• The Plant Pathology Journal
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• 제22권2호
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• pp.168-173
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• 2006

To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor $1{\alpha}(EF1{\alpha})$. This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

• 대한수의학회지
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• 제45권3호
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• pp.351-357
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• 2005

Here I describe a multiplex allele specific PCR-based approach for the rapid detection between Hanwoo and Holstein meat associated with Melanocortin 1 receptor (MC1R) gene. Specific and universal oligonucleotide primers were used in combination to detect the presence of a single nucleotide polymorphism within the bovine MC1R DNA sequence. The presence of the bovine MC1R gene is indicated by the production of a single control PCR product, whilst positive samples generate an alternative smaller specific product over the same region. The mutations in MC1R104 codon revealed depending on the presence or absence of an indicative fragment amplified from the wild-type allele of this codon. As little as 0.39 ng and 1.56 ng of genomic DNA of Hanwoo and Holstein could be detected by MAS-PCR assay, respectively. This technique, which is widely used in human genetic screening, provides a reliable and sensitive result that has not been documented for the identification of bovine coat color. The MAS-PCR assay approach was proven to be useful in complementing routine beef DNA analysis for differentiation of these MC1R variants and it would facilitate the screening of deceiving sales of Holstein meat in the butcher shop.

• Annals of Clinical Microbiology
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• 제22권1호
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• pp.9-13
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• 2019

배경: 다양한 임상검체에서 카바페넴분해효소 생성 장내세균(carbapenemase-producing Enterobacteriace, CPE)의 분리가 증가하고 있다. CPE 감염증은 치사율이 높고, 집단 발병의 가능성이 높아 지속적인 감시가 필수적이다. 저자들은 CPE 주요 유전자들을 한 번에 검출하기 위한 multiplex PCR을 개발하고 이를 평가하고자 하였다. 방법: 총 7종의 내성 유전자를 동시에 검출하기 위한 시발체를 새롭게 디자인하고 PCR 조건을 설정하였다. 주요 카바페넴분해효소인 KPC, IMP, VIM, NDM-1, GES, OXA-23 및 OXA-48 유전자를 대상으로 하였다. 각 유전자의 증폭 산물은 100 bp 이상의 차이가 나도록 고안하였다. 개발된 multiplex PCR은 총 69주의 CPE 양성 임상분리균주를 이용하여 평가하고, 비특이적 증폭에 의한 위양성 가능성의 배제를 위해 71주의 카바페넴 감수성 균주로 확인하였다. 결과: 본 연구에서 개발한 시발체 및 PCR 조건을 이용하여 실험한 결과 CPE 내성 유전자인 KPC 양성 14주, IMP 양성 13주, OXA-23 양성 12주, OXA-48 양성 11주, VIM 양성 9주, GES 및 NDM 양성 각 5주 등 총 69주 모두에서 CPE 유전자의 검출이 가능함을 확인하였다. 카바페넴 감수성 균주를 이용한 특이성 확인 시험에서 Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa 등을 포함한 총 71주의 다양한 그람양성 및 음성균주 모두에서 음성임을 확인하였다. 결론: 본 연구에서 개발된 multiplex PCR은 총 7종의 CPE 유전형을 한 번에 검출할 수 있어 CPE 확인 시험에 유용할 것으로 생각한다.

• 대한수의학회지
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• 제52권1호
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• pp.39-43
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• 2012

A multiplex PCR was developed for the simultaneous detection and differentiation of Mycoplasma (M.) hyopneumoniae and M. hyorhinis in clinical samples. Improved sensitivity is advantage of this technique over the previously reported multiplex assay. It was capable of detecting as little as 125 fg genomic DNA from M. hyopneumoniae and 62.5 fg genomic DNA from M. hyorhinis. Application of this multiplex PCR method to field isolates showed that M. hyopneumoniae and M. hyorhinis were present in 29% (107 of 370) of lung specimens and no mycoplasmas were detected in 56% (208 of 370) of the slaughtered pigs' lungs. At the farm level, M. hyopneumoniae and M. hyorhinis were detected in 34 of 36 (94.4%) randomly selected farms. We conclude that this assay would prove itself a value tool for monitoring these mycoplasmal infections and both M. hyopneumoniae and M. hyorhinis have been widely spread in swine herds of Korea.

