이 작품은 Profileringsfonds azM (PF245)에 의해 지원되었다.

부 I : 병원균 식별 1. 샘플 준비 참고 : 다음과 같은 프로토콜에서 설명하는대로 전체 분자 흐름이 분자 진단에있어서의 품질 보증 3에 대한 권장 사항에 따라 수행되어야한다. <ol><li> 1시 10분 희석 샘플이 될 1.5 ML 반응 관에 0.9 % NaCl 0.9 ML에 혈액 문화의 0.1 ML의 나누어지는을 추가합니다. (희석 qPCR 억제를 방지하기 위해서). </li><li> 펠렛 5 분 박테리아 DNA를 13,400 XG에서 샘플을 원심 분리기. </li><li> 100 μl 멸균 demineralised H 2 O를 안에 세균 펠릿을 Resuspend </li><li> 4 ° C까지 추가로 사용에서 DNA 샘플을 보관합니다. </li></ol> 2. 식별 분석 : 실시간 16 rDNA PCR <ol><li> 다음과 같이 반응 섞어 준비합니다. 분석은 시료 당 4 개의 별도의 반응으로 구성되어 있습니다. 각 혼합물은 12.50 μl 마스터를 포함믹스, 0.9 μm의 순방향 프라이머 (5 TCCTACGGGAGGCAGCAGT-3) 7, 0.6 μm의 역방향 프라이머 (5 GGACTACCAGGGTATCTAATCCTGTT-3) 8 프로브의 패널. 프로브의 금액은 아래에있는 네 개의 별도의 반응 각각에 대해 제공됩니다. </li><li> 첫 번째 반응은 다음과 같습니다 <ul><li> 0.2 μm의 범용 프로브 (5-식구들-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) 8 </li><li> 0.2 μm의 P. 녹농균이라는 박테리아 프로브 (5 조-CCAAAACTACTGAGCTAGAGTACG-3-BHQ1) </li></ul></li><li> 두 번째 반응은 다음과 같습니다 <ul><li> 0.2 μm의 E. 대장균 프로브 (5 조-GGAGTAAAGTTAATACCTTTGCTCATT-3-BHQ1) </li><li> 0.2 μm의 모나스 종. 프로브 (5-NED-CCTTCCTCCCAACTTAAAGTGCTT-3-MGBNFQ) </li></ul></li><li> 세 번째 반응이 포함됩니다 : <ul><li> 0.2 μm의 포도상 SPP. 프로브 (5-NED-AATCTTCCGCAATGGGCGAAAGC-3-MGBNFQ) </li><li> 0.2 μm의 S. 구균 프로브 (5-식구들-AGATGTGCACAGTTACTTACACATAT-3-BHQ1) </li><li> 0.2 μm의 Enterococcus 종. (5 - 죠 - TCCTTGTTCTTCTCTAACAACAGAG-3-BHQ1) </li></ul></li><li> 네 번째 반응이 포함됩니다 : <ul><li> 0.2 μm의 범용 프로브 (5-식구들-CGTATTACCGCGGCTGCTGGCAC-3-BHQ1) </li><li> 0.3 μm의 연쇄상 구균 SPP. 프로브 (5-NED-CCAGAAAGGGACSGCTAACT-3-MGBNFQ) </li><li> 0.2 μm의 S. pneumoniae 프로브 (5 조-CCAAAGCCTACTATGGTTAAGCCA-3-BHQ1) </li></ul></li><li> 20 μl의 총 볼륨에 도달 멸균 demineralised H 2 O를 추가합니다. 96 - 웰 PCR 플레이트의 우물 각 반응 혼합물 20 μl를 추가합니다. </li><li> 각도에 샘플의 5 μl를 추가합니다. </li><li> 96 - 웰 PCR 플레이트를 밀봉하는 접착제 필름을 사용합니다. </li><li> 다음과 같은 최적의 온도 사이클 조건을 사용하여 ABI 프리즘 7900HT 리얼 타임 PCR 시스템에 접시를 실행합니다 : <ul><li> 10 분 대한 ° C에서 50 미리 가열 </li><li> 15 분 95 ° C에서 초기 변성 </li><li> 42주기 <ul type="circle"><li> 15의 95 ° C에서 변성 </li><li> 1 분 동안 60 ° C에서 풀림 </li></ul></li></ul></li></ol> 3. 결과의 분석 <ol><li> 탭 분석 설정에서 0.1 CT 분석의 한계를 조정합니다. 6 월말 (주기) : 15 시작하는 기준 구성 (주기)를 좁힐. </li><li> 모든 샘플에 대한주기 임계값 (CT) 값을 기록합니다. 긍정으로서 PCR 결과를 고려할 컷 - 오프 값은 35 CT-값으로 설정할 수 있습니다. 