聚合酶連鎖反應技術檢測沙門氏桿菌之評估

Evaluation of the Polymerase Chain Reaction Technique for Detection of Salmonella

王貞懿(Jan-Yi Wang) ； 黃翠萍(Tsui-Ping Huang) ； 卓憲駿(Hsien-Chun Cho) ； 張育彰(Yu-Chang Chang) ； 施養志(Yang-Chih Shih)

《臺灣農業化學與食品科學》 48卷3期 (2010/06) Pp. 147-154

https://doi.org/10.6578/TJACFS.2010.018

摘要

以聚合酶連鎖反應(PCR)方法的四組引子檢驗沙門氏桿菌的三段基因(sefA、invA、fimA)及ITS區域，sefA為纖毛蛋白基因，invA為內膜蛋白基因，fimA為纖毛次單元的基因，ITS區域為16S和23S rDNA間的序列。藉由測試不同溫度條件對各組引子測定沙門氏桿菌DNA專一性的影響，進一步測試食品檢體，以比較不同引子的專一性。在沙門氏桿菌的測試中，invA及ITS引子的專一性測試結果為100% (95/95)，fimA引子只有83% (79/95)，而sefA引子專一性不佳。其檢測極限分別為invA引子1ng、ITS引子100pg、fimA引子1ng；實際運用在食品檢體測試方面，invA及ITS引子對沙門氏桿菌檢出率亦均為100%，但ITS引子會檢測出非目標菌株，因此invA引子的專一性最佳，所以在本研究中invA引子適用於沙門氏桿菌的PCR快速檢測。

關鍵字

聚合酶連鎖反應 ； 沙門氏桿菌

並列摘要

A polymerase chain reaction (PCR) method was used to detect Salmonella with 4 primer pairs to amplify 3 different genes (sefA, invA, fimA) and ITS regions. These 4 primer pairs amplify 4 different fragments included in (1) the sefA gene encoding the fimbrial protein, (2) the invA gene encoding an inner membrane protein, (3) the fimA gene encoding a major fimbrial subunit, and (4) the ITS region located between 16S and 23S rDNA. We studied whether different annealing temperatures affect the specificity of the 4 primer pairs for detection of Salmonella DNA and compared the specificity of the 4 primer pairs by testing food samples. The results for detection of Salmonella DNA showed that the invA and the ITS primers detected 100% (95/95) of the positive samples, the fimA primer detected 83% (79/95) of the positive samples, whereas the sefA primer was the least specific. The detection limits of this method were 1 ng, 100 pg and 1 ng for invA, ITS and fimA primers. In the results of testing food samples, the invA and ITS primers detected 100% of the positive samples, too. However, the ITS primer detected non-Salmonella. In this study, the invA primer is the most specific for detection of Salmonella. It is concluded that using the invA primers, PCR could be a rapid and reliable diagnostic tool for detection of Salmonella.

並列關鍵字

Polymerase chain reaction PCR ； Salmonella