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다중 중합효소 연쇄반응을 이용한 DNA 바이러스의 동시검출
성혜란,주진영,이종길,정연복,송석길,Sung, Hye-Ran,Joo, Jin-Young,Lee, Chong-Kil,Chung, Yeon-Bok,Song, Suk-Gil 한국미생물학회 2007 미생물학회지 Vol.43 No.1
Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis B virus (HBV), parvovirus B19 (B19)등 4종의 바이러스는 인체에 감염을 일으키는 병원체로서 DNA를 유전물질로 함유한다. 각 바이러스 유전자의 염기서열을 분석하여 EBV CMV, HBV의 pol 유전자와 B19의 ns 유전자에 특이적으로 결합할 수 있는 primer를 설계 제작하고 단일 시험으로 4종의 바이러스를 동시에 검출할 수 있는 다중 중합효소 연쇄반응(Multiplex PCR)법을 확립하였다. Primer 염기서열, PCR 반응조성물의 농도, PCR 반응시간 및 온도조건을 최적화하여 민감도를 증대시킴으로써, 단일 시험으로 5-10 분자수의 유전물질까지 검출이 가능하였다. 또한 4종의 바이러스 사이에 교차반응이 일어나지 않았으며 생체시료를 이용한 시험에서도 특이성과 민감도가 유지됨을 확인하였다. 그러므로 본 연구에서 확립한 다중 중합효소 연쇄반응은 세포배양액 또는 생체 시료에 감염된 4종 DNA 바이러스진단에 효율적으로 이용할 수 있을 것이라고 판단된다. 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.
성혜란,김일회,김지연,이종길,정연복,한상배,송석길,Sung, Hye-Ran,Kim, Il-Hoi,Kim, Jee-Youn,Lee, Chong-Kil,Chung, Yeon-Bok,Han, Sang-Bae,Song, Suk-Gil 한국미생물학회 2008 미생물학회지 Vol.44 No.2
미생물 자동배양기를 이용한 감염성 물질의 검출은 시험적 오차의 경감은 물론 분리율 향상과 시간 단축을 가능하게 하였다. BacT/ALERT 3D 자동배양기는 미생물성장 시 발생되는 이산화탄소를 비색법으로 검출하는 장비로서 임상시료를 이용한 미생물 배양과 검출에 이용되어왔다. 자동배양기의 효율성을 검증하고 의약품의 무균 시험에 적용가능한지를 평가하기 위하여 6종의 세균을 이용하여 중식 및 검출특성을 분석하였다. 3종의 호기성세균 Pseudomonas aeruginosa, Micrococcus Iuteus, Bacillus subtilis와 Staphylococcus aureus는 1 CFU가 31.44 시간 내에 검출되었고, 혐기성균인 Clostridium sporogenes는 15.96시간에 균의 중식이 감지되었다. 저성장 혐기성세균인 Propionibacterium acnes는 $10^4$ CFU의 균수에서 검출에 129.36시간이 소요되었다. 이 같은 결과는전통적 직접 배양법보다 검출감도에 있어서 $10\sim10^5$배 높고 검출시간을 $2\sim10$10시간 단축한 것으로서 자동배양법이 미생물 증식과 검출에 효율적임을 말해준다. 따라서 본 시험에서 이용한 자동배양법은 임상시료에서의 감염원 진단뿐 아니라 생물의약품의 무균시험에 이용 가능하다고 판단된다. Modern automated culture systems have increased the isolation rate of microorganisms and shortened the time to detection, reducing experimental errors in diagnosis of infecting agents. BacT/ALERT 3D system is based on the colorimetric detection of $CO_2$ produced by the growing microorganisms. In order to evaluate the efficiency of the detection system, sterility test were performed using 6 bacteria. With standard aerobic and anaerobic bottles containing the liquid media, both three aerobic bacteria (P. aeruginosa, M. luteus, B. subtilis) and a facultative bacterium S. aureus were detected up to 1 CFU in 31.44 hr. In addition, growth of anaerobic C. sporogenes was recognized up to 1 CFU in 15.96 hr. The slowly growing bacteria P. acnes was detected up to 10,000 CFU in 129.36 hr. In comparison with conventional culture method, BacT/ALERT 3D automated culture system was more sensitive and saved detection time up to$2\sim10$ hr. Therefore, this automated culture system enables to efficiently detect bacteria in clinical samples and biological medicines.
대장균에서 D - Ribose 의 저친화성 수송에 관여하는 유전자
김창훈,박찬규,송석길 한국유전학회 1996 Genes & Genomics Vol.18 No.4
Mutants defective in the high-affinity transport of D-ribose are able to utilize ribose as a sole carbon and energy source, suggesting that other low-affinity transport systems for the sugar exist in the cells. In order to search for these transport systems, transposon mutagenesis was performed. Mutants with different phenotypes on ribose minimal growth were observed and classified into five groups. The precise locations of insertions on the chromosome were determined by in vivo cloning and analysis using polA mutation. Group I insertions revealed an operon encoding a new ABC-type transporter, located in 92.8 min. Group II insertions lie in rbs operon with defective growth on ribose due to a polar inactivation of rbsK, the gene for ribokinase. Group III insertion was found in the open reading frame, f547 coding for a potential integral membrane protein similiar to the proton symporter XylE. Insertions in xylA and its promoter region (Group IV) enhanced the growth on ribose, which was abolished by secondary insertion (Group V) in xylG gene that is divergently transcribed from xylA. XylG protein appears to be a component of the high affinity transporter for D-xylose. This indicates that the high affinity D-xylose transport system also supports the uptake of D-ribose.
이문규, 임선아, 송석길, 이청길 충북대학교 약품자원개발연구소 2013 약학논문집 Vol.28 No.-
Myeloid-derived suppressor cells (MDSCs) accumulate in cancer patients and tumor-bearing mice and suppress the host immune system. MDSCs represent a group of immature myeloid cells that express CD11b and Gr-1. Recently, we showed that the TLR agonist resiquimod, which binds to TLR7 and TLR8, induces the differentiation of MDSCs into mature macrophages and dendritic cells (DCs). Here we report that the differentiation-inducing activity of resiquimod is blocked by granulocyte mammary carcinoma 4T1 cells, and then cultured with resiquimod for three days in the presence or absence of GM-CSF. Phenotypic analysis showed that the MDSCs cultured in the presence or resiquimod differentiated into F4/80+ macrophages, and CD11c+/Ⅰ-Ad+ DCs. The resiquimod-induced differentiation of MDSCs into mature myeloid cells, however, was almost completely adjuvant for vaccines for patients with cancers in whom MDSCs play a major role in the suppression of anti-tumor immune responses.