• 한국어병학회지
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• 제10권1호
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• pp.39-44
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• 1997

Metallothionein은 세균에서 척추동물에 이르기 까지 모든 생명체에 존재하며, 중금속의 세포내농도를 조절하는 중요한 단백질이다. 현재까지 metallothionein의 기능 및 유도기작에 관한 연구는 많이 진척되지는 않았으나, 여러 metallothionein 유전자의 구조가 밝혀져 있는 실정이다. 특히 어류의 metallothionein은 여러종류의 중금속과 환경적인 자극에 의하여 유도되고 정량적인 RT-PCR의 방법으로 metallothionein 유전자의 RNA transcript를 측정함으로써 환경적인 자극의 정도와 중금 속의 상대적인 양을 측정할 수 있기 때문에 중요한 단백질로 인식되고 있다. 본 연구에서는 유전자내부의 특이적 primer와 통상적인 3`말단의 primer를 이용하여 PCR에 의해 450 bp에 해당하는 metallothionein 유전자의 일부의 특성을 조사하였다. 챠넬메기의 cDNA library로부터 PCR에 의해 증폭된 450 bp의 PCR 절편은 다른 어류의 metallothionein 유전자와는 유사성을 보이지 않았다.

• 식물병연구
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• 제13권3호
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• pp.137-144
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• 2007

거봉 포도나무에 혹병을 일으키는 A. vitis 균주간의 DNA 다양성 평가를 하기 위하여 12종류의 URP primer 적용 성을 조사한 결과 URP1F, URP2F, URP2R, URP4R, URP17R primer가 균주 간 DNA 다형성검정에 유용하였다. 국내외에서 분리한 59 A. vitis 균주를 URP-PCR 증폭하였던 바 균주간의 매우 다양한 PCR 다형성 밴드를 형성 하였으며 12 strain type으로 나눌 수 있었으며 거봉포도로부터 분리된 A. vitis 균주는 4 strain type의 비교적 단순한 유전적 다양성으로 나타났으나, 거봉이외의 다른 포도 품종이나 국외에서 도입된 A. vitis 균주는 8 strain type의 많은 유전적 다양성을 보여 거봉품종 유래 국내 균주와는 PCR 다형성 type에 있어 차이점을 보였다. URP-PCR 다형성 밴드를 집괴 분석하여 UPGMA dendrogram을 작성한 결과 7개의 대 Group으로 분류할 수 있었으며, 그룹 간에는 $62{\sim}100$%까지 다양한 유전적 유사성이 나타났다.

• Journal of Plant Biotechnology
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• 제5권2호
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• pp.83-86
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• 2003

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two specific 22-mer primers located in coat protein gene of SPFMV. A 411 bp PCR-product was detected in virus infected plants as well as tissue culture raised sweet potato but not in healthy plants. For optimization of RT-PCR protocol, the optimum crude nucleic acid concentration, annealing temperature, primer concentration and numbers of PCR-cycle for maximum sensitivity and specificity were determined. The optimum condition for RT-PCR was as follows: RT-PCR reaction mixture was one-step mixture, containing 50 pmol of primer, 30 units of reverse transcriptase, 5 units of RNasin, and the crude nucleic acid extracts (200 ng). In RT-PCR, cDNA was synthesized at $42^{\circ}C$ for 45 min before a quick incubation on ice after pre-denaturation at $95^{\circ}C$ for 5 min. The PCR reaction was carried out for 40 cycles at $96^{\circ}C$ for 30 see, $63^{\circ}C$ for 30 sec, $72^{\circ}C$ for 1 min, and finally at $72^{\circ}C$ for 10 min. The viral origin of the amplified product was confirmed by sequencing, with the sequence obtained having $95-98\%$ homology with published sequence data for SPFMV. The benefits of this RT-PCR based detection of SPFMV would be simple, rapid and specific.

• 대한약학회:학술대회논문집
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• 대한약학회 2003년도 Proceedings of the Convention of the Pharmaceutical Society of Korea Vol.2-2
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• pp.85.1-85.1
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• 2003

Previously, we found the operon random primer (OP-5A) that is characteristic the genus Panax by randomly amplified polymorphic DNA (RAPD) analysis. However, OP-5A primer is limited to apply on the differentiation of only crude herbal plants. To construct more sensitive and unique primers on the genus Panax, ginseng-specific DNA profile (350 bp) that was amplified by OP-5A primer were inserted in a plasmid vector in the TA cloning method and sequenced. We designed the PCR primers (Forward: 5＂-AGGGGTCTTGCTAT AGCGGAAC-3＂, Reverse: 5＂-AGTCTTAATTTCATATTTTCGTATG-3＂) and identified the unique ginseng band (350 bp) in commercial granule products including ginseng extracts as well as crude ginseng plants by nascent PCR.(omitted)

• 환경생물
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• 제19권4호
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• pp.321-324
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• 2001

일반적으로 지하수환경은 세균의 수가 적기 때문에 세균의 핵산을 추출하기 위해서는 지하수 시료의 여과를 통하여 대량의 세균을 획득하는 것이 우선적으로 요구되어왔다. 그러나 이러한 여과법은 많은 시간과 인력의 낭비 뿐만 아니라 실험 과정의 특수성으로 인하여 오염의 위험성이 높다. 따라서 본 논문에서는 구아니딘 열탕법을 이용하여 소량의 지하수 시료로부터 핵산증폭실험에 적용할 수 있는 충분한 양의 핵산의 추출을 시도하였다. 지하수 시료는 서울시 내의 질소화합물 요염지역, 오염 예상지역, 청정지역으로 구분하여 각각 2개의 정점을 선별하여 총 6개의 정점으로부터 채수하였다. 구아니딘 열탕법을 이용하여 얻은 핵산으로 지하수에서 탈질화세균의 존재 여부를 검정한 결과 청정지역을 제외한 4개의 정점에서 탈질화 세균의 존재를 확인하였다.

