이 작품은 부여 RC3의 AI089106에서 건강 (NIH)의 국립 연구소에 의해 지원되었다. MDR-TB 핵산은 열대 질병 (TDR), 제네바, 스위스에서 연구와 교육을위한 국제 연합 아동 기금 / 유엔 개발 계획 / 세계 은행 / 세계 보건기구 (WHO) 특별 프로그램에 의해 제공되었다. 우리는 프로토 타입 분석에 포함 할 수있는 특정 유전자 돌연변이에 대한 지침에 대한 미국 질병 통제 예방 센터의 박사 톰 Shinnick 감사합니다.

보편적 인 PCR주의 사항을 준수 실험실의 경우, 여러 가지 증폭 마이크로 어레이 기판 당 가스켓을 포함하고 여기에 설명 된대로, 벌크 용기에 모두를 동시에 증폭 마이크로 어레이를 세척하는 운영 체제는 더 효율적입니다. 다른 30,33보고 소모품 형식은, 완전히 밀봉 폐쇄의 증폭 검사에서 사후 증폭 마이크로 어레이 세척 단계를 수행 할 수 있습니다. 1. 설치 <ol><li> 추출하고 선택하는 방법과 적절한 바이오 안전성 조건에서 시료로부터 핵산을 정화. 요구 사항은 핵산 비대칭, 멀티 플렉스 PCR 증폭을위한 충분한 순도가 될 것입니다. </li><li> 오염 제거 솔루션과 표면 및 장비를 닦아 작업 공간을 준비합니다. </li><li> 얼음이나 차가운 블록을 증폭 시약을 놓습니다. </li><li> 증폭 마스터 믹스에 대한 멸균 1.5 ML의 미세 원심 분리 튜브를 레이블 및 멸균 0.2 ML의 microfuge TU 레이블각 시료에 대한 수. </li><li> 시약은 짧게 소용돌이를 해동 미니 원심 분리기 펄스 스핀 튜브의 바닥에 내용을 수집 한 후. DNA 형성 촉매 효소 와동하지 마십시오. 조심스럽게 혼합하고 미니 원심 분리기 펄스 스핀 따라 튜브를 가볍게. </li><li> 표 1에 나타낸 당 샘플 반응 부피를 이용하여 증폭 마스터 믹스를 준비한다. 가능한 피펫의 부정확성을 고려하여, 처리되고있는 샘플들의 총 수보다 적어도 하나 더 반응 부피를 준비한다. 마스터 믹스 추가 DNA 형성 촉매 효소 이상과 muliplex PCR 버퍼와 함께 제공되는 것 이상을 포함합니다. 단지 다른 모든 시약이 결합 된 후 마스터 믹스로 증폭 / 억제 컨트롤을 추가하고, 주식 시약 튜브는 저장에 돌려 보냈다. </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 시약 </td><td> 별 샘플 볼륨 (μL) </td><td> &lt;강한&gt; 결승 [] </td></tr><tr><td> HotStar의 Taq 플러스와 Mulitplex PCR 버퍼 </td><td> 25 </td><td> 1X </td></tr><tr><td> 소 혈청 알부민 (BSA) </td><td> 0.55 </td><td> 0.6 ㎎ / ㎖ </td></tr><tr><td> 포름 아미드 </td><td> 3.8 </td><td> 7.6 % </td></tr><tr><td> 추가 DNA 형성 촉매 효소 </td><td> 0.8 </td><td> 단위 / μL (4 대 총) </td></tr><tr><td> MDR-TB 프라이머 믹스 </td><td> 17,325 </td><td> - </td></tr><tr><td> RNA 분해 효소가없는 H 2 O </td><td> 2.1 </td><td> - </td></tr><tr><td> 증폭 / 억제 제어 </td><td> 1 </td><td> 5.0 FG / μL </td></tr><tr><td> 합계 </td><td> 49 </td><td></td></tr></tbody></table> 표 1. MDR-TB 증폭 마이크로 어레이 마스터 믹스 조성. <ol start="7"><li> 소용돌이 마스터 믹스와 미니 원심 분리기 펄스 스핀 튜브의 바닥에 내용을 수집합니다. </li><li> 마스터 믹스 49 μL와 M.