• Title/Summary/Keyword: 다중 중합효소연쇄반응

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Rapid Detection of Vancomycin Resistant Enterococci Using Multiplex Polymerase Chain Reactions (다중 중합효소 연쇄반응을 이용한 반코마이신 내성 장구균의 신속 검출)

  • 김종배;김근희;송혜원;박성언;엄용빈;박상욱;김양수;박수진
    • Biomedical Science Letters
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    • v.5 no.1
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    • pp.95-100
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    • 1999
  • It is generally difficult, time-consuming, and expensive for the clinical laboratory to detect vancomycin resistant enterococci (VRE). The aim of this study was to develop and evaluate the multiplex polymerase chain reaction (PCR) assay system as a diagnostic tool for the rapid detection of VRE from clinical samples and/or for the identification of VRE from the bacterial strains isolated from clinical specimens. Specific primers, designed from the nucleotide sequences respectively encoding the vanA, vanB, vanC-1, vanC-2/3 genes in enterococci, were coupled in a multiplex PCR assay system. With this multiplex PCR assay system, we investigated the incidence rates and types of VRE isolated from clinical samples. A total of 75 strains of enterococci were isolated in 3 general hospitals in Korea. Of these isolates, 36 strains showed a pattern of high-level vancomycin resistance which associated with vanA gene, whereas 18 strains showed low-level vancomycin resistance associated with vanC-1 or vanC-2/3 gene. Thus, multiplex PCR assay method established in this study could be applied for the rapid detection of VRE.

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Development of a Panel of Multiplex Real-Time Polymerase Chain Reaction Assays for Simultaneous Detection of Canine Enteric Bacterial Pathogens (개의 장내 병원균의 동시 검출을 위한 다중 실시간 중합효소연쇄반응분석 패널개발)

  • Jang, Hye-Jin;Han, Jae-Ik;Kang, Hyo-Min;Na, Ki-Jeong
    • Journal of Veterinary Clinics
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    • v.32 no.2
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    • pp.154-157
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    • 2015
  • A major cause of diarrhea in a dog is an infection with bacteria which include Salmonella spp., Campylobacter (C.) spp., and Clostridium (Cl.) spp.. It is fastidious to identify these bacteria by the culture. The purpose of this experiment is to devise the method for detecting Cl. perfringens, C. jejuni, C. coli, and Salmonella spp. with rapid and high sensitivity. The fecal samples collected from 71 normal and 66 diarrheic dog feces were used to compare the prevalence of the enteric pathogens and to develop a multiplex real-time polymerase chain reaction (PCR) assay for clinical use. Detection of Cl. perfringens, C. coli, and C. jejuni in diarrhea feces was higher than normal feces. A developed multiplex real-time PCR is useful for determining the presence and quantity of pathogen-specific or other unique sequences with in a fecal sample.

Simultaneous Detection of Cytomegalovirus, Epstein-Barr Virus, Hepatitis B Virus, and Parvovirus by a Multiplex PCR (다중 중합효소 연쇄반응을 이용한 DNA 바이러스의 동시검출)

  • Sung, Hye-Ran;Joo, Jin-Young;Lee, Chong-Kil;Chung, Yeon-Bok;Song, Suk-Gil
    • Korean Journal of Microbiology
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    • v.43 no.1
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    • pp.1-6
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    • 2007
  • We describe a multiplex PCR method that can detect and differentiate simultaneously four different kinds of DNA viruses, Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis B virus (HBV) and parvovirus B19 (B19). Primers for the multiplex PCR reaction were designed to amplify specific regions of the EBV (pol), CMV (pol), HBV (pol) and B19 (ns) viral genomes and used to simultaneously detect individual viruses. In order to achieve optimal sensitivity and specificity for multiplex PCR, the thermo-cycling parameters, primer sequences, and concentration of each reaction components were optimized systematically. The sensitivity of the detection method ranged between 5 and 10 copies of viral genome with a mixture of multiple primer pairs. Furthermore, this highly sensitive test showed no cross-reactivity among the four viruses. Thus, the results obtained in this study provide evidence that the assay system is a good tool for supporting the diagnosis of viral infection and contamination.