혈액 배양에 존재하는 박테리아의 양은 CT-값을 35 이하로 발생, 10 7 10 11 CFU / ML로 원거리. </li></ol> 부 II : 항생제 자화율 시험 4. 긍정적인 혈액 배양 9 시부터 박테리아의 분리 <ol><li> 긍정적인 혈액 배양 병에서 국물의 5 ML을 기음과 혈청 분리관에 그 파일을 전송합니다. </li><li> 10 분 2000 XG에서 혈청 분리관을 원심 분리기. </li><li> 혈청 분리관에서 뜨는 폐기하십시오. </li><li> 전송 BACteria는 멸균 면봉으로 튜브 겔 층에서 0.9 %의 식염수에 0.5 맥팔랜드 표준 정지 얻을 때까지. </li></ol> 5. 마이크로 Titre 플레이트의 접종 <ol><li> 5 × 10 5 CFU / ML의 현탁액을 형성하는 두 농축 뮐러 Hinton II 국물의 0.5 맥팔랜드 서스펜션을 희석. </li><li> 항생제 (표 1)의 선택을 포함하는 마이크로 titre 플레이트의 우물이 서스펜션을 추가합니다. </li><li> 6 H에 대해 37 ° C에서 마이크로 titre 판을 품어. </li><li> 4 ° C (마이너스 성장 제어 등)에 정지 나누어지는을 저장합니다. </li><li> 부화 6 H 후, 멸균 튜브에 각 우물의 콘텐츠를 전송할뿐만 아니라, 부정적인 성장 제어 샘플 4 ° C.에 저장된되었다 그 </li><li> 5 분 용 16,000 XG에 튜브를 원심 분리기. </li><li> 조심스럽게 세균성 펠렛을 방해하지 않고, 표면에 뜨는를 제거합니다. </li><li> 멸균 demineralised H에 펠릿을 Resuspend <sUB&gt; 2 O. </li><li> 멸균 demineralised H 2 O에 동안 10 배 샘플을 희석 </li></ol> 6. 실시간 16 rDNA PCR 10 <ol><li> 다음과 같이 PCR 혼합물 준비 : <ul><li> 12.50 μl IQ SYBR 녹색 Supermix </li><li> 앞으로 0.5 μm의 프라이머 16-1 (5 TGGAGAGTTTGATCCTGGCTCAG-3) 11 </li><li> 0.25 μm의 역방향 프라이머의 16-2 (5 TACCGCGGCTGCTGGCAC-3) 11 </li><li> 20 μl의 총 볼륨에 무균 demineralised H 2 O </li></ul></li><li> 96 - 웰 PCR 플레이트의 우물에 PCR 혼합물의 20 μl를 추가합니다. </li><li> 각도에 샘플의 5 μl를 추가합니다. </li><li> 96 - 웰 PCR 플레이트를 밀봉하는 접착제 필름을 사용합니다. </li><li> 다음과 같은 최적의 온도 사이클 조건을 사용하여 MyiQ 단일 컬러 리얼 타임 PCR 검출 시스템에 접시를 실행합니다 : <ul><li> 4 분 95 ° C에서 초기 변성 </li><li> 초기는 ° C ~ 30의 65에서 풀림 </li><li> 35주기 <ul><li> 9시 변성15의 5 ° C </li><li> 1 분 동안 60 ° C에서 풀림 </li></ul></li><li> (° C ~ 0.57 ° C 단위로 20 분 안에 60-95에서) 곡선 분석을 용융 </li></ul></li></ol> 7. 결과의 분석 <ol><li> 다음 수식 (항생제의 종류에 따라) 중 하나를 사용하여 절단 CT 값을 계산합니다. <ul><li> 일반적으로 : 컷 - 오프 CT 값 = CT 값 긍정적인 성장을 제어 + 0.5 X (CT 값 마이너스 성장 관리 - CT 값 긍정적인 성장을 제어) </li><li> Piperacillin, piperacillin / tazobactam과 그람 음성 막대의 ceftazidime, Amoxicillin, oxacillin과 트리 메소 / S.aureus의 설파 메 톡 사 졸, Enterococcus SPP의 Amoxicillin :. 컷 - 오프 CT 값 = CT 값 긍정적인 성장을 제어 + 0.25 X (CT 값 마이너스 성장 제어 - CT 값 긍정적인 성장을 제어) </li></ul></li><li> 긍정적인 성장 관리로 멸균 demineralised H 2 O와 incubated 샘플을 사용하십시오.</li><li> 미생물에 따라 적절한 제외어 성장 제어를 사용합니다 : <ul><li> 그람 음성 막대 샘플은 항생제의 혼합으로 incubated </li><li> Enterococcus SPP. 