以PCR之普遍性引子增殖混合酵母菌體的核醣體RNA基因之靈敏度   全文下載

Effect on Sensitivity of PCR Amplification of Ribosomal DNA Gene Using Universal Primer with a Mixture of Yeast Species

使用PCR之普遍性引子增殖酵母菌之核糖體RNA基因之內轉錄區，以檢測單一或混合二種或三種酵母菌之靈敏度。以Novozyme分解三個屬之酵母菌的細胞壁抽取DNA，DNA的抽取量隨菌種不同而有很大的差異。以一對普遍性PCR引子增殖單一和混合酵母菌的核醣體RNA基因的內轉錄區ITS 1，PCR之靈敏度在Saccharomyces exiguus為2.5 pg的抽取DNA（約102個細胞），Candida mogii為12 pg（約103個細胞），Saccharomyces cerevisiae為20 pg（約103個細胞），由於三株菌經由PCR增殖產物的片段長度皆不同，因此以PCR增殖二種或三種混合酵母菌之DNA，在同樣PCR條件下，其靈敏度較單一酵母菌降低約1000倍（約105～106個細胞）。 To detect the sensitivity of PCR using universal primers to amplify the RNA gene of internal transcribed spacer (ITS I) with a mixture of yeast species. The amounts of extracted DNA obtained from three yeast genera using Novozyme to lyse the cell walls differed significantly. When a set of universal primers were used in pure yeast cultures, PCR sensitivity for Saccharomyces exiguus was 2.5 pg (approx. 102 cells), for Candida mogii 12 pg (approx. 103 cells), and for Saccharomyces cerevisiae 20 pg (approx. 103 cells). In mixed yeast cell cultures, there was a 1000-fold (approx. 105~106 cells) decrease in sensitivity under the same PCR conditions."

To detect the sensitivity of PCR using universal primers to amplify the RNA gene of internal transcribed spacer (ITS I) with a mixture of yeast species. The amounts of extracted DNA obtained from three yeast genera using Novozyme to lyse the cell walls differed significantly. When a set of universal primers were used in pure yeast cultures, PCR sensitivity for Saccharomyces exiguus was 2.5 pg (approx. 102 cells), for Candida mogii 12 pg (approx. 103 cells), and for Saccharomyces cerevisiae 20 pg (approx. 103 cells). In mixed yeast cell cultures, there was a 1000-fold (approx. 105~106 cells) decrease in sensitivity under the same PCR conditions.