Detection of Enterohemorrhagic Escherichia coli O157:H7 Strains Using Multiplex Polymerase Chain Reaction (Multiplex PCR을 이용한 장출혈성 대장균 O157:H7의 검출)

  • 엄용빈;김종배
    • Biomedical Science Letters
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    • v.4 no.1
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    • pp.43-56
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    • 1998
  • A multiplex PCR method was designed by employing primers specific for the eaeA gene, conserved sequences of Shiga-like toxins (SLT-I.II), and the 60-MDa plasmid of enterohemorrhagic E. coli (EHEC) O157:H7 strain. A set of six synthetic oligonucleotide primers derived from sequences of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 were used in a multiplex PCR amplification procedure to detect these genes in the same enteric pathogens. In two enterohemorrhagic E. coli O157:H7 (ATCC 35150, ATCC 43894) reference strains, PCR products of 317bps (eaeA), 228bps (SLT-I.II), and 167bps (60-MDa plasmid) were successfully amplified simultaneously in a single reaction. However, the specific PCR products were not amplified in control strains of other enteric bacteria. The sensitivity of the multiplex PCR assay for detection of the SLT-I.II, eaeA, and 60-MDa plasmid genes of E. coli O157:H7 was found to be 2.5$\times$10$^{6}$ of bacteria in diarrheal stool to amplify all three bands. The multiplex PCR technology will allow large-scale screening of many clinical specimens or contaminated foods, and will be a very useful method for the detection of a wide range of microorganisms present in the environment, including EHEC O157:H7 in various types of specimens. The multiplex PCR assay has the potential to be used as a specific and rapid method for clinical diagnosis of disease caused by EHEC O157:H7.

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Multiplex Quantitative Real-time Polymerase Chain Reaction Assay for Rapid Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples (분변 시료에서 Mycobacterium avium subsp. paratuberculosis 의 빠른 검출을 위한 다중 실시간 중합효소연쇄반응기법의 개발)

  • Han, Jae-Ik;Jung, Young-Hun;Choe, Changyong;Yoo, Jaegyu;Kang, Seog-Jin;Yoo, Hansang;Park, Hongtae;Kwon, Eung-Gi;Cho, Yong-Il
    • Journal of Veterinary Clinics
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    • v.32 no.3
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    • pp.219-223
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    • 2015
  • Mycobacterium avium subsp. paratuberculosis (MAP) causes paratuberculosis or Johne's disease, an intestinal granulomatous infection in domestic and wild animals. The study aimed to develop and evaluate a panel of multiplex quantitative real-time polymerase chain reaction (mqPCR) assay for simultaneous detection of three MAP-specific genes (IS900, F57 and ISMAP02 genes). The analytic sensitivity (i.e., limit of detection, expressed as cells per 1 ml) was 150 for IS900, 1500 for F57, and 50 for ISMAP02. The specificity of the method was determined by testing 152 bovine fecal samples. Based on the test, it showed that the assay simultaneously detected the target genes in short period of time and at lower cost compared to laboratory routine tests. The test agreement between the assay and routine test was 94%. The discrepancy in the results was due to samples that were tested positive by the panel but negative by the routine tests, suggesting that the assay has higher sensitivity than the routine tests. In conclusion, the mqPCR assay could be a rapid and accurate testing tool for investigating paratuberculosis or Johne's disease cases in domestic and wild animals.

Multiplex PCR-aided Differential Diagnosis of Taeniid Species (Multiplex PCR을 이용한 조충류의 감별진단)

  • Lee, Hye-Jung;Seo, Min;Kwak, Sahng-June
    • Journal of Life Science
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    • v.20 no.6
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    • pp.955-959
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    • 2010
  • Differential diagnosis of the taeniid proglottids between Taenia asiatica and T. sagniata is a daunting task due to their close morphological similarity. However, to correctly diagnose them on time is important in managing infected patients, as well as for reducing serious complications such as cysticercosis. Currently, DNA-based methods for the dissection of genomic information of parasites are being employed to make accurate and rapid diagnoses in the field of parasitology. In this study, multiplex PCR was established and exploited to identify exact species of taeniid adult worms recovered from Korean people. To discriminate one from the other other, primers-Ta4978F, Ts5058F, Tso7421F, and Rev7915- were used for the multiplex PCR, which provided swift and precise identification of the taeniid worms being observed. Also,having instituted PCR methodology, we ascertained that easiness would be achieved to reassess and re-evaluate Korean endemic data on human taeniid cestodes.