샘플은 4 ° C에 보관 </li><li> S.aureus 샘플은 4 ° C에 보관 </li></ul></li><li> 자화율 (S) 또는 다음과 같이 테스트 항생제에 대한 변형의 저항 (R) 결정 : <ul><li> 컷 - 오프 CT 값보다 높은 CT 값은 느끼기 쉬운 상태를 나타냅니다 </li><li> 컷 - 오프 CT 값보다 낮은 CT 값은 저항을 나타냅니다 </li></ul></li></ol> 8. 대표 결과 두 모델 생물은 그람 음성 E. 즉 대장균 및 그람 양성 S. 구균은 세균성 병원균과 그 항균 프로파일의 결정 탐지 및 식별을위한 통합 절차를 시각화하기 위해 선택됩니다. 프로토콜의 첫 번째 부분은 병원균 식별로 구성되어 있습니다. Specific 프로브들은 8 임상 관련 미생물의 검출을 위해 설계되었습니다. 세균성 패널에 포함된 대상의 존재에 증폭 곡선이 생성되고 CT 값 (그림 2) 계산됩니다. 긍정으로서 PCR 결과를 고려할 컷 - 오프 값은 35 CT 값으로 설정됩니다. 그림 2A에서 E.coli에 감염된 혈액 문화의 신분 프로필이 표시됩니다. 16 범용 프로브라는 두 개의 독립된 반응 혼합물에 포함되어 있으며 결과적으로이 증폭 곡선 (25.20 및 25.95의 CT) 생성됩니다. 세 번째 신호는 E.위한 프로브 특정에서 파생됩니다 대장균 (27.04 중 CT). S.의 식별 구균에 감염된 혈액 문화는 그림 2B에 표시됩니다. 16 범용 프로브는 33.35과 33.71의 증폭 신호를 가지고 있습니다. 남은 두 신호는 황색 포도상 SPP 특정 프로브에서 파생됩니다. 그리고 S. 구균 (32.48 및 30.59의 CT). 그림 3도 표시되었다 E.coli 변형을 나타내는 항생제 자화율 시험 증폭 플롯의 예입니다 그림 2A 인치 각 라인은 세균 샘플로 incubated되었다 싶고, 항생제를 나타냅니다. 낮은 CT 값을 가진 샘플은 성장이 테스트 항생제에 대한 항생제, 나타내는 저항의 면전에서 오류가 발생하였습니다있는 샘플입니다. 반대로, 높은 CT 값이 더 성장 때문에 테스트 항생제에 대한 항생제, 표시 자화율의 효과적인 작동 중에 발생하지 않은있는 샘플을 나타냅니다. 표 1 중에 항균 프로필의 결정을 보여줍니다 대장균 및 S. 구균의 분리합니다. 모든 CT 값은보고하고, 프로토콜 텍스트 (7.1)에서 설명한 수식을 사용하여, 두 컷 - 오프 CT 값은 calcu 있습니다저항 및 자화율 구별 lated. 보고된 CT 값이 계산 컷 - 오프 CT 값 (및 그 반대)보다 낮으면 변형은 항생제에 강한 것입니다. <img alt="그림 1" src="/files/ftp_upload/3254/3254fig1.jpg" /> rDNA PCR 실시간 기가 사용 병원균 식별 및 항생제 자화율 시험 절차의 그림 1. 플로우 차트. <img alt="그림 2" src="/files/ftp_upload/3254/3254fig2.jpg" /> 그림 2 인식 분석 :. 증폭 플롯과 사이클 문턱 값 (CT 값) 특정 프로브는 인과 병원체의 식별에 사용되는 반면 긍정적인 혈액 문화, 유니버설 16 rDNA 탐침에 의해 감지된다.. 혈액 문화의 A.의 증폭 줄거리는 엑스타시를 포함하는 대장균, S.를 포함하는 혈액 문화의 바실러스 증폭 줄거리 구균, Pseu AE, Pseudomon박테리아와 같은, 유니, 16 범용 프로브, Ecoli, 에스 대장균 프로브, Pseu SP, 모나스 종. 프로브, S. pneu, 연쇄상 구균 pneumoniae 프로브, Strep SP, 연쇄상 구균 SPP. 프로브, 힘들어, Enterococcus 종. 프로브, S. 구균, 포도상 구균 프로브, 포도상 SP, 포도상 SPP. 프로브. <img alt="그림 3" src="/files/ftp_upload/3254/3254fig3.jpg" /> 중에 항생제 자화율 테스트 그림 3. 증폭 줄거리 대장균은 (예제 1) 분리. 각 곡선이 변형이 함께 incubated되었다 싶고, 항생제를 나타냅니다. 초기 신호는 변형이 테스트 항생제의 면전에서 성장하고 항생제에 대한 내성 따라서는 것을 의미 높은 세균 하중에 의해 발생합니다. 늦은 신호 변형율은 항생제의 면전에서 성장하지 않은 것을 의미, 즉, 그것은 쉽습니다. <table border="1"><tbody&gt; <tr><td> 예제 1 : E. 대장균 </td><td></td><td></td><td> 예제 2 : S. 