臺灣博碩士論文加值系統

中文摘要
本研究選用16S rRNA、virA、tpl、及H1d 等四種目標基因做為鑑定食品媒介之致病性細菌，包括Escherichia coli、Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus；及Staphylococcus aureus等七種試驗細菌。實驗顯示九對本研究所使用的引子均能對目標基因具有特異性，而七種試驗細菌之16S rRNA 基因產物均經定序比對確認，另外，再以示差性PCR分別對E. coli / Shigella及Citrobacter / Salmonella做進一步之鑑別，並以限制酵素AfaI切割分析V. cholerae、V. parahaemolyticus 與 Staphylococcus aureus 16S rRNA之基因產物，確認本研究設計於這三種細菌之引子具備種特異性。實驗顯示本研究複合式聚合酵素連鎖反應之最適引子煉合溫度為59 ℃，因此選用合適之目標基因、特異性高之引子、PCR反應條件及參數使得本研究在32個循環複製之後，複合式聚合酵素連鎖反應之靈敏度可達102 CFU，顯示此方法能在單一次PCR反應中鑑定食品媒介之致病性細菌，且具有靈敏度高、特異性強、及快速偵測之特點。

英文摘要
Multiplex PCR amplification of 16S rRNA gene、virA、tpl、and H1d genes was developed enabling simultaneous detection in Escherichia coli，an indicator of fecal contamination and food-borne microbial pathogens，Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus、and Staphylococcus aureus。Each of the nine pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene。The optimized multiplex PCR reaction utilized a primer annealing temperature of 59 ℃and used agarose gel electrophoresis for detection of the PCR-amplified products。Selection of appropriate target genes、oligonucleotide primers 、PCR reaction、and cycling parameters resulted in the amplification of four target genes simultaneously in a single PCR reaction with the sensitivity of detection was 102 CFU after 32 cycles。Multiplex PCR amplification followed by differential PCR for E. coli / Shigella， and Citrobacter / Salmonella，sequenced for the PCR-amplified products of 16S rRNA gene of the seven pathogens in this study，and used restriction endonuclease AfaI to confirm the PCR-amplified products of V. cholerae，V. parahaemolyticus and Staphylococcus aureus，has been shown to be an sensitive，specific，and rapid method to detect food-borne bacterial pathogens。

目錄

誌謝……………………………………………………………………...I
中文摘要………………………………………………………………..II
英文摘要…………………………………………………………….....III
目錄…………………………………………………………………….IV
一、緒論…………………………………………………………………1
(1)、前言…………………………………………………………...1
　　(2)、大腸桿菌（Escherichia coli）………………………………1
　　(3)、志賀氏桿菌（Shigella flexneri）……………………………2
　　(4)、枸櫞酸桿菌（Citrobacter freundii）…………………………2
(5)、傷寒桿菌（Salmonella typhi）………………………………3
(6)、霍亂弧菌（Vibrio cholerae）………………………………..3
(7)、腸炎弧菌（Vibrio parahaemolyticus）………………………3
(8)、金黃色葡萄球菌（Staphylococcus aureus）………………..4
　　(9)、16S核糖體核糖核酸基因（16S ribosomal RNA gene）…..4
(10)、毒力基因（Virulence gene）…………………………………5
(11)、酪氨酸裂解酵素（Tyrosine phenol lyase）…………………5
(12)、鞭毛抗原基因（Flagellin gene）……………………………6
(13)、複合式聚合酵素連鎖反應（Multiplex PCR）……………..6
(14)、差異性聚合酵素連鎖反應（Multiplex PCR）……………..7
(15)、限制片段長度多型性（Restriction fragment length
polymorphism，RFLP）……………………………………8
二、研究目的……………………………………………………………9
三、材料與方法…………………………………………………………10
　　(1)、菌株來源………………………………………………………10
　　(2)、冷凍乾燥管之開封及菌株之保存與培養…………………..10
　　(3)、細菌基因組DNA（genomic DNA）之製備……………….10
(4)、16S核糖體核糖核酸基因序列之比對（alignment）分析..10
(5)、引子（primer）之選擇與特異性（specificity）………….10
(6)、Differential PCR之試驗方法……………………………….10
(7)、Multiplex PCR之特異性試驗………………………………10
(8)、Multiplex PCR之靈敏度（sensitivity）試驗……………..11


－IV－


(9)、限制酵素之切割（Digestion）分析………………………11
四、結果………………………………………………………………..16
(1)、引子的特異性……………………………………………….16
(2)、Differential PCR之特異性………………………………...16
(3)、限制表現型（restriction phenotype）之特異性…………17
(4)、Multiplex PCR之特異性…………………………………..17
(5)、Multiplex PCR之靈敏度…………………………………..19
五、討論………………………………………………………………..20
六、結論………………………………………………………………..26
七、參考文獻…………………………………………………………..27
八、表…………………………………………………………………..31
九、圖…………………………………………………………………..36

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