Supraclavicular BCG Lymphadenitis Noted at 21 Months after BCG Vaccination Confirmed by a Molecular Method (분자생물학적 방법으로 확진한 접종 21개월 후에 발생한 BCG 쇄골상부 림프절염 1례)

  • Lee, Min Hyun;Chae, Moon-Hee;Park, Kyoung Un;Cho, Hye-Kyung
    • Pediatric Infection and Vaccine
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    • v.21 no.2
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    • pp.139-143
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    • 2014
  • Bacille Calmette-$Gu\acute{e}rin$ (BCG) lymphadenitis is the most common complication of BCG vaccination. It commonly occurs in infants aged <6 months involving ipsilateral axillary lymph nodes. We described BCG lymphadenitis in a 22-month-old boy presenting swelling of left supraclavicular lymph node that was confirmed by real-time polymerase chain reaction (PCR) and the multiplex PCR targeting the region of difference (RD).

Development of real-time PCR Detections against 11 Pathogens of Bombus Species (뒤영벌 병원체 11종에 대한 실시간 중합효소 연쇄반응 검출법 개발)

  • Min, Sang-Hyun;Kim, Jung-Min;Lim, Su-Jin;Kim, Byoung-Hee;Lee, Chil-Woo;Yoon, Byoung-Su
    • Journal of Apiculture
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    • v.32 no.2
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    • pp.99-109
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    • 2017
  • The multiple real-time PCRs against pathogens of Bombus species including DWV, IAPV, KBV, SBV, BQCV, kSBV, SBPV and Paenibacillus larvae, Mellisococcus plutonius, Lysinibacillus fusiformis, and Klebsiella oxytoca have been developed. One extracted nucleic acid from Beesample could be applied to 11 different PCRs in same time and condition. Specific PCR-products were amplified qualitative and quantitative manner inner 20 minutes successfully, when each 1000 molecules of pathogen-specific target DNA is existed as template, respectively. The multiple PCR detection that we propose would be expected to apply to quarantine test for international exchange of Bombus species.

Multiple Genetic Marker Analysis with Using Quantitative RT-PCR in Gastric Cancer (위암에서 정량적 역전사 중합효소연쇄반응을 이용한 다중 표지자 분석)

  • Yoo, Moon-Won;Lee, Hyuk-Joon;Choi, Soo-Min;Yu, Ji-Eun;Hur, Keun;Kim, Young-Kook;Yang, Han-Kwang
    • Journal of Gastric Cancer
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    • v.7 no.2
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    • pp.59-66
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    • 2007
  • Purpose: This study was aimed at evaluating the diagnostic validity of peritoneal dissemination of gastric cancer cells by performing multiple genetic marker analysis via quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in gastric cancer cell lines and gastric cancer tissues. Materials and Methods: Quantitative RT-PCR was performed on 12 human gastric cancer cell lines and 10 gastric cancer tissues with four mRNAs of carcinoembryonic antigen (CEA), Cytokeratin 20 (CK20), dopa decarboxylase (DDC), and L-3-phosphoserine phosphatase (L3PP). Results: Out of the 12 human gastric cancer cell lines we tested, CEA was overexpressed in four cell lines (33%), CK20 in one (8%), DDC in six (50%) and L3PP was expessed in all the lines (100%). Out of the 10 gastric cancer tissues we tested, CEA was overexpressed in nine tissues, CK20 in eight, DOC in nine and L3PP was overexpressed in all the tissues. L3PP was overexpressed in all the gastric cancer cell lines and tissues, but the levels of overexpression were lower than those of CEA and DDC. Conclusion: Multiple genetic marker analysis can compensate for the weak points of single marker analysis when testing gastric cancer, and three mRNAs of CEA, DDC and L3PP can be used as candidate genes.

