細菌辨識與抗藥性基因偵測之整合性平台建置

Abstract

生活在我們身體週遭的微生物大多數是無害的，這些無害的微生物可以幫助我們消化食物、分解毒素、以及擊退致病微生物。但仍然有些許微生物，會影響人類健康、造成疾病，人類常見細菌造成的疾病如：敗血症及細菌性陰道炎等等。 細菌性疾病的治療上，常針對造成該疾病的主要致病菌投以抗生素做治療，而目前臨床上常見的方法為做血液細菌培養，此實驗方法從開始至得知結果需要約三天的時間，另外臨床上需要時亦會進行抗生素感受性試驗，此實驗過程亦需耗費不少時間，故若能將上述二者試驗過程時間縮短，將能讓醫生在更短的時間內施予病患正確的藥物。 本研究提供一個特定物種與抗藥性基因的引子及探針設計之平台，可用於建立細菌性疾病專用的檢測套組，目標在於設計出之檢測套組能使用最少量的引子與探針，達到準確預測之需求。期許檢測套組能有效縮短菌種辨識及抗藥性基因偵測的時間，以增加診斷價值。 在引子設計上，使用者可自行選擇產物的長度，引子的長度、溫度範圍、GC 含量範圍、引子集結構之∆ G等，而探針的設計上則可自行選擇探針的長度、前後忽略的核苷酸數、最大可接受的核苷酸錯誤配對數目、溫度範圍等等。為考慮到實驗上的方便性，本平台亦可做多組引子間的引物二聚體之檢查及確認，可避免單一實驗進行多重聚合酶鏈式反應或多重生物鑑定時產生引子或探針出現非預期的雜交反應。 敗血症目前排在台灣死因第十二位，我們根據台北榮民醫院2011年1月至2013年6月的血液培養細菌結果，鎖定37個細菌物種以及9種抗藥性基因透過本研究開發的平台設計出敗血症之檢測套組。

The vast majority of the microbes which live in human body are not harmful. Furthermore, these microbes without harmful can help for the digestion, breaking down toxins, and fighting off the harmful microbes. In the other hand, there are still some microbes may cause disease in human body (e.g., sepsis and bacterial vaginosis), which called pathogen. The bacterial diseases caused by the pathogen are usually used antibiotic for the treatment. Nowadays, blood culture is the most common way to find out pathogens, request a period of time. If we can provide an improvement for the time-consuming blood culture, doctors could gave the drug in shorter time precisely. Besides, antibiotic sensitivity testing is also needed to ensure whether the pathogen has antibiotic resistance gene. This research provided a platform for primer and probe design to detect the specific bacteria and antibiotic resistance gene. This platform can use to create panels for bacterial disease detection. We expect that this panel can be served as a time-saving method for bacteria identification and antibiotic resistance gene detection, adding diagnostic value on detecting causative pathogens. In primer design, user can decide the parameters, which include the length of primer and PCR product, melting temperature, GC content, and the value of ∆G. In addition to the melting temperature and the GC content, the amount of ignored first, last base(s), and mismatch can also be decided by the users in probe design. For the convenience of experiment, this platform can also check the dimer, preventing the primer or probe hybridized to each other in multiplex PCR or multiplex bioassays from causing unexpected results. Sepsis is ranked twelfth cause of death in Taiwan last year, is one of the bacterial disease. We focused on 37 bacteria and 9 antibiotic resistance genes to design the specific detection panel for sepsis according to the number of strain by blood culture in Taipei Veterans General Hospital from 2011 January to 2013 June.