• 한국식품영양과학회지
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• 제39권4호
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• pp.595-601
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• 2010

본 연구는 국내 주요 식중독 원인균인 Staphylococcus aureus, Salmonella enterica subsp., Vibrio parahaemolyticus를 동시에 검출 및 동정할 수 있는 simultaneous multiplex PCR방법을 개발하고자 하였다. S. aureus의 23s rRNA 유전자(482 bp), V. Parahaemolyticus의 toxR 유전자(368 bp), S. enterica subsp.의 invA 유전자(284 bp)를 특이적으로 검출 및 동정할 수 있는 3개 primer set 즉, STA-5F/STA-5R, ToxR-F/ToxR-R, 139/141을 구축하였으며, 그 결과 정제되어진 각 식중독 원인균의 genomic DNA를 template로 하여 세 균주 모두 10 pg까지 다중동시검출이 가능하였다. 생균수(CFU)와 상응되는 검출한계 결과로써 $10^1\sim10^2$ CFU/reaction의 검출한계를 보였으며 이는 즉, S. aureus $6.0\times10^4$ CFU/mL, S. enterica subsp. $9.5\times10^4$ CFU/mL, V. parahaemolyticus $6.1\times10^5$ CFU/mL의 검출한계를 나타내었다. 균체회수부터 agarose gel 상에서 검출 및 동정까지 3~4 hr의 시간 소요로 single tube 반응으로 세 식중독 원인균의 다중동시검출이 가능하였다. 또한 추가적인 연구를 통하여 세 식중독 원인균주의 검출을 위한 향상된 민감도를 가지는 multiplex PCR법 및 real time PCR을 이용한 다중동시검출법 개발을 위한 기초자료로서 활용 가능할 것이라 사료된다.

• Pediatric Infection and Vaccine
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• 제25권2호
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• pp.91-100
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• 2018

목적: 본 연구는 30일 이상 90일 미만의 발열이 있는 영아에서 경험적 항생제 사용에 미치는 요소를 조사하였다. 방법: 2010년 1월부터 2016년 12월까지 부산대학교 어린이병원에 발열이 있는 이전에 건강했던 영아를 대상으로 임상양상, 검사소견, 항생제 사용에 대해 의무기록을 후향적으로 분석하였다. 호흡기 바이러스는 다중 역전사 중합 연쇄반응검사로 검출하여 1-3일 후 보고되었고, enterovirus는 중합 연쇄반응검사로 검출하여 수시간 만에 보고되었다. 결과: 366명의 대상자 중 129명은 경험적 항생제를 사용하였고 237명은 경험적 항생제를 사용하지 않았다. 입원 전 발열기간이 긴 경우와 호흡기 증상이 있을 때, 아파보일 때, CRP 상승 시 경험적 항생제 사용이 많았다. 경험적 항생제를 사용하지 않은 환자의 재입원율이 낮았다. Enterovirus polymerase chain reaction (PCR)이 검출된 대부분의 환자는 경험적 항생제를 사용하지 않았다. Respiratory virus multiplex reverse transcriptase (RT)-PCR 결과는 경험적 항생제 사용에 차이를 보이지 않았다. 결론: 본 연구에서 respiratory virus multiplex RT-PCR은 항생제 처방에 영향을 주지 않았고 enterovirus PCR은 항생제 처방에 영향을 주었다. Respiratory virus multiplex RT-PCR 결과가 신속하게 보고된다면 항생제 사용에 영향을 줄 것이다.

• Fisheries and Aquatic Sciences
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• 제26권11호
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• pp.678-688
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• 2023

Flatfish are one of the largest families in the order Pleuronectiformes and are economically important edible marine fish species. However, they have similar morphological characteristics leading to challenges in classifying correctly, which may result in mislabeling and illegal sales, such as fraudulent labeling of processed food. Therefore, accurate identification is important to ensure the quality and safety of domestic markets in Korea. Species-specific primers were prepared from the mainly consumed eleven species of the order Pleuronectiformes. To rapidly identify the 11 flatfish species, a highly efficient, rapid, multiplex polymerase chain reaction (PCR) with species-specific primers was developed. Species-specific primer sets were designed for the mitochondrial DNA cytochrome c oxidase subunit I gene. Species-specific multiplex PCR (MSS-PCR) either specifically amplified a PCR product of a unique size or failed. This MSS-PCR analysis is easy to perform and yields reliable results in less time than the previous Sanger sequencing methods. This technique could be a powerful tool for the identification of the 11 species b the family Pleuronectidae and can contribute to the prevention of falsified labeling and protection of consumer rights.

• 현장농수산연구지
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• 제5권1호
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• pp.57-63
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• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• 대한의생명과학회지
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• 제13권1호
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• pp.71-73
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• 2007

For the rapid detection of objective pathogenic bacteria from colon biopsy specimens, multiplex PCR (polymerase chain reaction) method was developed. The major objective bacteria in this study were Shiga-like toxin producing E. coli O157:H7, Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Shigella spp. Salmonella spp. and Yersinia spp.. To detect simultaneously 7 kinds of pathogenic bacteria in single reaction tube, multiplex PCR system was executed using 6 sets of primers used in single PCR system for the respective bacteria. The results in this research might be applied for the detection of pathogenic bacteria form colon biopsy samples.

• 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.

• 미생물학회지
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• 제49권3호
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• pp.292-296
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• 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
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• 제30권4호
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• pp.527-532
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• 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.

• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제27권2호
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• pp.95-103
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• 2024

Purpose: Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. Methods: This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. Results: The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. Conclusion: The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

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