구균 </td><td></td><td></td></tr><tr><td> 대서양 표준시 </td><td> CT </td><td> R / S </td><td> 대서양 표준시 </td><td> CT </td><td> R / S </td></tr><tr><td> Amoxicillin 8 MG / L </td><td> 16,83 </td><td> R </td><td> Amoxicillin 0.25 MG / L </td><td> 21,03 </td><td> R </td></tr><tr><td> Amoxicillin-clavulanate 4분의 8 MG / L </td><td> 17,36 </td><td> R </td><td> Oxacillin이 MG / L </td><td> 25,80 </td><td> S </td></tr><tr><td> Piperacillin 16 MG / L </td><td> 16,67 </td><td> R </td><td> Vancomycin이 MG / L </td><td> 25,20 </td><td> S </td></tr><tr><td> Piperacillin-tazobactam 16 /4 MG / L </td><td> 24,15 </td><td> S </td><td> Gentamicin 4 MG / L </td><td> 25,86 </td><td> S </td></tr><tr><td> * 시프 로플 록 사신 1 MG / L </td><td> 29,72 </td><td> S </td><td> 트리 메소 - 설파 메 톡 사 졸 38분의 2 MG / L </td><td> 24,62 </td><td> S </td></tr><tr><td> Ceftazidime 1 MG / L </td><td> 24,03 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Ceftazidime 8 MG / L </td><td> 26,58 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> Gentamicin 4 MG / L </td><td> 29,83 </td><td> S </td><td></td><td></td><td></td></tr><tr><td> 트리 메소 - 설파 메 톡 사 졸 38분의 2 MG / L </td><td> 27,60 </td><td> S </td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 마이너스 성장 제어 (항생제의 혼합물) </td><td> 30,41 </td><td></td><td> 마이너스 성장 제어 (샘플은 4 ° C에 보관) </td><td> 27,42 </td><td></td></tr><tr><td> 긍정적인 성장 제어 </td><td> 16,90 </td><td></td><td> 긍정적인 성장 제어 </td><td> 20,22 </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 컷 - 오프 CT-값 1 * </td><td> 21,76 </td><td></td><td> 컷 - 오프 CT-값을 1 *** </td><td> 23,82 </td><td></td></tr><tr><td> 컷 - 오프 CT-값 2 ** </td><td> 18,75 </td><td></td><td> 컷 - 오프 CT-값 2 **** </td><td> 22,02 </td><td></td></tr><tr><td> * amoxicillin, amoxicillin-clavulanate, * 시프 로플 록 사신, gentamic 위해트리 메소 - 설파 메 톡 사 졸에서 ** piperacillin, piperacillin-tazobactam, ceftazidime에 대한 </td><td></td><td></td><td> vancomycin과 gentamicin을 위해 *** **** amoxicillin, oxacillin과 트리 메소 - 설파 메 톡 사 졸의 경우 </td><td></td><td></td></tr></tbody></table> 표 1. 두 샘플 (E.coli와 S.aureus)의 항생제 자화율 시험의 결정. PCR-검정의 CT 값이 자동으로 프로토콜 텍스트에 표시된 수식을 사용하여 긍정적이고 부정적인 성장 컨트롤에서 두 컷 - 오프 CT의 alues​​을 계산 수있는대로이 Excel 파일에 복사되었습니다. 항생제는 컷 - 오프 CT 값보다 CT 값을 낮게 나타나면 CT 값이 절단보다 높은 아니었다면, 변형은 항생제에 강한이며, 변형은 쉽습니다.

