 yst와 Y16S 2종에만 증폭을 보였다 반면에 기타 비병원성 Yersinia균인 Y. frederikseni, Y. intemedia, Y kri-steneni, Y pseudotuberculosis 등은 Y16S에만 DNA밴드를 나타내었으나 기타 세균인 E. colt ATCC 25392, Shi. dysen-teri, S. aureus ATCC 25923, L. monocytognes ATCC 19111 균에서는 어떤 primer에서도 특이 DNA 밴드를 확인할 수 없었다 PCR 민감도는 다른 유전자에 비하여 yst 유전자가 Y. enterocolitica균에 가장 높은 민감도를 나타내었고, Y16S 유전자는 속과 종에 관계없이 높은 민감도를 나타내었다. 따가서, 먹샘물이나 야채류에 대한 병원성 Yersinia균의 지표유전자자로서 속을 대표하는 Y16S유전자와 종을 대표하는 yst유전자를 선정하였다. 수질 중 Y. enferocolitica의 검출한계를 알아보기 위하여 일반세균수가 3600 CFU/mL정도 함유된 먹는 샘물의 경우, DNA를 분리하지 않고 단순 열처리한 배양액을 DNA주형으로 사용하여 multiplex-PCR을 실시한 결과, Y16S 와 yst 유전자 모두 7$\times$$10^1$ CFU/mL 수준가지 검출할 수 있었으며, 일반세균수가 2.5$\times$$10^{5}$CFU/g 정도 함유된 상치는 7 및 7$\times$$10^1$CFU/g, 일반세균이 7.1$\times$$10^4$CFU/g정도 함유된 양송이버섯은 7 및 7$\times$$10^1$CFU/g 수준까지 검출할 수 있었다. 본 연구에서 나타난 바와 같이, 수질이나 야채류에서 Y16S와 yst 유전자를 혼합하여 Y. enterocolitica를 검출할 경우, DNA를 분리하지 않고 직접 whole cell을 lysate하여 DNA주형으로 사용하여도 2차 PCR을 수행할 경우에는 증균과정을 하지 않고도 민감도를 증진시키면서 신속하게 효율적으로 원하는 대상균을 검출할 수 있는 것으로 나타났다.다.