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研究生:黃鉉瑋
研究生(外文):Hsuan-wei Huang
論文名稱:即時定量聚合酶鏈鎖反應法快速檢測及鑑定沙門氏桿菌之研究
論文名稱(外文):Studies on Rapid Detection and Identification of Salmonella spp. with Real-time PCR
指導教授:黃小萍黃小萍引用關係
指導教授(外文):Shiao-ping Huang
學位類別:碩士
校院名稱:輔英科技大學
系所名稱:醫事技術系碩士班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:61
中文關鍵詞:wzx基因SBG增菌肉湯內部控制組即時定量聚合酶鏈鎖反應invA基因
外文關鍵詞:real-time PCRSBG enrichment brothwzx geneinvA geneInternal control
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沙門氏桿菌為革蘭氏陰性桿菌,屬於腸內菌科。而沙門氏桿菌感染所引起的臨床症狀相當多樣化,包括了腸胃炎、菌血症以及敗血症等。臨床診斷沙門氏桿菌的感染,主要是利用培養後分離鑑定的方法。已有許多的增菌肉湯被應用在增加沙門氏桿菌的分離上。在我們過去的研究中發現,SBG增菌肉湯可以有效的增加沙門氏桿菌的檢出。本研究的目的為建立沙門氏桿菌的分子檢測及鑑定法。為了增加沙門氏桿菌分離率,本研究使用SBG增菌肉湯進行增菌。為了使用real-time PCR進行臨床檢體中沙門氏桿菌的偵測,所以本研究使用invA基因作為偵測的標的基因。另外,為了避免偽陰性的出現,所以本研究使用M13噬菌體DNA作為內部控制組。為了更進一步的分析沙門氏桿菌血清型之分子基礎,所以在研究中使用與體抗原相關的wzx基因,進行B群與D群的特異性引子設計,並使用real-time PCR進行檢測。經由結果可以得知,使用real-time PCR可以有效率的搭以invA基因進行臨床沙門氏桿菌進的偵測並且以wzx基因進行血清型的鑑定。一、使用invA進行沙門氏桿菌的檢測,M13噬菌體DNA可以成功的被應用成為內部控制組。二、使用SBG-real-time PCR進行241個臨床檢體的偵測後發現,分子檢測法的靈敏度(46/241)為Direct-plating XLD與SBG-XLD的657% (7/241)與135% (34/241)。三、利用腸道的致病菌以及腸道常在菌評估invA基因的引子特異性,發現特異性為100%。四、測量以real-time PCR進行檢驗invA基因的偵測極限得知,在10 CFU/assay的時候偵測率為100% (10/10),1 CFU/assay偵測率為100% (10/10)。五、使用real-time PCR進行63個沙門氏桿菌株的wzx基因檢測後發現,本研究使用的方法可以正確的鑑別出B群沙門氏桿菌(44/44)、D群沙門氏桿菌(10/10)及9株非B非D群沙門氏桿菌(9/9)。六、利用2株常見的腸道致病菌以及8株常在菌進行B群以及D群的引子特異性試驗,發現皆為100%陰性結果。七、測量以real-time PCR進行檢驗wzx基因的偵測極限得知,B群在10 CFU的時候偵測率為100% (10/10),1 CFU偵測率為60% (10/10),D群在10 CFU的時候偵測率為100% (10/10),1 CFU偵測率為60% (6/10)。總之,本研究提供了一個可以從臨床檢體偵測及鑑定沙門氏桿菌的方法,兼具高效率、靈敏度以及特異性等優點。
Salmonellae are gram-negative bacilli belonging to the Enterobacteriaceae family. The clinical syndromes caused by the infection of Salmonella spp. vary to a large degree and may include gastroenteritis, bacteremia, and septicemia. Clinical diagnosis of Salmonella infection is on the basis of recovery and identification of bacteria from the specimens. Several kinds of enrichment broth have been applied to increase the recovery of Salmonella. In particular, SBG enrichment broth was demonstrated to increase the recovery in our previous study. The aim of this study is to investigate the method for molecular detection and identification of Salmonella spp.. To increase the recovery of Salmonella, SBG enrichment broth was included in this study. To detect Salmonella spp. in clinical samples with real-time PCR, the invA gene was chosen as the target gene. To avoid false-negative in entire process including DNA extraction and amplification steps, the M13 phage DNA was applied as an internal control. To further identify