<ol>
	<li>Wallet, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preliminary+clinical+study+using+a+multiplex+real-time+PCR+test+for+the+detection+of+bacterial+and+fungal+DNA+directly+in+blood.">Preliminary clinical study using a multiplex real-time PCR test for the detection of bacterial and fungal DNA directly in blood.</a> Clin. Microbiol. Infect. 16, 774 (2010).</li><li>Beekmann, S. E., Diekema, D. J., Chapin, K. C., Doern, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+rapid+detection+of+bloodstream+infections+on+length+of+hospitalization+and+hospital+charges.">Effects of rapid detection of bloodstream infections on length of hospitalization and hospital charges.</a> J. Clin. Microbiol. 41, 3119 (2003).</li><li>Raymaekers, M., Bakkus, M., Boone, E., de Rijke, B., Housni, H. E. l., Descheemaeker, P., De Schouwer, P., Franke, S., Hillen, F., Nollet, F., Soetens, O., Vankeerberghen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Diagnostics+working+group.+Reflections+and+proposals+to+assure+quality+in+molecular+diagnostics.">Molecular Diagnostics working group. Reflections and proposals to assure quality in molecular diagnostics.</a> Acta. Clin. Belg. 66, 33 (2011).</li><li>Peters, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+developments+in+the+diagnosis+of+bloodstream+infections.">New developments in the diagnosis of bloodstream infections.</a> Lancet Infect. Dis. 4, 751 (2004).</li><li>Hansen, W. L., Beuving, J., Bruggeman, C. A., Wolffs, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+probes+for+the+diagnosis+of+clinically+relevant+bacterial+infections+in+blood+cultures.">Molecular probes for the diagnosis of clinically relevant bacterial infections in blood cultures.</a> J. Clin. Microbiol. 48, 4432-4432 (2010).</li><li>Beuving, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+susceptibility+testing+of+grown+blood+cultures+by+combining+culture+and+real-time+polymerase+chain+reaction+is+rapid+and+effective.">Antibiotic susceptibility testing of grown blood cultures by combining culture and real-time polymerase chain reaction is rapid and effective.</a> PLoS ONE. 6, (2011).</li><li>Rolain, J. M., Mallet, M. N., Fournier, P. E., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+for+universal+antibiotic+susceptibility+testing.">Real-time PCR for universal antibiotic susceptibility testing.</a> J. Antimicrob. Chemother. 54, 538 (2004).</li><li>Nadkarni, M. A., Martin, F. E., Jacques, N. A., Hunter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+bacterial+load+by+real-time+PCR+using+a+broad-range+(universal)+probe+and+primers+set.">Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.</a> Microbiol. 148, 257 (2002).</li><li>Waites, K. B., Brookings, E. S., Moser, S. A., Zimmer, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+susceptibility+testing+with+positive+BacT/Alert+blood+cultures+by+using+MicroScan+overnight+and+rapid+panels.">Direct susceptibility testing with positive BacT/Alert blood cultures by using MicroScan overnight and rapid panels.</a> J. Clin. Microbiol. 36, 2052 ( ).</li><li>Vliegen, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+identification+of+bacteria+by+real-time+amplification+and+sequencing+of+the+16S+rRNA+gene.">Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene.</a> J. Microbiol. Meth. 66, 156 (2006).</li><li>Hall, L., Doerr, K. A., Wohlfiel, S. L., Roberts, G. D. <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+MicroSeq+system+for+identification+of+mycobacteria+by+16S+ribosomal+DNA+sequencing+and+its+integration+into+a+routine+clinical+mycobacteriology+laboratory.">Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory.</a> J. Clin. Microbiol. 41, 1447 (2003).</li></ol>