의 1 μl를 결합 위의 단계 1.4에서 샘플 튜브의 각 결핵 DNA. 외부의 경우, 템플릿 컨트롤 (NTC)는 M. 대신에 분자 생물학 등급 물 1 μl를 사용하지 결핵 DNA 샘플. 소용돌이와 펄스 스핀 완료 튜브 (들). </li></ol> 2. 증폭 마이크로 어레이를로드 <ol><li> 마이크로 어레이 자체를 만지지주의하면서, 각각의 마이크로 어레이의 중심에 각 마스터 믹스 / 샘플의 48 μl를 추가합니다. </li><li> 마이크로 어레이 챔버에서 공기 방울없는 함정에주의하면서, 마이크로 어레이 가스켓 및 씰의 상단에 커버 슬립을 놓습니다. 기포가 증폭 효율에 부정적인 영향을 미칠 수있다과 유물 또는 이후의 마이크로 어레이 이미지의 노이즈를 만들 수 있습니다. </li></ol> 3. 열 사이클링 <ol><li> 일단 모든 마이크로 어레이는 평면 블록 열 자전거 타는 사람에 대한 샘플, 장소 기판으로로드됩니다. 가열 된 뚜껑 옵션이 &quot;Off&quot;로 설정되어 있는지 확인합니다. Akonni 생물계에서 얻은 장비는 이미 사용 prequalified됩니다. 그렇지 않으면, 평면 블록 열 자전거 타는 사람 (들)의 모든 마이크로 어레이 기판에 걸쳐 (들)을 균일하고 일관된 난방을 제공하는지 확인하는 것이 매우 중요합니다. </li><li> , 열 자전거 타는 소프트웨어를 열고 해당 프로그램을 선택, 반응 볼륨에 대해 &quot;50 μl를&quot;입력하고 실행을 시작합니다. 여기에 설명 된 열 순환 프로토콜 구성 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 열 순환 단계 </td></tr><tr><td> 1 </td><td> 88 ° C </td><td> 5 분 </td></tr><tr><td> 2 </td><td> 88 ° C </td><td> 30 초 </td></tr><tr><td> 3 </td><td> 55 ° C </td><td> 1 분 </td></tr><tr><td> 4 </td><td> 65 ° C </td><td> 30 초 </td></tr><tr><td> 5 </td><td> 50 사이클 단계 (2-4)를 반복 </td></tr><tr><td> 6 </td><td> 65 ° C </td><td> 3 분 </td></tr><tr><td> 7 </td><td> 55 ° C </td><td> 3 시간 </td></tr></tbody></table></li><li> 총 열 사이클의 수와 55 °의 C 포스트 증폭 대기 시간이 필요하거나 특정 실험에 적합한 사용자가 수정할 수 있음을 독립 변수입니다. MDR-TB의 마스터 믹스 비대칭 (주로 단일 가닥) 증폭 산물을 생성하기 때문에, 그렇지 않으면 기존의 지수 PCR 증폭 프로토콜에 활용 될 수있는 것보다 더 많은 열 사이클을 포함하는 것이 도움이됩니다. </li><li> 열 순환 및 하이브리드 프로그램이 완료되면, 프로그램을 종료 증폭 마이크로 어레이를 제거 소프트웨어를 닫고 열 자전거 타는 사람 (들)을 끕니다.</li></ol> 4. 세척 및 건조 <ol><li> 0.1 % 트리톤 X-100 세척 버퍼를, 적어도 250 ㎖의 탈 이온수 또는 밀리 Q 등급의 물을 각각 두 개의 컨테이너 - 동시에 24 기판까지 세척을 위해, 적어도 250 ㎖의 1X SSPE 준비합니다. 세척 버퍼는 사전에 제조 할 수있다. </li><li> 조심스럽게 평면 엔드 집게를 사용하여 각 증폭 마이크로 어레이 실에서 커버 슬립과 가스켓을 제거합니다.