• 한국식품영양학회지
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• 제17권3호
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• pp.254-259
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• 2004

Primer 20가지로 Listeria spp.에 대해 screening을 하여 병원균인 L. monocytogenes를 구별하게 하는 RAPD-PCR의 band pattern을 나타내는 10-mer random primer가 OPG-13이 라는 것을 알았다. OPG-13은 GC%가 70%이므로 계산상으로는 annealing 온도가 34$^{\circ}C$이다. 32～36$^{\circ}C$까지 다섯 가지의 온도로 annealing한 결과 L. monocytogenes만이 갖는 특정한 크기의 band가 역시 34$^{\circ}C$에서 형성됨을 알아냈고, 34$^{\circ}C$를 annealing 온도로 정하였다. Line 1부터 4까지 1. monocytogenes ATCC15313, 19111, 19112, 19113은 2개의 700 bp와 1500 bp band를 형성하였고 그 밖의 Listeria spp.들 Line 6부터 11까지 L. ivanovii ATCC 19119, L. grayi ATCC19120, L. murrayi ATCC25401, L. innocua ATCC33090, L. welshimeri ATCC35897, L. seeligeri ATCC35967은 약 2,000 ～ 2,300 bp크기 한 개의 band를 보여 병원성 균과 비병원성 균이 매우 확실하게 구분되는 band pattern이 나타나는 이러한 결과로 10-mer random primer인 OPG-13을 이용한 RAPD-PCR 방법이 병윈균인 L. monocytogenes를 검색하는데 이용될 수 있음을 확인할 수 있었다.

• 한국초지조사료학회지
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• 제37권2호
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• pp.168-175
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• 2017

옥수수 품종판별 마커 개발을 위하여 SNAP 방법을 변형하여 2bp 불일치 SNP PCR 방법을 옥수수 품종판별에 적용하였다. SNP 마커개발을 위하여 MaizeGDB 웹사이트(www.maizegdb.org)를 통해서 200 SNP 위치를 확인하였으며, 표준맵으로 알려진 B73 옥수수 게놈서열을 바탕으로 2bp 불일치 Primer을 디자인하였다. PCR 생성물은 200-500bp 사이에서 결정되었으며 SNP site가 있을시 PCR 생성물이 생성되지 않게 디자인 되었다. 선행연구에서 선발된 16개의 Primer조합을 이용해서 농촌진흥청에서 개발된 사료용 옥수수 10품종(강다옥, 광평옥, 다평옥, 안다옥, 양안옥, 신광옥, 장다옥, 청다옥, 평광옥, 평안옥)과 수입 사료용 옥수수 40품종과의 판별 가능성을 검정하였다. SNP PCR 결과를 바탕으로 한 Cluster분석에서 신광옥과 PI1395 그리고 몇몇 수입 사료용 옥수수를 제외하고는 모두 판별 가능한 것으로 검정되었다. SNP 통한 품종판별 최소조합수를 선발한 결과 강다옥은 IBM911과 IBM1798, 장다옥은 IBM440과 IBM549, 평강옥은 IBM440과 IBM1269, 평안옥은 IBM795와 IBM1601였다. 이는 SNP 마커 개발을 통해 빠르고, 손쉽게 품종판별이 가능한 마커로 활용가능하다는 것을 보여준다.

• 한국가금학회지
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• 제20권4호
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• pp.177-188
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• 1993

닭에서 적절한 성감별 방법을 개발하고 닭의 성분화 기작의 기초자료를 얻기 위하여 배아의 섬유아세포의 염색체를 분석하고, W 염색체 특이적인 반복염기서열과 50∼60％의 유사성을 보이는 random primer로 PCR 증폭을 실시하여 성을 판별하는 방법이 이용되었으며, 닭에서 성분화에 관련된 유전자를 분리하기 위해 W 염색체 특이적인 반복염기서열을 클로닝하였고, PCR을 이용하여 ZFY와 SRY 염기서열을 증폭하였다. 닭의 배아섬유세포의 염색체 분석 결과 Z 염색체와 W 염색체를 구분함으로써 배아의 성을 직접적으로 판별하는 것이 가능하였으며 , 암닭의 DNA를 Xho Ⅰ와 Eco RI로 절단하여 생성되는 band를 이용하여 성을 판별하는 것이 가능하였다. Xho Ⅰ와 Eco RI family를 클로닝하고, colony hybridization을 통해 Xho Ⅰ과 염기서열이 유사한 80∼100개의 clone을 동정하여, 이들 두 그룹간 DNA homology는 매우 유사하였다. 150개의 random primer 중 W 염색체 특이적인 반복 염기서열과 유사성을 보이는 primer 7개를 screening하였으며, 이 중 3개의 primer는 닭에서 자성과 웅성간의 차이를 나타내었다. 닭에서 성분화에 관련된 유전자를 동정하기 위하여 포유류의 ZFY와 SRY유전자의 PCR증폭을 실시하였다. ZFY를 증폭한 결과, 자성과 웅성간의 차이를 발견할 수 없었으며, 이는 닭에서 ZFY는 상염색체 또는 Z 염색체에 존재함을 시사한다. SRY의 증폭에서는 성간의 차이가 확인되었으나, 이 유전자가 Z 염색체에 존재하는지 W 염색체에 존재하는지 혹은 상염색체 존재하는지 여부는 연구가 필요하리라 사료된다.