우리는 공개 할게 없다.

여기에서 설명한 프로토콜은 병원체의 급속한 식별을 가능하게함으로써 혈류 감염 환자의 예후를 개선하는 적절한 항생제의 조기 관리로 이어질 수있는 기능성 항균 프로파일을 제공합니다. 시험 요청한 조건에 따라 최소 즉 저렴한 비용, 높은 처리량은 설정 - 주변의 시간, 테스트 조건 조정할 수 있습니다. 모든 절차가 하나 근무일 이내에 수행할 수 있습니다. 또한 프로토콜의 두 부분이 크게 이정표 주위에 시간을 단축하는 동시에 수행할 수 있습니다. 여기에 제시된대로 식별 패널은 우리 병원에서 가장 임상적으로 관련성이 높은 박테리아의 선택입니다. 주요 원칙은 16 유전자 영역을 목표로하고 있기 때문에, 다른 미생물에 대한 구체적인 프로브 설계 및 분석에 추가할 수 있습니다. 완전한 분석은 원래 혈액 배양의 신속한 분석을위한되었다뿐만 아니라 O의 처리에 사용될 수 있습니다군터 샘플 자료. 이것은 또한 항생제 자화율 시험에 사용된 항생제의 경우 : 자세한 또는 다른 항생제는 로컬 저항 패턴 및 가이드라인을 바탕으로 추가할 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 나트륨 염화물 (NaCl) </td><td> 머크 화학 </td><td> 106,404 </td><td> 물에 0.9 % </td></tr><tr><td> Vacutainer SST 혈청 분리관 5 ML </td><td> BD 진단 시스템 </td><td> 367,986 </td><td></td></tr><tr><td> 뮐러 Hinton II 국물 </td><td> BD 진단 Dystems </td><td> 212,322 </td><td> 44g / 물의 패 </td></tr><tr><td> Rotixa 50 RS를 원심 분리기 </td><td> 안드레아스 Hettich 게엠베하 &amp; 주식 KG </td><td> 4910 </td><td></td></tr><tr><td> 5415 D를 원심 분리기 </td><td> Eppendorf </td><td> 중단 </td><td></td></tr><tr><td> Primers </td> &lt;TD&gt; 시그마 - 올드 리치 </td><td> NA </td><td></td></tr><tr><td> 프로브 </td><td> Sigma-Aldrich/Applied Biosystems </td><td> NA </td><td></td></tr><tr><td> TaqManEnvironmental 마스터 믹스 2.0 </td><td> 응용 Biosystems </td><td> 4,396,838 </td><td></td></tr><tr><td> IQ SYBRGreen Supermix </td><td> 바이오 방사선 연구소 BV </td><td> 170-8880 </td><td></td></tr><tr><td> MicroAmp 광학 96 자 반응 플레이트 </td><td> 응용 Biosystems </td><td> N8010560 </td><td></td></tr><tr><td> MicroAmp 광학 접착제 필름 </td><td> 응용 Biosystems </td><td> 4,311,971 </td><td></td></tr><tr><td> IQ 96 자 PCR 플레이트 </td><td> 바이오 방사선 연구소 BV </td><td> 223-9441 </td><td></td></tr><tr><td> Microseal B 접착제 씰 </td><td> 바이오 방사선 노동atories BV </td><td> MSB-1001 </td><td></td></tr><tr><td> 실시간 PCR 탐지 시스템 </td><td> 응용 Biosystems </td><td> ABI 프리즘 7900HT </td><td></td></tr><tr><td> 리얼 타임 PCR 탐지 시스템 </td><td> 바이오 방사선 연구소 BV </td><td> MyiQ 단일 색상 </td><td></td></tr></tbody></table>