이 단계는 물리적으로 손상 젤 요소 배열의 가장 큰 위험이되는 지점이다. 증폭 된 핵산의 확산을 방지하기 위해 적절한 PCR 오염 컨트롤을 사용하여 작업 환경에서. </li><li> 조직학 슬라이드 홀더에 마이크로 어레이 기판을 배치하고 세척 버퍼를 포함하는 세척 함으로 모든 슬라이드를 놓습니다. 뚜껑이있는 용기를 커버. </li><li> 부드러운 교반과 함께 실온에서 10 분 동안 마이크로 어레이를 씻으십시오. </li><li> 마이크로 어레이 세 탈의 두 개의 연속 세척의 배 각각의 이상을 찍어밀리 Q 등급 물, 공기 건조. </li></ol> 5. 영상 비대칭 MDR-TB 프라이머 믹스를 Cy3 - 라벨 증폭 산물을 생성한다. 젤 요소 마이크로 어레이 이미지를 Cy3 할 수있는 표준 마이크로 어레이 이미 저와 이미지가 될 수있다. 다음 이미지를 만드는 절차가 Akonni에서 제공하는 MD​​R-TB의 마스터 믹스, Dx2100 필드 휴대용 화상 카메라 및 자동 분석 소프트웨어에 대한 특정이다. <ol><li> 이미 저에서 이더넷 케이블이 컴퓨터에 연결되어 있는지 확인합니다. </li><li> 열 화상 카메라의 전원을 켭니다. 전원 스위치는 기기의 후면 패널에 있습니다. 화상 카메라가 켜져있을 때 화상 카메라의 전면에 블루 LED가 점등됩니다. </li><li> 컴퓨터를 켭니다. </li><li> 컴퓨터 바탕 화면에서 두 번 자동 이미지 분석 소프트웨어를 실행하는 소프트웨어의 아이콘을 클릭합니다. </li><li> 이미 저 카메라와 연결을 설정하는 소프트웨어를 기다립니다. 연결이 설정되면, 분석 단추를 사용할 수 있습니다, 그리고메시지 &quot;성공적인 연결&quot;화면의 왼쪽 하단 모서리에 표시됩니다. </li><li> 마이크로 어레이는 사용자로부터 멀어 직면, 대물 렌즈 방향과 건조 증폭 마이크로 어레이 (microarray)를 삽입합니다. 마이크로 어레이는 부적절 렌즈 및 카메라에 대해 배향되는 경우, 자동 분석 소프트웨어는 형광 이미지 격자를 배치하는 데 실패 할 것이다. </li><li> 액세스 도어를 닫습니다. 미리보기 이미지가 컴퓨터 화면에 사용할 수 있습니다. </li><li> 드롭 다운 메뉴에서 MDR-TB 분석 스크립트를 선택하고 (밀리 초) 원하는 노출 시간을 입력합니다. 겔 소자 증폭 마이크로 어레이에 대한 전형적인 출발점 100-500 밀리 초이다. </li><li> 포화 픽셀은 빨간색으로 미리보기에 표시됩니다. 개별 스팟 안에 포화 픽셀의 수를 최소화하기 위해, 노광 시간을 조정한다. </li><li> 형광 이미지를 획득하고 분석하는 분석을 클릭합니다. 이 소프트웨어는 자동으로 배치됩니다이미지 위에 MDR-TB 분석 특정 그리드 통합 강도와 배경 값을 추출하고, 약제 내성 및 유전자형 결과를보고합니다. 자동 분석은 일반적으로 몇 초 정도 걸릴 것입니다. 흠집, 먼지, 형광 유물, 또는 마이크로 어레이 기능에 물리적 손상을 포함하는 도전적인 이미지의 소프트웨어는 분석을 완료하는 데 1 분까지해야 할 수도 있습니다. </li><li> 분석이 완료되면 저장을 클릭 파일 이름에 대상 폴더 유형을 선택하고 확인을 클릭합니다. 원하는 경우 기본 마이크로 어레이 데이터가. xls 파일로 저장 및 오프 라인 내보낼 수 있습니다, 분석한다. </li><li> 분석이 완료되면, 이미 저로부터 증폭 마이크로 어레이를 제거하고, 다음의 분석을 시작하기 위해 새로운 클릭. </li><li> 데이터 수집이 완료되면 소프트웨어를 닫고 컴퓨터를 종료하고 화상 카메라를 끄십시오. </li></ol>

