• The Plant Pathology Journal
• /
• 제29권3호
• /
• pp.338-343
• /
• 2013

A detection system based on a multiplex reverse transcription (RT) polymerase chain reaction (PCR) was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV), lily mottle virus (LMoV), lily symptomless virus (LSV). Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single- or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV) by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

• 농업과학연구
• /
• 제48권4호
• /
• pp.1051-1065
• /
• 2021

Recent times have seen sustained increases in genetically modified (GM) crops not only for cultivation but also for the utility of food and feed worldwide. Domestically, commercial planting and the accidental or unintentional release of living modified (LM) crops into the environment are not approved. Many detection methods had been devised in an effort to realize effective management of the safety of agricultural genetic resources. In order to develop event-specific polymerase chain reaction (PCR) markers for LM crops, we analyzed the genetic information of LM crops. Genetic components introduced into crops are of key importance to provide a basis for the development of detection methods for LM crops. To this end, a total of 18 varieties from four major LM crop species (maize, canola, cotton, and soybeans) were subjected to an analysis. The genetic components included introduced genes, promoters, terminators and selection markers. Thus, if proper monitoring techniques and single or multiplex PCR strategies that rely on selection markers can be established, such an accomplishment can be regarded as a feasible solution for the safe management of staple crop resources.

• 미생물학회지
• /
• 제49권3호
• /
• pp.292-296
• /
• 2013

본 연구는 multiplex real-time PCR을 이용하여 Actinobacillus actinomycetemcomitans, Campylobacter rectus, Porphyromonas gingivalis, Tannerella forsythus, Treponema denticola, Prevotella intermedia 등과 같은 6종의 주요 치주 질환 원인 미생물들을 동시에 검출할 수 있는 분석 방법에 관한 것이다. 4개의 형광 염료를 사용하여 internal control과 함께 3개의 균종씩 나누어 분석하였으며, 분석 대상 균종 간 그리고 다른 종류의 구강 미생물 균종과의 간섭과 교차 반응이 없음을 확인하였다. 본 연구의 multiplex real-time PCR은 타액과 플라그 등의 다양한 샘플에 포함되어 있는 각 미생물들을 정성, 정량적으로 분석할 수 있었으며, 치주염 환자와 건강한 사람들에 대한 비교 분석 결과 분명한 차이를 발견 할 수 있었다.

• 한국동물위생학회지
• /
• 제30권4호
• /
• pp.527-532
• /
• 2007

In present study, we described the reliability of the dual priming oligonucleotide (DPO) multiplex polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis complex (MTC) and non-Mycobacterium tuberculosis (NMT) in blood samples of the Korea native cattle, Hanwoo. Among 340 samples 22 (6.5%) were positive in using DPO multiplex PCR, 21 (6.2%) were positive in PCR. The relative agreement between 2 tests was 99.7%, and the agreement quotient (kappa), was 0.95 (excellent). In these results, we demonstrated the successful application of DPO multiplex PCR for the diagnosis of bovine tuberculosis in Hanwoo. Multiplex PCR, using DPO primers, can be useful for the simple diagnosis of bovine tuberculosis in bovine blood samples.

• 한국발생생물학회지:발생과생식
• /
• 제23권4호
• /
• pp.367-375
• /
• 2019

Pufferfish (Takifugu spp.) are economically important edible marine fish. Mistakes in pufferfish classification can lead to poisoning; therefore, accurate species identification is critical. In this study, we used the mtDNA cytochrome c oxidase subunit I gene (COI) to design specific primers for six Takifugu species among the 21 domestic or imported pufferfish species legally sold for consumption in Korea. We rapidly and simultaneously identified these pufferfish species using a highly efficient, multiplex polymerase chain reaction (PCR) system with the six species-specific primers. The results showed that species-specific multiplex PCR (multiplex species-specific polymerase chain reaction; MSS-PCR) either specifically amplified PCR products of a unique size or failed. MSS-PCR yielded amplification fragment lengths of 897 bp for Takifugu pardalis, 822 bp for T. porphyreus, 667 bp for T. niphobles, 454 bp for T. poecilonotus, 366 bp for T. rubripes, and 230 bp for T. xanthpterus using the species-specific primers and a control primer (ca. 1,200 bp). We visualized the results using agarose gel electrophoresis to obtain accurate contrasts of the six Takifugu species. MSS-PCR analysis is easily performed and provides identification results within 6 h. This technique is a powerful tool for the discrimination of Takifugu species and will help prevent falsified labeling, protect consumer rights, and reduce the risk of pufferfish poisoning..

• 대한의생명과학회지
• /
• 제5권1호
• /
• pp.95-100
• /
• 1999

일반적으로 임상검사실에서 vancomycin resistant enterococci (VRE)를 검출하는 일은 어렵고, 시간이 많이 들며, 검체처리 비용도 많이 든다. 따라서 본 실험은 임상검체에서 분리된 세균으로부터 VRE를 신속하게 확인하고, 진단하기 위한 방법으로서 다중 중합효소 연쇄반응을 확립하였다. 본 실험에 사용된 primer는 장구균에 특이 한 유전자인 vanA, vanB, vanC-1, vanC-2/3 각각의 염기서열을 기초로 primer를 제작하고, 다중 중합효소 연쇄 반응을 실시하여 임상검체로부터 분리된 VRE 유전자의 type및 분포율을 조사하고자 하였다. 국내에서 분리된 75주의 장구균을 대상으로 다중 중합효소 연쇄반응을 실시한 결과 36주의 분리균주에서 vancomycin에 대해 높은 저항성을 보이는 vanA 유전자를 가진 것으로 나타났다. 그리고 18주에서는 vancomycin에 낮은 저항성을 내성을 보이는 vanC-1 또는 vanC-2/3 유전자를 보유한 것으로 나타났다. 따라서 본 실험에서 확립한 다중 중합효소 연쇄 반응 기법은 신속한 VRE 진단 방법으로 이용할 수 있을 것이다.