the serogroups of Salmonella spp. with molecular basis of O antigen, the specific primers of wzx genes for serogroup B and serogroup D were applied in the molecular identification with real-time PCR. The results demonstrated the performance of molecular detection of Salmonella with invA as a target gene and molecular serotyping of Salmonella with wzx gene in real-time PCR. First by, the M13 phage DNA was successfully applied as an internal control in the molecular detection of Salmonella with invA gene. Second by, following SBG enrichment the recovery of Salmonella from 241 clinical samples in by real-time PCR amplification of invA gene (46 positives) were 657% and 135% of those by direct-plating XLD (7 positives) and SBG-XLD (34 positives), respectively. Third by, the specificity of molecular detection of invA gene was demonstrated to be 100% with common pathogens and normal flora in enterogastric specimens. Fourth by, the limit of detection of invA gene was demonstrated to be 1 CFU (100%, 10/10). Fifth by, the molecular serotyping of 63 Salmonella strains based on wzx genes was demonstrated to be serogroup B (44/44), serogroup D (10/10), and non-B-non-D serogroup (9/9), respectively. The results were consistent with that obtained by serological method. Sixth by, the specificity of molecular serotyping of serogroup B and serogroup D were found to be 100%, by using with 2 species of common pathogens and 8 species of normal enteric flora found in enterogastric specimens. Seventh by, the limit of detection and of the wzx genes were 60% (6/10) at a concentration of 1 CFU and 100% (10/10) at a concentration of 10 CFU for both serogroup B and D. In conclusion, the study provides a useful method for detection and identification of Salmonella in clinical samples in terms of performance, sensitivity, and specificity.
摘 要 i
Abstract iii
目 錄 v
圖 目 錄 vii
表 目 錄 viii
簡 稱 說 明 ix
第一章 前言 1
第一節 沙門氏桿菌簡介 2
一. 沙門氏桿菌命名 2
第二節 增菌培養及培養基 5
一. 革蘭氏陰性菌肉湯 5
二. 亞硒酸鹽肉湯 5
三. 亞硒酸煌綠磺胺鹽增菌肉湯 6
第三節 分子檢測法 8
一. 聚合酶連鎖反應 8
二. 即時定量聚合酶連鎖反應 8
第四節 研究動機與目的 11
第二章 材料與方法 12
第一節 實驗材料 12
一. 檢體收集及處理 12
二. 參考菌株 12
三. 培養基 13
四. 藥品與試劑耗材 13
五. 儀器 13
第二節 實驗方法 14
一. 內部控制組 14
二. 菌液調配與DNA製備 14
三. 核酸萃取 14
四. 引子設計與合成 15
五. 即時定量聚合酶連鎖反應 16
六. 解離曲線分析 16
七. 統計分析 17
第三章 研究結果 18
第一節 以M13作為內部控制組的應用 18
第二節 invA基因分子生物檢測法的引子特異性試驗 19
第三節 分子生物檢測法測invA基因之檢測極限 20
第四節 分子生物檢測法測invA基因之檢出率及靈敏度 21
第五節 分子生物血清型鑑定法所使用的引子特異性試驗 23
第六節 分子生物血清型鑑定法的檢測極限 24
第七節 以wzx基因鑑定法進行血清型檢驗 25
第四章 討論 27
參考文獻 51
附錄一即時定量PCR操作步驟 56
附錄二 研討會紀錄 57
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疫情報導第23卷第12期, 2007
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