one-day workflow scheme for bacterial pathogen detection and antimicrobial resistance testing from blood cultures

혈류 감염 때문에 패혈증의 가능성이 발현, 심한 패혈증 및 패혈증에 의한 쇼크 1의 높은 사망률 속도와 관련된. 따라서 적절한 항생제 치료의 급속한 행정부는 혈류 감염의 치료에 제일 중요합니다. 이 과정에서 중요한 요소는 박테리아 식별 및 항생제 자화율 시험의 결과에 크게 의존 타이밍입니다. 이러한 매개 변수 모두가 일상적으로 시간이 많이 소요되며 평균 24~48시간 2, 4를 취한다 문화 기반 테스트에 의해 얻을 수 있습니다. 연구의 목적은 DNA를 기반​​ 혈류 감염의 신속한 식별을위한 assays뿐만 아니라, 신속한 항균 자화율 시험을 개발하는 것이었습니다. 첫 번째 분석은 종 또는 속 특정 프로브 5 보완 rDNA 기반 실시간 PCR 분석 eubacterial 16입니다. 이러한 프로브, 모나스 종을 포함한 그람 음성 박테리아를 사용하여., 녹농균이그리고 대장균뿐만 아니라 포도상 SPP., 포도상 구균, Enterococcus SPP., 연쇄상 구균 SPP. 및 연쇄상 구균 pneumoniae를 포함한 그람 양성 세균은 구분할 수 있습니다. 이 multiprobe 분석을 사용하여 원인이되는 미생물의 첫번째 식별은 2 시인데 후에 주어졌다. 둘째, 우리는 S.의 항생제 자화율 테스트를위한 세미 분자 분석을 개발 구균, Enterococcus 종. 그리고 (통성) aerobe 그람 음성 막대 6. 이 분석은 PCR은 세균 7의 성장을 측정하는 데 사용된하는 연구를 기반으로했다. 혈액 배양에서 직접 수확한 박테리아는 항생제의 선택 6 H 위해 incubated 및 Sybr 그린 기반 실시간 PCR 분석을 따라하는 것은 성장 억제를 결정합니다. 이 두 방법의 조합은 같은 날 (그림 1)에 대한 적절한 항생제 치료의 선택을 안내할 수 있습니다. 결론적으로신분증과 항생 자화율 모두, 분자 분석은 병원균 검출을위한 빠른 대안을 제공하고 혈류 감염의 진단을 향상시킬 수 있습니다.

Watch this Scientific Journal Video about One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures at JoVE.com

One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

세균성 병원체 진단을위한 간단 하루 워크플로우 방식의 디자인은 혈류 감염의 급속한 인식을 가능하게합니다. 여덟 임상적으로 관련성이 높은 세균성 대상과 항생제 저항 프로파일을 포함하는 더 적절한 치료로 이어질 수있는 당일에 임상 초기 통찰력을 제공합니다.

혈액 배양에서 세균 병원체 탐지 및 항균 저항 테스트를위한 하루 워크플로 계획

Bloodstream infections are associated with high mortality rates because of the probable manifestation of sepsis, severe sepsis and septic shock1. ...

one-day-workflow-scheme-for-bacterial-pathogen-detection

Research

JoVE Journal

Immunology and Infection

25.2K 조회수.  Maastricht University Medical Center. 세균성 병원체 진단을위한 간단 하루 워크플로우 방식의 디자인은 혈류 감염의 급속한 인식을 가능하게합니다. 여덟 임상적으로 관련성이 높은 세균성 대상과 항생제 저항 프로파일을 포함하는 더 적절한 치료로 이어질 수있는 당일에 임상 초기 통찰력을 제공합니다.

비디오: One-day Workflow Scheme for Bacterial Pathogen Detection and Antimicrobial Resistance Testing from Blood Cultures

Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.

This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.

재조합 RBD 기반 SARS 백신에 대한 프로토콜 : 단백질 준비, 동물 예방 접종 및 중화 감지

Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide ...

연구

면역학 및 감염병학

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium. Because resistance to ampicillin and vancomycin in E. faecalis is still uncommon compared to resistance in E. faecium, the development of rapid tests allowing differentiation between enterococcal species is important for appropriate therapy and resistance surveillance. The E. faecalis OE PNA FISH assay (AdvanDx, Woburn, MA) uses species-specific peptide nucleic acid (PNA) probes in a fluorescence in situ hybridization format and offers a time to results of 1.5 hours and the potential of providing important information for species-specific treatment. Multicenter studies were performed to assess the performance of the 1.5 hour E. faecalis/OE PNA FISH procedure compared to the original 2.5 hour assay procedure and to standard bacteriology methods for the identification of enterococci directly from a positive blood culture bottle.

A 1.5 Hour Procedure for Identification of Enterococcus Species Directly from Blood Cultures

A rapid protocol for the direct identification of Enterococcus faecalis and other Enterococcus species from a positive blood culture using a Peptide Nucleic Acid fluorescent in situ hybridization assay (PNA FISH).

의 식별을위한 1.5 시간 절차 Enterococcus</em직접 혈액 배양에서> 종

Enterococci are a common cause of bacteremia with E. faecalis being the predominant species followed by E. faecium.  Because resistance to ampicillin and ...

