應用多套式PCR（Multiplex PCR）快速檢測病原性大腸桿菌及沙門氏菌   全文下載

Rapid and Specific Detection of Enterotoxigenic Escherichia coli and Salmonella Strains by Multiplex PCR Systems

本研究目的主要發展快速且具特異性之多套式PCR，可同時檢測食品中熱不穩定腸毒性大腸桿菌（LT ETEC）及沙門氏菌（Salmonella sp.），多套式PCR 使用二組引子應用於熱不穩定腸毒性大腸桿菌及沙門氏菌檢測，皆可分別得到425 bp 及163 bp 大小之PCR 產物。SCLB（selenite cystinelactose broth）培養基應用於食品及糞便的預培養，對Salmonella 及LT ETEC 其靈敏度為101-102 cells/g。進一步以多套式PCR 檢測160 種天然樣品中之熱不穩定腸毒性大腸桿菌及沙門氏菌。由結果顯示，多套式PCR 方法於家禽肉及糞便檢出熱不穩定腸毒性大腸桿菌，而傳統方法（BAM）則於糞便檢出熱不穩定腸毒性大腸桿菌。

Escherichia coli and Salmonella are two of the most important food-borne bacterial pathogens. Classical identification for these strains is laborious, time-consuming, and may generate erroneous results. The purpose of this study was to develop a rapid and specific multiplex PCR (m-PCR) method to simultaneously detect heat labile enterotoxin gene of E. coli (LT ETEC) and oriC of Salmonella sp. Multiplex PCR using two pairs of primers produced specific amplicons of expected sizes of 163 bp and 425 bp from mixed populations of Salmonella sp. and LT ETEC bacterial strains, respectively. These primers were then used for the detection of food and feces with 101-102 cells/g of Salmonella and LT ETEC, followed by SCLB (selenite cystine-lactose broth, selenite cystine / lactose broth, 5/3, w/w) incubation. The presence of these two pathogens in food and feces was detectable. Finally, we used this method for the detection of 160 kinds of market-available foods and feces, and found that LT ETEC bacterial strains were detected in 2 samples (poultry and feces), and one sample (feces) by the BAM (Bacteriological Analytical Manu) method.