<ol>
	<li>Lemaire, J. -. F., Casenghi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+diagnostics+for+tuberculosis:+fulfilling+patient+needs+first.">New diagnostics for tuberculosis: fulfilling patient needs first.</a> J. Int. AIDS Soc. 13, 40 (2010).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Tuberculosis diagnostic technology landscape. 42, (2012).</li><li>Notomi, T., Okayama, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucl. Acids Res. 28, e63 (2000).</li><li>Vincent, M., Xu, Y., Kong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicase-dependent+isothermal+DNA+amplification.">Helicase-dependent isothermal DNA amplification.</a> EMBO Rep. 5, 795-800 (2004).</li><li>Pritchard, C. G., Stefano, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+detection+of+viral+nucleic+acid+at+subattomole+levels+using+Q+beta+replicase.">Amplified detection of viral nucleic acid at subattomole levels using Q beta replicase.</a> Ann. Biol. Clin. 48, 492-497 (1990).</li><li>Compton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+sequence-based+amplification.">Nucleic acid sequence-based amplification.</a> Nature. 350, 91-92 (1991).</li><li>Guatelli, J. C., Whitfield, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isothermal,+in+vitro+amplification+of+nucleic+acids+by+a+multienzyme+reaction+modeled+after+retroviral+replication.">Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication.</a> Proc. Natl. Acad. Sci. U.S.A. 87, 1874-1878 (1990).</li><li>Shi, X. C., Liu, X. Q., Xie, X. L., Xu, Y. C., Zhao, Z. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+chip+array+for+differentiation+of+mycobacterial+species+and+detection+of+drug+resistance.">Gene chip array for differentiation of mycobacterial species and detection of drug resistance.</a> Chin. Med. J. , 3292-3297 (2012).</li><li>Troesch, A., Nguyen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mycobacterium+species+identification+and+rifampin+resistance+testing+with+high-density+DNA+probe+arrays.">Mycobacterium species identification and rifampin resistance testing with high-density DNA probe arrays.</a> J. Clin. Microbiol. 37, 49-55 (1999).</li><li>Caoili, J. C., Mayorova, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+TB-biochip+oligonucleotide+microarray+system+for+rapid+detection+of+rifampin+resistance+in+Mycobacterium+tuberculosis.">Evaluation of the TB-biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis.</a> J. Clin. Microbiol. 44, 2378-2381 (2006).</li><li>Waddell, S., Laing, K., Senner, C., Butcher, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+analysis+of+defined+Mycobacterium+tuberculosis+populations+using+RNA+amplification+strategies.">Microarray analysis of defined Mycobacterium tuberculosis populations using RNA amplification strategies.</a> BMC Genomics. 9, 94 (2008).</li><li>Fukushima, M., Kakinuma, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+identification+of+mycobacterium+species+isolates+by+DNA+microarray.">Detection and identification of mycobacterium species isolates by DNA microarray.</a> J. Clin. Microbiol. 41, 2605-2615 (2003).</li><li>Park, H., Jang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+genotyping+of+Mycobacterium+species+from+clinical+isolates+and+specimens+by+oligonucleotide+array.">Detection and genotyping of Mycobacterium species from clinical isolates and specimens by oligonucleotide array.</a> J. Clin. Microbiol. 43, 1782-1788 (2005).</li><li>Yao, C., Zhu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rpoB,+katG+and+inhA+gene+mutations+in+Mycobacterium+tuberculosis+clinical+isolates+from+Chongqing+as+determined+by+microarray.">Detection of rpoB, katG and inhA gene mutations in Mycobacterium tuberculosis clinical isolates from Chongqing as determined by microarray.</a> Clin. Microbiol. Infect. 16, 1639-1643 (2010).</li><li>Sun, A. H., Fan, X. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+rpoB+gene+mutations+in+rif-resistant+M.+tuberculosis+isolates+by+oligonucleotide+microarray.">Rapid detection of rpoB gene mutations in rif-resistant M. tuberculosis isolates by oligonucleotide microarray.</a> Biomed. Environ. Sci. 22, 253-258 (2009).</li><li>Volokhov, D. V., Chizhikov, V. E., Denkin, S., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+detection+of+drug-resistant+Mycobacterium+tuberculosis+with+a+scanning-frame+oligonucleotide+microarray.">Molecular detection of drug-resistant Mycobacterium tuberculosis with a scanning-frame oligonucleotide microarray.</a> Methods Mol. Biol. 465, 395-417 (2009).</li><li>Fu, L., Shinnick, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+action+of+INH+on+a+highly+INH-resistant+Mycobacterium+tuberculosis+strain+using+Genechips.">Understanding the action of INH on a highly INH-resistant Mycobacterium tuberculosis strain using Genechips.