• 농업과학연구
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• 제47권3호
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• pp.673-681
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• 2020

Human enteric Adenovirus 41 (HueAdV-41) is a major waterborne virus that causes human gastroenteritis and is classified as a viral group I double-strand DNA virus, Adenoviridae. HueAdV-41 has been detected with the polymerase chain reaction (PCR) in various samples such as ground water. However, the PCR-based diagnostic method has problems such as reaction time, sensitivity, and specificity. Thus, the loop-mediated isothermal amplification (LAMP) assay has emerged as an excellent method for field applications. In this study, we developed a LAMP system that can rapidly detect HueAdV-41 with high specificity and sensitivity. HueAdV-41 specific LAMP primer sets were tested through a specific, non-specific selection and sensitivity test for three prepared LAMP primer sets, of which only one primer set and optimum reaction temperature were selected. The developed LAMP primer set condition was confirmed as 63℃, and the sensitivity was 1 copy. In addition, to confirm the system, a LAMP positive reaction was developed with the restriction enzyme Taq I (T/GCC). The developed method in this study was more specific, rapid (typically within 2 - 3 hours), and highly sensitive than that of the conventional PCR method. To evaluate and verify the developed LAMP assay, an artificial infection test was done with five cDNAs from groundwater samples, and the results were compared to those of the conventional PCR method. We expect the developed LAMP primer set will be used to diagnose HueAdV-41 from various samples.

• 농업과학연구
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• 제47권1호
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• pp.173-182
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• 2020

Human Astrovirus (HuAstV), known as a waterborne virus, is a group IV positive-sense single-stranded RNA that belongs to Astroviridae. The first outbreak of HuAstV was reported in England in 1975. HuAstV can exist not only among clinical patients but also in various water environments, such as water for agriculture and vegetables. For diagnosis of HuAstV from water samples, a polymerase chain reaction (PCR) system has been developed. However, the PCR-based diagnostic method has problems in field application, such as reaction time, sensitivity and specificity. For this reason, in this study we developed the loop-mediated isothermal amplification assay (LAMP) system, aimed specifically at HuAstV. Three prepared LAMP primer sets were tested by specificity, non-specificity and sensitivity; one LAMP primer set was selected with optimum reaction temperature. The developed LAMP primer set reaction conditions were confirmed at 62℃, and detection sensitivity was 1 fg/μL. In addition, restriction enzyme HaeIII (GG/CC) was introduced to confirm that the LAMP reaction was positive. As a result, selected LAMP primer set was 100 - 1000 times more specific, rapid, and sensitive than conventional-nested PCR methods. For verification of the developed LAMP assay, twenty samples of cDNA from groundwater samples were tested. We expect that the developed LAMP assay will be used to diagnose HuAstV from various samples.

• Mycobiology
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• 제29권1호
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• pp.7-10
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• 2001

Based on the rDNA ITS sequences data, specific primer set for PCR detection of wood-decaying fungus Phellinus linteus was designed. The length of PCR products using designed primer set(SHF and SHR) was about 540 bp. Among 11 species, 17 isolates of Phellinus spp. including Phellinus linteus, P. pomaceus, P. spiculosus, P. baumi, P. pini, P. igniarius, P. gilvus, P. biscuspidatus, P. weirii, P. johnsonianus, P. robutus, and P. igniarius, seven isolates of Phellinus linteus showed about 540 bp-sized single band. This molecular technique could offer a useful tool for detecting and identifying Phellinus linteus.

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

• 한국응용곤충학회지
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• 제37권1호
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• pp.23-30
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• 1998

DNA의 제한요소단편 다형현상(RFLP)이 유전변이 연구에 널리 이용되고 있다. 본 연구는 파밤나방(Spodoptera exigua(H bner)) 미토콘드리아 DNA(mtDNA)의 RFLP방법을 개발하기 위해 게놈 크기 측정 및 PCR primer들을 선발하였다. 파밤나방의 mtDNA 전체크기는 약 16kb였다. 대부분 곤충 mtDNA에 적합하게 구성된 (Simon et al., 1994)29개 promer들중 21개가 파밤나방의 mtDNA증폭에 적합했다. 이들 primer들을 이용하여 여러 유전자 영역(CO-I, CO-II, Cyt-B, ND-1, 12S rRNA, 16S rRNA 및 일부 tRNA)의 일분 또는 전체를 포함하는 유전자 절편을 증폭시켰다. 일반적으로 다형을 보이는 primer조합을 중심으로 4염기 제한부위를 인식하는 8종의 제한 효소를 통해 분석된 PCR-RFLP는 서로 다은 지역(안동, 경산, 순천) 집단들간에 제한부위에 있어서 차이가 없었으나 일부 영역에서는 길이 차이를 보여 유용한 유전지표로서의 가능성을 제시했다.