本文關鍵詞： 流式細胞術(FCM) 碘化丙啶陽性百分率(PI%) 平均熒光強度(MFI) 流式細胞熒光術抗生素敏感試驗(FCST) 銅綠假單胞菌(PA)　出處：《蘇州大學》2006年碩士論文　論文類型：學位論文

【摘要】：目的：傳統(tǒng)的抗生素敏感試驗包括：稀釋法(肉湯稀釋法和瓊脂稀釋法)、紙片擴散法和E-試驗，均需要將細菌在藥物作用下培養(yǎng)過夜才能觀察結果，相當費時費力。目前多采用比濁法目測結果，提供的結果代表細菌生長的均數，對亞菌群的敏感性無法判斷。流式細胞儀可同時、快速、多參數地分析單個細胞的生物學特性，已經被應用于生物醫(yī)學的許多領域。銅綠假單胞菌(Pseudomonas Aeruginosa PA)是醫(yī)院感染的三大病原菌之一，探索一種快速檢測此類細菌及其藥物敏感性的方法，對于重癥感染性疾病的快速診斷和治療，預防和治療醫(yī)院感染，減少多重耐藥菌株的產生與傳播，減少患者和醫(yī)院的負擔有重要的經濟效益與社會效益。 本試驗選擇能夠通過破損的細胞膜進入細胞并與核酸相結合的熒光染料碘化丙啶(PI)，使其著染于經過抗生素處理后的細菌，用流式細胞儀檢測細菌熒光信息的變化，推斷細菌對抗生素的敏感性和藥物的最低抑菌濃度(Minimal inhibitory concentration，MIC)。并與傳統(tǒng)的試管稀釋法檢測結果進行比較，客觀判斷流式細胞熒光術抗生素敏感試驗(Flow cytofluorometric antibiotic susceptibility test，FCST)的優(yōu)缺點，為該方法在臨床上的應用積累實驗依據。 方法：我們收集了2003-2004年蘇州大學附屬兒童醫(yī)院外科及重癥監(jiān)護病房醫(yī)院感染患兒的臨床標本中分離出的銅綠假單胞菌共22株，并購買了質控標準菌株ATCC27853。將細菌轉種于血瓊脂平板，35℃孵育過夜，次日從平板上挑取1～2個菌落，接種于M-H肉湯培養(yǎng)基中，于35℃恒溫水浴箱中培養(yǎng)2小時，用VITEK比濁儀調整菌液濃度至0.5麥氏單位(相當于1.5×10~8CFU／ml)。在測定管中加入亞胺培南1.0ml使其終濃度為32.0、16.0、8.0……0.25、0.125ug／ml，然后在測定管中加入細菌懸液1.0ml，調整使其終濃度為7.5×10~6CFU／ml，37.0℃孵育3小時后加入染料PI 10ul(最終濃度為10ug／m)室溫避光5分鐘后，以流式細胞儀檢測10~4以上個細菌。根據熒光信息的變化判斷細菌對藥物的敏感性。隨著藥物濃度的增加，如果碘化丙啶熒光陽性百分率(PI％)或平均熒光強度(MFI)均在狹窄范圍內，無明顯上升，判斷為菌株耐藥：若PI％逐漸增至75％或以上，則此時的最低藥物濃度為MIC，，
[Abstract]:Objective: traditional antibiotic sensitivity tests include: broth dilution and Agar dilution test, paper diffusion method and E- test, bacteria need to be cultured overnight in order to observe the results. It is rather time-consuming and laborious. At present, most of the results are measured by turbidimetry. The results provided represent the mean number of bacteria growth, and the sensitivity to subflora can not be judged. Flow cytometry can be used simultaneously and quickly. The biological characteristics of single cell were analyzed with multiple parameters. Pseudomonas Aeruginosa PAA is one of the three major pathogens of nosocomial infection. To explore a method for rapid detection of these bacteria and their drug sensitivity, for the rapid diagnosis and treatment of severe infectious diseases, to prevent and treat nosocomial infections, and to reduce the production and transmission of multidrug resistant strains. Reducing the burden of patients and hospitals has important economic and social benefits. In this experiment, we selected a fluorescent dye, pyridine iodide, which can enter the cell through damaged cell membrane and bind to nucleic acid, so that it can be infected with bacteria treated with antibiotics. The changes of bacterial fluorescence information were detected by flow cytometry. The sensitivity of bacteria to antibiotics and the minimum inhibitory concentration (MEC) of bacteria to antibiotics were inferred. The results were compared with the traditional test tube dilution method. Objective judgement of antibiotic sensitivity test by flow cytometry (FCM). Flow cytofluorometric antibiotic susceptibility test. The advantages and disadvantages of FCSTs provide experimental basis for the clinical application of FCSTs. Methods: from 2003 to 2004, 22 strains of Pseudomonas aeruginosa were isolated from surgical and intensive care unit patients with nosocomial infection in the Children's Hospital affiliated to Suzhou University. The standard strain ATCC27853 was purchased. The bacteria were transferred to the blood Agar plate at 35 鈩

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