Sensitivity of PCR
For monitoring purposes, PCR detection of indicator and pathogenic organisms requires not only specificity, but also sensitivity to ensure the safety of the potable water. In order to evaluate the minimal detectable number of S. flexneri organisms, viable CCRC 10772 cells were diluted 10-fold in sterile distilled water, enumerated by dilutional plating and template DNA was prepared by the boiling method described herein. Aliquots of the lysate supernatants were first assayed for sensitivity with the single primer pair H8 and H15. The detection limit was at least 1.6×104 cfu of S. flexneri per assay, corresponding to 1.6×106 cfu/ml of lysate. To reach a lower detection limit with simultaneous confirmation of the reaction product, the samples were subjected to the seminested PCR assay. After seminested PCR, the last dilution step amplified corresponded to approximately 1.6×103 cfu per assay (1.6×105 cfu/ml of lysate). Thus, detection levels were increased 10-fold in seminested PCR assays. The sensitivity of the PCR assays following enrichment of pure cultures in GN broth was also investigated. PCR reactions were performed on crude lysates after 0, 2, 4, and 6 h of enrichment. The obtained results demonstrated that 1.6 cfu (160 cfu/ml) S. flexneri organisms could be detected in the seminested PCR assay after 6 h of incubation (Fig. 1). In contrast, the limit of detection in the single-step PCR reaction using primers H8 and H10 was 16 cfu (1.6×103 cfu/ml), which gave a faintly stained band (Fig. 1).
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為監視目的, 顯示的PCR 偵查和致病性有機體要求不僅特異性, 而且敏感性保證飲用水的安全。為了評估S. flexneri 有機體的最小的可發現的數量, 可實行的CCRC 10772 細胞被稀釋10-fold 在不育的蒸餾水裡, 由dilutional 鍍層列舉並且模板DNA 由煮沸的方法準備了此中被描述。lysate supernatants 的整除數第一次被檢驗了為敏感性以底漆對H8 和H15 。檢測極限是至少1.6X04 S. flexneri cfu 每分析用試樣, 對應於1.6X06 cfu/ml lysate 。到達一個更低的檢測極限以反應產品的同時確認, 樣品被服從對seminested PCR 分析用試樣。以後seminested PCR, 最後稀釋步被放大對應了於近似地1.6X03 cfu 每分析用試樣(1.6X05 cfu/ml lysate) 。因而, 偵查水平10-fold 被增加了在seminested PCR 分析用試樣。PCR 分析用試樣的敏感性跟隨純文化的充實在GN 湯裡並且被調查了。PCR 反應執行了在粗暴lysates 在0, 2, 4, 和6 h 以後充實。被獲得的結果顯示出, 1.6 cfu (160 cfu/ml) S. flexneri 有機體能被查出在seminested PCR 分析用試樣在6 h 以後孵出(圖1) 。相反, 偵查極限在single-step PCR 反應使用底漆H8 和H10 是16 cfu (1.6X03 cfu/ml), 給一條微弱地被弄髒的帶(圖1) 。Source(s): 查的