</a> Tuberculosis. 87, 63-70 (2007).</li><li>Fu, L. M., Shinnick, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+exploration+of+the+drug+action+of+capreomycin+on+Mycobacterium+tuberculosis+using+Affymetrix+oligonucleotide+GeneChips.">Genome-wide exploration of the drug action of capreomycin on Mycobacterium tuberculosis using Affymetrix oligonucleotide GeneChips.</a> J. Infect. 54, 277-284 (2007).</li><li>Denkin, S., Volokhov, D., Chizhikov, V., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+pncA+genotyping+of+pyrazinamide-resistant+strains+of+Mycobacterium+tuberculosis.">Microarray-based pncA genotyping of pyrazinamide-resistant strains of Mycobacterium tuberculosis.</a> J. Med. Microbiol. 54, 1127-1131 (2005).</li><li>Gryadunov, D. A., Mikhailovich, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+hybridisation+on+oligonucleotide+microarrays+for+analysis+of+drug-resistant+Mycobacterium+tuberculosis.">Evaluation of hybridisation on oligonucleotide microarrays for analysis of drug-resistant Mycobacterium tuberculosis.</a> Clin. Microbiol. Infect. 11, 531-539 (2005).</li><li>Tang, X., Morrix, S. L., Langone, J. J., Bockstahler, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+and+allele+specific+PCR+detection+of+point+mutations+in+Mycobacterium+tuberculosis+genes+associated+with+drug+resistance.">Microarray and allele specific PCR detection of point mutations in Mycobacterium tuberculosis genes associated with drug resistance.</a> J. Microbiol. Methods. 63, 318-330 (2005).</li><li>Wade, M. M., Volokhov, D., Peredelchuk, M., Chizhikov, V., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+mapping+of+mutations+of+pyrazinamide-resistant+Mycobacterium+tuberculosis+strains+with+a+scanning-frame+oligonucleotide+microarray.">Accurate mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains with a scanning-frame oligonucleotide microarray.</a> Diagn. Microbiol. Infect. Dis. 49, 89-97 (2004).</li><li>Yue, J., Shi, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+rifampin-resistant+Mycobacterium+tuberculosis+strains+by+using+a+specialized+oligonucleotide+microarray.">Detection of rifampin-resistant Mycobacterium tuberculosis strains by using a specialized oligonucleotide microarray.</a> Diagn. Microbiol. Infect. Dis. 48, 47-54 (2004).</li><li>Kim, S. Y., Park, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+CombiChip+Mycobacteria+Drug-Resistance+detection+DNA+chip+for+identifying+mutations+associated+with+resistance+to+isoniazid+and+rifampin+in+Mycobacterium+tuberculosis.">Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis.</a> Diagn. Microbiol. Infect. Dis. 54, 203-210 (2006).</li><li>Brown, T. J., Herrera-Leon, L., Anthony, R. M., Drobniewski, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+macroarrays+for+the+identification+of+MDR+Mycobacterium+tuberculosis.">The use of macroarrays for the identification of MDR Mycobacterium tuberculosis.</a> J. Microbiol. Methods. 65, 294-300 (2006).</li><li>Brossier, F., Veziris, N., Truffot-Pernot, C., Jarlier, V., Sougakoff, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+the+genotype+MTBDR+line+probe+assay+for+detection+of+resistance+to+rifampin+and+isoniazid+in+strains+of+Mycobacterium+tuberculosis+with+low-+and+high-level+resistance.">Performance of the genotype MTBDR line probe assay for detection of resistance to rifampin and isoniazid in strains of Mycobacterium tuberculosis with low- and high-level resistance.</a> J. Clin. Microbiol. 44, 3659-3664 (2006).</li><li>Padilla, E., Gonzalez, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+the+new+version+of+the+INNO-LiPA+Mycobacteria+and+GenoType+Mycobacterium+assays+for+identification+of+Mycobacterium+species+from+MB/BacT+liquid+cultures+artificially+inoculated+with+mycobacterial+strains.">Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and GenoType Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with mycobacterial strains.</a> J. Clin. Microbiol. 42, 3083-3088 (2004).</li><li>Barnard, M., Gey van Pittius, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnostic+performance+of+the+GenoType+MTBDRplus+Version+2+line+probe+assay+is+equivalent+to+that+of+the+Xpert+MTB/RIF+assay.">The diagnostic performance of the GenoType MTBDRplus Version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.</a> J. Clin. Microbiol. 50, 3712-3716 (2012).</li><li>Sekiguchi, J. -. I., Nakamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+a+line+probe+assay+for+rapid+identification+of+pncA+mutations+in+pyrazinamide-resistant+Mycobacterium+tuberculosis+strains.">Development and evaluation of a line probe assay for rapid identification of pncA mutations in pyrazinamide-resistant Mycobacterium tuberculosis strains.</a> J. Clin. Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