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생쥐 초기 배아에서 Aquaporin 8과 9의 발현에 관한 연구

  • 신현상;계명찬;강수만;이성은;이지원;강한승;김문규
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.68-68
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    • 2003
  • Aquaporin은 막관통 통로 단백질(transmembrane channel protein)로서, 삼투압의 농도구배에 따라 세포막을 가로질러 물분자를 이동시키는 기능을 하고 있다. 포유류 초기배아에서 포배강 형성은 영양외배엽세포에서 $Na^+ / K^+$ATPase에 의한 이온 농도 구배가 형성되면 auqaporin에 의해 물이 포배강으로 유입되면서 이루어진다. 본 연구에서는 생쥐 초기배아에서 반정량적인 역전사 중합효소 연쇄반응 방법(semi-quantitative RT-PCR)과 실시간 역전사 중합효소 연쇄반응 방법(real-time RT-PCR)을 통하여 AQP8과 9의 mRNA발현을 조사하고 다중 면역형광현미경 방법(confocal immunofluorescence microscopy)을 통해 단백질 발현양상을 분석하였다. AQP8 mRNA는 상실기까지 발현되지 않다가 포배기에 이르러 발현되었고 AQP9 mRNA는 수정란에서부터 발현되어 포배기에는 유의할 정도로 증가하였다. 따라서 AQP8 mRNA는 배아유전자가 활성화되어 나타나는 것이고 AQP9 mRNA는 모계유전자 기원임을 알 수 있었다. AQP8 단백질은 상실배 단계까지 발현되지 않다가 포배시기에 영양외배엽세포사이의 접합면에 발현되었고 AQP9 단백질은 상실배 시기에 할구 사이의 인접 부위에서 강하게 발현되었다가 포배시기에는 세포간의 접합면에 약하게 발현하는 경향을 나타내었다. 실시간 역전사 중합효소 연쇄반응 방법으로 조사한 결과 포배에서 물과 글리세롤을 통과시키는 AQP9는 mRNA의 발현양이 AQP8보다 약 4배 정도 많았다. 또한 포배기에 이르러서야 물만을 통과시키는 AQP8의 발현이 나타나는 것을 보아 포배강 형성시 외부에서 영양외배엽을 통해 포배강으로 유입되는 물의 이동(trans- trophectodermal water movements)에 AQP9보다 AQP8이 더 중요하게 관여할 것으로 사료된다., K, Pb, Cd, Cr, Co, Cu, Ni)을 측정하였다. 실험 조건1의 결과로서 각 국의 유아용 일회용 기저귀의 중금속 함량은 거의 유사한 경향을 나타내었으며 Cr, Zn, Pb, Ni, Mn, Mg, Li, K는 detection limit(2 ppm) 이하였고, Cd, Fe, Co, Cu, Ca, Al, Sr는 검출되었지만 기준치 이하였다. 실험 조건2의 결과로서 측정 항목(Cr, Sb, Cd, Pb, Ni, Co, Cu)중 Cr, Cd, Ni, Cu는 detection limit(0.1 ppm) 이하였고, Sb, Pb, Co는 검출되었지만 기준치 이하였다.았다. 4%의 경우에는 8$0^{\circ}C$이하로 온도를 낮추는 것이 좋은 상태를 나타내었다. 이와 같은 결과는 일반적으로 화학적 레팅을 4%, 7%에서한 선행결과와 상당히 다른 결과이다.염 농도가 증가할수록 감소 현상을 보였다.X>, 75BG30은 8.6$\mu\textrm{m}$, 75BG40은 7.02$\mu\textrm{m}$로 나타났다. 따라서 경화제 양에 관계없이 10$\mu\textrm{m}$ 이하로 나타나, 경화제 10$m\ell$만으로 미세한 크기를 얻을 수 있음을 알 수 있다. 젤리 강도 변화에 따른 차이는 300BF는 78.09$\mu\textrm{m}$ 300BG는 56.32$\mu\textrm{m}$로, 75BF나 75BG에 비하여 현저히 증가하여, 젤라틴의 젤리 강도는 캡슐 제조 조건의 주요한 변수임을 알 수 있다.추출물 투여시 혈당강하 및 혈중콜레스테롤 강하가 나타났으며, 상엽복합추출물 투여와 운동을 병행시 이러한 감소 효과가 더 뚜렷하게 나타났다.교육의 적임자로 보는 시각이 비교적 높았고 약 1/2정도는 영양교육에 참여하겠다는 의지를 가지고 있을 뿐만 아니라 실제로 영양지도를

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