• Journal of Microbiology and Biotechnology
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• 제25권10호
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• pp.1714-1727
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• 2015

This work describes a new strategy for optimal design of Multiplex-PCR primer sequences. The process is based on the Particle Swarm Optimization-Simplex algorithm (Mult-PSOS). Diverging from previous solutions centered on heuristic tools, the Mult-PSOS is selfconfigured because it does not require the definition of the algorithm's initial search parameters. The successful performance of this method was validated in vitro using Multiplex-PCR assays. For this validation, seven gene sequences of the most prevalent bacteria implicated in urinary tract infections were taken as DNA targets. The in vitro tests confirmed the good performance of the Mult-PSOS, with respect to infectious disease diagnosis, in the rapid and efficient selection of the optimal oligonucleotide sequences for Multiplex-PCRs. The predicted sequences allowed the adequate amplification of all amplicons in a single step (with the correct amount of DNA template and primers), reducing significantly the need for trial and error experiments. In addition, owing to its independence from the initial selection of the heuristic constants, the Mult-PSOS can be employed by non-expert users in computational techniques or in primer design problems.

• Journal of Microbiology
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• 제39권3호
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• pp.226-228
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• 2001

Human lymphotropic herpesvirus is known to be a major pathogen associated with various diseases in bone marrow transplantation (BMT) recipients. A multiplex nested-polymerase chain reaction (PCR) method was developed for the simultaneous detection of human lymphotropic herpesviruses, including Ebstein-Barr virus (EBV), cytomegalovirus (CMV), and human herpesvirus 6 variants A and B (HHV6-A, HHV6-B). To demonstrate the usefulness of multiplex PCR for the analysis of clinical samples, peripheral blood mononuclear cells and serum from BMT recipients were analysed. The results skewed that a clear detection could be made between EBV, HCMV and HHV-6. This multiplex PCR assay is an efficient and cost-effective approach to the analysis of large numbers of samples to determine the epidemiological importance of EBV HCMV and HHV-6.

• 한국양봉학회지
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• 제32권1호
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• pp.27-39
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• 2017

꿀벌 6종 주요 감염성 질병을 동시 진단하기 위한 PCR-chip 기반 초고속 다중 PCR 진단법을 개발 하였다. 6종 주요 꿀벌 감염성 병원체들은, 세균성 질병인 미국부저병의 원인균, Paenibacillus larvae와 유럽부저병의 원인균인 Melissococcus plutonius, 또한 진균인 Ascosphaera apis(백묵병), Aspergillus flavus(석고병)와 Nosema apis, Nosema ceranae(노제마병)를 선발하였다. 개발된 PCR-chip 기반 초고속 다중 PCR은, 꿀벌 주요 병원체 6종에 대하여 각기 $10^3$ 분자이상이 존재할 경우 모두 성공적 증폭을 보였으며, 증폭여부의 확인에 걸린 시간(Ct-time)은 6종 중 4종은 9분 내외, 2종은 7분 내외이었으며, 총 40회전의 PCR은 11분 42초, 융점분석 1분 15초로 총 PCR분석에 소요된 시간은 12분 57초(40회전 및 융점분석)이었다. 표준 DNA 기질을 사용한 PCR-chip 기반 초고속 다중 PCR은 100%에 근접한 정확도를 보였으며, 꿀벌 genomic DNA를 사용한 실험에서 false-amplification은 발견되지 아니하였다. PCR-chip 기반 초고속 다중 PCR은 실험실 내 초고속 진단 뿐 아니라 양봉 현장에서도 신속하고 효율적인 병원체 검출법이 될 것으로 기대한다.

• Journal of Plant Biotechnology
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• 제43권1호
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• pp.91-98
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• 2016