• 한국축산식품학회지
• /
• 제31권5호
• /
• pp.658-662
• /
• 2011

In this study, 543 samples of press hams, sausages, processed ground meat and processed cheese acquired from retail markets in Seoul and Gyeonggi province in Korea from 2005 to 2010 were monitored using a one-step multiplex polymerase chain reaction (PCR) method that involves the amplification of specific soya or maize endogenous genes and the amplification of 35S promoter (p35S) and nopaline synthase terminator (tNOS) for GMO detection. Among the 543 samples, 477 samples were amplified for maize and/or soybean endogenous genes. Although one sausage sample collected in 2008 showed amplification of tNOS, the result was assumed to be false positive based on the results from further tests of other sausage samples of the same brand. Our results demonstrate the absence of GM soya and/or maze of livestock products in the Korean market during 2005-2010. In addition, the one-step multiplex PCR using previously constructed primer sets appears to be useful as a screening method for the detection of GMOs in processed livestock products. However, more specific methods should be established and employed to detect the event-specific GM gene for positive reaction samples by screening tests in processed livestock products.

• Journal of Applied Biological Chemistry
• /
• 제58권4호
• /
• pp.383-387
• /
• 2015

국내 유통 식품 수입 식품 중 토끼와 고양이 고기의 혼입 여부를 알아내고 불법 도축된 고양이 고기를 토끼 고기나 다른 고기로 속여 판매하는 것을 방지하기 위해 토끼와 고양이를 동시에 검출할 수 있는 polymerase chain reaction (PCR) 법을 개발하였다. 토끼와 고양이의 종 특이 프라이머는 미토콘드리아의 cytochrome b 유전자를 대상으로 하였고 개발된 프라이머를 가공식품에 활용하는 것을 고려하여 PCR 산물의 크기는 토끼 101 bp, 고양이 191 bp로 최소화 하였다. 프라이머의 특이성은 총 21종의 동물을 대상으로 검토하였다. 개발된 검출법의 검출 한계는 시료 DNA를 희석하여 PCR과 Bioanalyzer로 확인한 결과 토끼는 0.005 ng, 고양이는 0.0005 ng이었다.

• Parasites, Hosts and Diseases
• /
• 제60권4호
• /
• pp.295-299
• /
• 2022

Malaria elimination and control require prompt and accurate diagnosis for treatment plan. Since microscopy and rapid diagnostic test (RDT) are not sensitive particularly for diagnosing low parasitemia, highly sensitive diagnostic tools are required for accurate treatment. Molecular diagnosis of malaria is commonly carried out by nested polymerase chain reaction (PCR) targeting 18S rRNA gene, while this technique involves long turnaround time and multiple steps leading to false positive results. To overcome these drawbacks, we compared highly sensitive cytochrome oxidase gene-based single-step multiplex reaction with 18S rRNA nested PCR. Cytochrome oxidase (cox) genes of P. falciparum (cox-III) and P. vivax (cox-I) were compared with 18S rRNA gene nested PCR and microscopy. Cox gene multiplex PCR was found to be highly specific and sensitive, enhancing the detection limit of mixed infections. Cox gene multiplex PCR showed a sensitivity of 100% and a specificity of 97%. This approach can be used as an alternative diagnostic method as it offers higher diagnostic performance and is amenable to high throughput scaling up for a larger sample size at low cost.

• 대한수의학회지
• /
• 제43권2호
• /
• pp.231-237
• /
• 2003

Listeria (L.) monocytogenes in samples could not be detected occasioally by faster growth of other Listeria spp. especially L. innocua. The aim of this study was to develop the differential media and multiplex polymerase chain reaction (PCR) assays for the rapid detection of L. monocytogenes. L. monocytogenes colonies were characterized by their ${\beta}$-hemolysis with fluorescence under 366 nm UV light on the Listeria hemolysis agar (LHA). L. innocua, a species commonly present in foods, did not produce ${\beta}$-hemolysis on LHA. Therefore, one or more colonies of L. monocytogenes were easily distinguished from large populations of L. innocua. The multiplex PCR assays were developed to distinguish from L. monocytogenes and other Listeria spp. with two pairs of primers. The primers were designed in 16S rRNA and listeriolysin O gene for specific amplification of all members of the genus Listeria and L. monocytogenes, respectively. The multiplex PCR assays produced 560 and 938 bp products in L. monocytogenes; only 938 bp products in the genus Listeria. The multiplex PCR assays could detect as little as 50 pg of L monocytogenes DNA. These results indicated that the differential media and multiplex PCR assays might be useful diagnostic tools for the rapid detection of L. monocytogenes.