저자는 더 경쟁 재정적 이익을 선언하지 않습니다.

증폭 마이크로 어레이와 다중화의 정도는 최종적으로 비대칭 멀티 플렉스 PCR의 효율 아니라 마이크로 어레이에 의​​해 결정된다. 우리의 경험에 의하면, 10-12 독특한 프라이머 쌍은 쉽게 증폭 마이크로 어레이 형식으로 다중화 될 수있다. 기존의 프라이머 및 프로브 설계 기준에 따라서 그 중 하나는 또한 솔루션 상 핵산과 고정 된 마이크로 어레이 프로브, PCR 버퍼에 열 열 자전거 타는 사람의 ...

초기 증상 감지 및 신속한 치료가 결핵균 (MTB) 전송 1을 줄일 수있는 가장 효과적인 제어 전략으로 간주하고 치료 (POC) 또는 POC 시험 근처의 지점이 동시에 TB를 진단하는 TB 커뮤니티의 폭 넓은 합의가 지금이됩니다 및 약제 내성 (DR)가 필요합니다. 이러한 세 페이드의 GeneXpert 유용한 핵산 증폭 실험 같은 기술은 많은 TB 환자에 대한 진단 시간을 단축하고, 리팜핀 또는 다른 제 또는 제 2 라인 약물이 내성을 부여 선택된 돌연변이에 대한 내성을 나타내는 빠른 판독을 제공한다. 실시간 및 등온 핵산 증폭 검사가 MDR-TB 초래할 약물 내성 돌연변이를 식별하도록 설계되어 있지만, 그들이 검출 돌연변이 스펙트럼은, 환자의 약물 내성 프로파일에 대응하는 개별 약물 요법을 디자인하는 것이 부적절 및 관련 기술 제약광학적 크로스 토크 (cross-talk) 또는 증폭 및보고 케미스트리의 복잡성 3-7 유전자좌 또는 검출 된 돌연변이의 수를 제한 할 수있다. 따라서, 높은 다중 용량 감지 기술은 MDR-TB의 POC 진단에 알려진 격차를 해결하기 위해 필요합니다. 마이크로 어레이 및 WHO 승인 한 Hain의 라인 프로브 분석은 MDR-TB 8-29 진단의 &quot;다중 유전자, 여러 변이&quot;문제를 해결할 수 있습니다. 불행하게도, 이러한 하이브리드 기반의 멀티 플렉스 감지 플랫폼은 다단계, 복잡한 분자 기술에 상당한 훈련과 능력을 필요로 오픈의 증폭 프로토콜을 사용합니다. 증폭 마이크로 어레이 (30)는 이러한 마이크로 어레이 작업 흐름 및 운영 문제 중 일부를 해결하기 위해 설계되었습니다. 단순화 유체 원리는, 증폭 혼성화 및 단일 마이크로 유체 챔버 내 표적 핵산을 검출한다. 사용자는 핵산 증폭 MAS를 소개터가 피펫 유체 챔버에 혼합 및 열 순환 프로토콜을 시작합니다. 여기에 도시 된 배치 프로세싱 방법을 위해, 마이크로 어레이이어서, 벌크 용액으로 세척하고, 건조하고, 이미지화된다. 이 연구는 rpoB에 대한 MDR-TB 마이크로 어레이 검사 (30 돌연변이), katG (2 돌연변이), 인하대 (4 돌연변이), RPSL (2 돌연변이), embB (1 돌연변이), IS1245, IS6110를 사용하여 증폭 마이크로 어레이의 기능을 보여줍니다 , 및 내부 증폭 및 하이브리드 제어 할 수 있습니다. 마이크로 어레이 프로브 (야생형 (WT) 및 단일 염기 변이 (MU)) 중 적어도 하나의 일치하는 쌍은 그 각각의 돌연변이 포함되어 있습니다. 다중 약물 내성 M.보기에서 정제 된 핵산 결핵은 TDR 결핵 균주 은행 (31)이다. 그 밖에 32 바와 같이 젤 요소 마이크로 어레이 우리 polymeriz 각 프로브 4 % 단량체 및 0.05 ㎜를 사용하는 것을 제외하고, 본질적으로 공중합하여 유리 기판 상에 제조된다ATION 혼합물. 배열은 사용하기 전에 50 ㎖ 가스​​켓으로 둘러싸여 있습니다. 열 사이클링, 하이브리드 및 세척 단계 후, 증폭 마이크로 어레이는 Akonni 휴대용 분석기에 몇 군데 있습니다. 배경 보정, 통합 된 신호 강도가 원료에서 얻을 수 있습니다. 고정 원 알고리즘을 사용하여 TIF 이미지가. 각 겔 요소 잡음은 로컬 스폿 배경의 세 배의 표준 편차로 산출된다. 신호대 잡음비 (SNR)에 신호 ≥ 3 값 유전자 대상은 통상적으로 검출 가능한 것으로 간주된다. 각 유전자 또는 코돈에 특이적인 변이의 존재 여부를 결정하기 위해, 판별 비율 (WT-MU) / (WT + MU)로서 SNR 값으로부터 계산된다. 판별 비율 0 야생형 시퀀스를 표시한다 &lt;비율 반면 0, 로커스에서 약물 - 내성 돌연변이를 표시한다&gt;는.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