면화(cotton)는 섬유를 수확하고, 면실유는 식용으로 가공 후 남은 것은 다시 사료로 이용되어 다방면으로 다양하게 활용되는 작물이다. LM 면화는 옥수수, 대두, 캐놀라와 달리 아시아권(중국, 인도 등)에서 가장 많이 재배되고 있으며, 우리나라는 2013년까지 세계 1위의 LM 면실(사료용) 수입국이며, 세계 4위의 면실박 수입국으로 해마다 증가하는 LM 면화의 수입량과 맞물려 유통 및 소비과정에서의 비의도적으로 유출 가능성이 증가함에 따라 LM 면화의 자연생태계 위해성 평가 및 안전관리가 요구된다. 본 연구에서는 국내 수입 승인 LM 면화 6개 이벤트(MON15985, MON531, GHB614, LLCOTTON25, MON88913, MON1445)의 동시증폭 검출법(multiplex PCR)을 개발하여 보다 신속하고 명확한 검출기법을 확립하고자 하였다. 최적 multiplex PCR 반응 조건은 2개 LM 면화 이벤트 MON15985 (214 bp), MON531 (270 bp)와 4개 LM 면화 이벤트 GHB614 (119 bp), LLCOTTON25 (164 bp), MON88913 (276 bp), MON1445 (389 bp)가 한번의 반응에 명확하게 검출되는 최적 반응 조건 및 primer 반응 농도의 조절을 통해 서로 다른 생성물 크기로 명확히 구분되도록 하였고, 최적 primer 농도는 반응액 최종농도 0.2~0.66 pmol로 primer 쌍 마다 각각 다른 최적 농도 조합의 cocktail을 만들어 활용하였다. Duplex PCR 반응 조건은 초기 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $55^{\circ}C$ 20초 15회 반응하고, 다시 $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 25회 반응하였을 때 최적 검출이 이루어졌고, tetraplex PCR 반응 조건은 $95^{\circ}C$ 5분 반응 후, $95^{\circ}C$ 15초, $60^{\circ}C$ 20초 50회 반응하였을 때 최적 검출이 이루어졌다. 본 연구에서 개발된 multiplex PCR 검출법은 국내 수입 유통 LM 면화의 자연환경 모니터링에 활용함에 있어 요구되는 연구인력, 시간 및 비용을 보다 효율적으로 개선하는데 적용될 수 있을 것으로 사료된다.

• Food Science and Biotechnology
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• 제17권4호
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• pp.761-765
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• 2008

Bacillus cereus causes two different types of food poisoning syndromes: diarrhea and emesis. The diarrheal syndrome is attributed to various enterotoxins, including nonhemolytic enterotoxin, hemolytic enterotoxin, and enterotoxin-T, whereas the emetic syndrome is caused by the dodecadepsipeptide toxin cereulide. A multiplex polymerase chain reaction (PCR) assay was developed to rapidly detect and identify B. cereus strains. Three primer pairs specific to regions within genes encoding nonhemolytic enterotoxin (nheA), molecular chaperonin (groEL), and cereulide synthetase (ces) were used to identify and differentiate between the enterotoxin-producing and emetic toxin-producing B. cereus strains. The cereulide-producing emetic B. cereus showed 3 PCR products of 325, 405, and 685 bp for the groEL, ces, and nheA genes, respectively, whereas the enterotoxin-producing B. cereus showed 2 PCR products without a ces gene specific DNA fragment. Specific amplifications and differentiations by multiplex PCR assay were obtained using 62 B. cereus strains and 13 strains' of other bacterial species. The detection limit of this assay for enterotoxin-producing strain and emetic toxin-producing strain from pure cultures were $2.4{\times}10^1$ and $6.0{\times}10^2\;CFU/tube$, respectively. These results suggest that our multiplex PCR method may be useful for the rapid detection and differentiation of B. cereus strains in foods.

• 대한임상검사과학회지
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• 제38권3호
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• pp.173-178
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• 2006

The production of extended-spectrum ${\beta}$-lactamases ($ESBL_S$) is the main mechanism of bacterial resistance to third-generation cephalosporins and monobactams, whose prevalence varies depending on the different geographical areas. In the last years it has increased notably to the point of being considered a health problem of great importance. The characterization of the ESBLs producing Klebsiella penumoniae strains present in clinical isolates is time-consuming. I describe here the development of a new system, which consists of a multiplex PCR. I found 51 K. pneumoniae strains to be presumptive strains ESBLs producers by clinical and laboratory standards institute (CLSI) guidelines. The double disc synergy test showed 47 positive K. pneumoniae, which were K. pneumoniae isolates. All ESBLs producing K. pneumoniae strains were resistant to antibiotic amikacin, gentamicin and ciprofloxacin. By multiplex PCR analysis, $bla_{TEM}$ gene in 17 strains 44 $bla_{SHV}$ genes and $bla_{CTX}$ genes in 33 strains were identified. In this study, the multiplex polymerase chain reaction (PCR) assay was a good method to detect and differentiate ESBLs producing K. penumoniae strains in clinical isolates.