Invasion of Human Cells by a Bacterial Pathogen

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be applied to the study of cell invasion by virtually any culturable bacterium. The stages at which specific aspects of invasion can be studied, such as the role of actin rearrangement or caveolae, will be highlighted. Host cells are grown in flasks and when ready for use are seeded into 24-well plates containing Thermanox coverslips. Using coverslips allows subsequent removal of the cells from the wells to reduce interference from serum proteins deposited onto the sides of the wells (to which S. aureus would attach). Bacteria are grown to the required density and washed to remove any secreted proteins (e.g. toxins). Coverslips with confluent layers of endothelial cells are transferred to new 24-well plates containing fresh culture medium before the addition of bacteria. Bacteria and cells are then incubated together for the required amount of time in 5% CO2 at 37&deg;C. For S. aureus this is typically between 15-90 minutes. Thermanox coverslips are removed from each well and dip-washed in PBS to remove unattached bacteria. If total associated bacteria (adherent and internalised) are to be quantified, coverslips are then placed in a fresh well containing 0.5% Triton X-100 in PBS. Gentle pipetting leads to complete cell lysis and bacteria are enumerated by serial dilution and plating onto agar. If the number of bacteria that have invaded the cells is needed, coverslips are added to wells containing 500 &mu;l tissue culture medium supplemented with gentamicin and incubation continued for 1 h, which will kill all external bacteria. Coverslips can then be washed, cells lysed and bacteria enumerated by plating onto agar as described above. If the experiment requires direct visualisation, coverslips can be fixed and stained for light, fluorescence or confocal microscopy or prepared for electron microscopy.

A general protocol for the study of invasion of host cells by a bacterial pathogen, focusing on Staphylococcus aureus and human endothelial cells.

세균성 병원체에 의해 인간의 세포의 침략

Here we will describe how we study the invasion of human endothelial cells by bacterial pathogen Staphylococcus aureus . The general protocol can be ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

인플루엔자 바이러스에 대한 높은 처리량 검출 방법

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7.&#160;We developed a new model1 which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly,&#160;revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.

Acquired resistance to antibiotics is a major public healthcare problem and is presently ranked by the WHO as one of the greatest threats to human life. Here we describe the use of cantilever technology to quantify antibacterial resistance, critical to the discovery of novel and powerful agents against multidrug resistant bacteria.

약물 타겟 상호 작용 및 항균 저항 검출 Nanomechanics

The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in ...

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

Staphylococcal Cassette Chromosome mec (SCCmec) typing is a very important molecular tool for understanding the epidemiology and clonal strain relatedness of methicillin-resistant Staphylococcus aureus (MRSA), particularly with the emerging outbreaks of community-associated MRSA (CA-MRSA) occurring on a worldwide basis. Traditional PCR typing schemes classify SCCmec by targeting and identifying the individual mec and ccr gene complex types, but require the use of many primer sets and multiple individual PCR experiments. We designed and published a simple multiplex PCR assay for quick-screening of major SCCmec types and subtypes I to V, and later updated it as new sequence information became available. This simple assay targets individual SCCmec types in a single reaction, is easy to interpret and has been extensively used worldwide. However, due to the sophisticated nature of the assay and the large number of primers present in the reaction, there is the potential for difficulties while adapting this assay to individual laboratories. To facilitate the process of establishing a MRSA SCCmec assay, here we demonstrate how to set up our multiplex PCR assay, and discuss some of the vital steps and procedural nuances that make it successful.

Multiplex PCR Assay for Typing of Staphylococcal Cassette Chromosome Mec Types I to V in Methicillin-resistant Staphylococcus aureus

We demonstrate a simple multiplex PCR assay for quick-screening and typing of Staphylococcal Cassette Chromosome mec (SCCmec) types I-V for methicillin-resistant Staphylococcus aureus, and provide some of the vital steps and procedural nuances that make it successful for adapting this assay to individual laboratories.

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Staphylococcal Cassette Chromosome mec (SCCmec) typing  is a very important molecular tool for understanding the epidemiology and  clonal strain ...

Preparation of a Blood Culture Pellet for Rapid Bacterial Identification and Antibiotic Susceptibility Testing

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.

A rapid bacterial pellet preparation from a positive blood culture can be used as a sample for applications such as identification by MALDI-TOF, Gram staining, antibiotic susceptibility testing and PCR-based test. The results can be rapidly communicated to clinicians to improve the outcome of patients suffering from bloodstream infections.

빠른 세균의 식별 및 항생제 감수성 테스트를위한 혈액 문화 펠렛의 제조

Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

의 종 특이 검출을위한 루프 - 매개 등온 증폭 (LAMP) 분석 실험<em&gt; 에이 메리아</em&gt; 닭을 감염이

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...