마이크로 어레이 워크 플로우를 간소화하는 것은 정기적으로 낮은 자원 환경에서 사용할 수있는 MDR-TB 마이크로 어레이 기반의 진단 프로그램을 만들기위한 필요한 첫 번째 단계입니다. 증폭 마이크로 어레이는 단일 솔루션 내의 단일 마이크로 유체 챔버로 비대칭 PCR 증폭, 타겟 사이즈 선택 대상 라벨링 및 마이크로 어레이 혼성화를 결합한다. 일괄 처리 방법은 다중 약물 내성 결핵균 (MDR-TB)을 유전형에 대한 9 복잡한 비대칭 마스터 믹스 저밀도 젤 요소 마이크로 어레이와 함께 보여줍니다. 여기에 설명 된 프로토콜은 6 시간에 완성 된 게놈 DNA의 적어도 1,000 셀 당량의 정확한 유전자형을 제공 할 수 있습니다. 온칩 세척 단계 통합하면 완전히 폐쇄 된 앰플 리콘 방법 및 시스템에 발생되는, 가능하다. 증폭 마이크로 어레이와 다중화의 범위는 궁극적으로 단일 마스터 m으로 결합 할 수있는 프라이머 쌍의 개수에 의해 제한된다IX 여전히 오히려 어레이에 고정화되는 프로브의 개수보다 원하는 민감도 및 특이도 성능 메트릭을 달성한다. 마찬가지로, 총 분석 시간은 구체적인 용도, 연구 문제 및 소망의 검출 한계에 따라 짧게 또는 길게 할 수있다. 그럼에도 불구하고, 일반적인 접근은 상당히 노동 집약적 및 시간 소모적 인 처리 단계의 수를 줄임으로써 최종 사용자에 대한 마이크로 어레이 플로를 간소화하고, 일상적인 임상 사례로 마이크로 어레이 기반 진단을 번역을 위해 단순화 된 생화학 적 및 미세 유동 경로를 제공한다.