• Journal of Genetic Medicine
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• 제9권2호
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• pp.73-77
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• 2012

Purpose: Fragile X carrier detection before or at early pregnancy through a wide screening program may not only confer a risk of having offspring with Fragile X syndrome (FXS), but may also confer a risk for Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome. However, prior to the implementation of such a program, the carrier prevalence in a population and the availability of effective screening test should be evaluated. The aim of our study was to determine the prevalence of premutation carriers and to evaluate the feasibility of screening test. Materials and Methods: The blood samples were obtained from 8,641 pregnant women with no family history of mental retardation. We performed a three-primer CGG repeat primed (RP) PCR using the AmplideX$^{TM}$ FMR1 PCR kit (Asuragen, Inc. Austin, TX, USA). Samples showing full mutation alleles were reflexed to Southern blot analysis for methylation status and sizing. Results: Among the 8,641 women, we found 8 premutation carriers (1:1,090, 0.09%) and 46 women with an intermediate allele (1:190, 0.53%). No woman was found to carry the fully mutated allele. All the detected alleles were within the CGG repeat range of 8-117. Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. The CGG RP PCR method provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis. Conclusion: This is the first study that has focused on the prevalence of FXS premutation carriers and FMR1 allele distribution in normal pregnant women. These data have important implications for population-based fragile X carrier screening in Korea.

• 대한수의학회지
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• 제48권2호
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• pp.191-195
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• 2008

This study was performed to detect Neospora caninum in blood of 61 Korean native cattle and 50 dairy cows in Chungnam province. All of them were healthy and did not show any clinical signs. DNA was isolated from blood samples and a 328 bp fragment was amplified by PCR using primer pair Np21 and Np6. The PCR positive rate was 14.8% in Korean native cattle and 0% in dairy cows. Cows with 15.6% were a little higher than bulls with 12.5% in gender. The detection rate of over 3-year-old Korean native cattle was 28.6% in age. The results demonstrate that N. caninum DNA can be detected in blood by PCR. PCR analysis in blood may be useful to annually screening test for N. caninum infection in clinically healthy cattle.

• 대한수의학회지
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• 제49권2호
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• pp.105-111
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• 2009

In this study, occurrence of canine brucellosis was surveyed in kennels, indoor dogs and stray dogs in Korea, and infrequent restriction site-polymerase chain reaction (IRS-PCR) was applied to analyze DNA polymorphism of Brucella canis (B. canis) isolates. Among a total of 501 dogs tested, B. canis antibodies by both rapid screening agglutination with 2-mercaptoethanol (2-ME RSAT) and immunochromatographic assay were detected in only 14.1% of kennel dogs. There were no seropositive cases in indoor dogs and stray dogs. DNA polymorphism was observed in 16 B. canis isolates by the IRS-PCR. Sixteen isolates were tested with primers, PsalA, PsalC, PsalG and PsalT, and different primers produced different DNA patterns. In regard to the IRS-PCR pattern of 16 isolates, 9 (56.3%) belonged to the IRS-PCR type I. The remaining 7 were differentiated as type II, III and IV. An application of the primer PsalC provided discrimination between B. canis isolated in 2005 and others.