정 성적 이미지 분석은 자동화 이미지 분석 소프트웨어에 의해 생성 된 데이터 테이블에서 식별되는 도전 실험 소음 또는 가변성의 소스에 대한 통찰력을 제공 할 수있다. 따라서, 1) 모든 젤 성분이 손상 및 손상되지, 2) 글로벌 배경 잡음 비율 (SNR) 값을 각각의 신호에 영향을 줄 수있는 형광 유물이없는 것을 확인 시각적으로 유용 할 수있다, 3)에 대한 증거는 없다 기포 형성 또는 불균일 증폭 / ?...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

증폭 마이크로 어레이 크게 최종 사용자를위한 마이크로 어레이 플로 간소화 단일 챔버로 비대칭 PCR 증폭 및 마이크로 어레이 혼성화를 결합한다. 마이크로 어레이 워크 플로우를 간소화하는 것은 정기적으로 낮은 자원 환경에서 사용할 수있는 마이크로 어레이 기반의 진단 프로그램을 만들기위한 필요한 첫 번째 단계입니다.

다중 약물 내성을 보여<em&gt; 결핵균</em&gt; 증폭 마이크로 어레이

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.5K 조회수.  Akonni Biosystems, Inc..  증폭 마이크로 어레이 크게 최종 사용자를위한 마이크로 어레이 플로 간소화 단일 챔버로 비대칭 PCR 증폭 및 마이크로 어레이 혼성화를 결합한다. 마이크로 어레이 워크 플로우를 간소화하는 것은 정기적으로 낮은 자원 환경에서 사용할 수있는 마이크로 어레이 기반의 진단 프로그램을 만들기위한 필요한 첫 번째 단계입니다. 

비디오: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

Microtiter 플레이트 형식의 맛을 희석하여 첫 번째와 두 번째 라인 의약품 Mycobacterium 결핵의 복합 항균 자화율 테스트

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

연구

면역학 및 감염병학

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

의 샘플 준비<em&gt; Mycobacterium 결핵</em핵 자기 공명 Metabolomic의 연구&gt; 압축을 풉니 다

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

의 성장 결핵은 Mycobacterium Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.

Prions의 단백질 Misfolding의 순환 증폭

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

자연의 전송에 결핵 말라리아 동시 감염을 연구하는 실험 모델 결핵균와 변형체 berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

복구하는 새로운 미세 절제 접근<em&gt; 결핵균</em폐 육아종 임베디드 포르말린 고정 파라핀에서&gt; 특정 성적 증명서

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

에 대한 기능성 연구를위한 3D 인간의 폐 조직 모델<em&gt; 결핵균</em&gt; 감염

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

세포 내 타겟팅 억제제의 효능 및 세포 독성 검사를위한 시스템<em&gt; 결핵균</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

높은 처리량을 지원 분석은 대식 세포 계대에 대한 약물 효능을 평가하기<em&gt; 결핵균</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

18FDG PET/CT 영상 공부 결핵균 감염 및 비 인간 영장류에서 치료 도구로 서의 분석

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...