• Journal of Microbiology
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• 제41권4호
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• pp.320-326
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• 2003

In order to identify Vibrio vulnificus in the Yellow Sea near Gunsan, Korea during the early and late summers, the efficiency of the real-time quantitative TaqMan PCR was compared to the efficiency of the conventional PCR and Biolog identification system^TM. Primers and a probe were designed from the hemolysin/cytolysin gene sequence of V. vulnificus strains. The number of positive detections by real-time quantitative TaqMan PCR, conventional PCR, and the Biolog identification system from seawater were 53 (36.8%), 36 (25%), and 10 strains (6.9%), respectively, among 144 samples collected from Yellow Sea near Gunsan, Korea. Thus, the detection method of the real-time quantitative TaqMan PCR assay was more effective in terms of accuracy than that of the conventional PCR and Biolog system. Therefore, our results showed that the real-time TaqMan probe and the primer set developed in this study can be applied successfully as a rapid screening tool for the detection of V. vulnificus.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.749-759
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• 2009

A survey of Bacillus thuringiensis (Berliner) strains isolated from Spanish citrus orchards has been performed, and the strains were tested for insecticidal activity against the Mediterranean fruit fly Ceratitis capitata (Wiedemann), a key citrus pest in Spain. From a total of 150 environmental samples, 376 isolates were selected, recording a total B. thuringiensis index of 0.52. The collection was characterized by means of phase-contrast microscopy, SDS-PAGE, and PCR analysis with primer pairs detecting toxin genes cry1, cry2, cry3, cry4, cry5, cry7, cry8, cry9, cry10, cry11, cry12, cry14, cry17, cry19, cry21, cry27, cry39, cry44, cyt1, and cyt2. Diverse crystal inclusion morphologies were identified: bipyramidal (45%), round (40%), adhered to the spore (7%), small (5%), and irregular (3%). SDS-PAGE of spore-crystal preparations revealed 39 different electrophoresis patterns. All primer pairs used in PCR tests gave positive amplifications in strains of our collection, except for primers for detection of cry3, cry19, cry39, or cry44 genes. Strains containing cry1, cry2, cry4, and cry27 genes were the most abundant (48.7%, 46%, 11.2%, and 8.2% of the strains, respectively). Ten different genetic profiles were found, although a total of 109 strains did not amplify with the set of primers used. Screening for toxicity against C. capitata adults was performed using both spore-crystal and soluble fractions. Mortality levels were less than 30%. We have developed a large and diverse B. thuringiensis strain collection with huge potential to control several agricultural pests; however, further research is needed to find out Bt strains active against C. capitata.

• Journal of Microbiology and Biotechnology
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• 제20권7호
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• pp.1107-1113
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• 2010

Because conventional methods for detecting emetic-toxin-producing B. cereus are laborious and costly, various PCR assays, which are easy and cheap, have recently been reported. Therefore, this study estimated and compared the ability of various PCR assays to detect emetic-toxin-producing B. cereus strains isolated in Korea. The PCR assays were performed on 160 B. cereus strains, including 40 emetic-toxin-producing strains. Although the species-specific PCR assays were all shown to be highly specific, the sensitivities varied greatly. The accuracies of the primers were 97.5% (CER), 95.6% (EM1), 96.3% (RE234), 89.4% (CES), and 83.1% (Ces3R/CESR2). Moreover, the CER primer had a higher sensitivity (100%) than all the other primers tested, and a specificity of 96.7%. Thus, the CER primer was shown to be the most effective for screening the emetic-toxin-producing B. cereus strains tested in this study. However, the ability of these PCR assays to identify emetic-toxin-producing B. cereus should also be confirmed using other methods.

• 생명과학회지
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• 제13권6호
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• pp.879-888
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• 2003

추에서 항균 phytoalexin으로 알려진 capsidiol의 생합성을 촉매하는 5-epi-aristolochene hydroxylase는 cytochrome P450 (P450) 억제제인 ancymidol과 ketocornazol에 의해 그 활성이 특이적으로 억제되어, 이 효소가 P450계 효소임을 알 수 있었다. P450 효소가 공통으로 보유하는 염기서열을 지닌 primer를 이용하여 RT-PCR과 cDNA screening을 실시한 결과 고추배양세포에서 elicitor 처리에 의해 강하게 유도되는 cDNA (P450Hy01)를 cloning하였다. 배양세포에 cellulase, arachidonic acid, jasmonic acid, 자외선 등을 처리하여 capsidiol을 생합성하는 량과 P450Hy01 mRNA의 발현정도는 밀접한 유사성이 있었다. P450Hy01의 염기서열은 담배에서 밝혀진 5-epi-aristolochene-1,3-hydroxylase와 98%의 유사성이 있었으며, P450 효소가 공통으로 지니는 heme-binding domain인 FxxGxRxCxG를 보유하고 있었다 이러한 결과는 본 연구에서 cloning된 P450Hy01이 고추의 세포에서 capsidiol의 생합성을 촉매하는 5-epi-aristolochene hydroxylase 효소를 coding 하고 있음을 시사한다.