發展以多重聚合連鎖反應方法檢測六株常見食源性病源菌   全文下載

Development of a Multiplex PCR Method for the Detection of Six Common Foodborne Pathogens

本研究開發了一種多重聚合?連鎖反應（multiplex PCR）方法，用以篩選和檢測六株澳門常見的食源性致病菌。此方法使用六對引子對，可自混有數株參考菌株之食物樣本或單一菌種之純培養中，擴增產生特定大小之複製子（amplicon）。多重聚合?連鎖反應方法所檢測的六個目標基因包括：大腸桿菌O157: H7 之verocytotoxin（stx）基因、李斯特菌之hemolysin（hly）基因、沙門氏桿菌之invasion（invA）基因、霍亂弧菌之cholera toxin（ctx）基因、副溶血性弧菌之thermolabile hemolysin（tlh）基因，以及金黃色葡萄球菌之thermostable nuclease（nuc）基因。此檢測法之靈敏度為1~100 CFU/mL。此多重聚合?連鎖反應方法可檢測下列三類食物所含之病菌：肉類及肉類產品中之沙門氏桿菌、李斯特菌和大腸桿菌O157: H7；海鮮及海鮮產品中之霍亂弧菌和副溶血性弧菌；即時食品中之金黃色葡萄球菌。綜上所述，本研究結果顯示，多重聚合?連鎖反應為ㄧ快速之食源性致病菌檢測方法，可用於例行監測食物之安全，或評估其風險。

This study developed a multiplex PCR method for the screening and detection of six common foodborne pathogens in Macao. The m-PCR procedure, which uses six pairs of primers, produced specific amplicons of the expected sizes from mixed populations of reference bacterial strains in food samples and from pure cultures. The verocytotoxin (stx) gene of Escherichia coli O157: H7, the hemolysin (hly) gene of Listeria monocytogenes, the invasion (invA) gene of Salmonella spp., the cholera toxin (ctx) gene of Vibrio cholerae, the thermolabile hemolysin (tlh) gene of V. parahaemolyticus, and the thermostable nuclease (nuc) gene of Staphylococcus aureus were used as target genes for m-PCR detection. The detection limit of the assay for the bacterial targets was 1-100 cfu per mL. The m-PCR analysis was designed for three main food clusters; meat and meat products testing for Salmonella spp., L. monocytogenes, and E. coli O157: H7, seafood and seafood products testing for V. cholerae and V. parahaemolyticus and ready-to-eat foods testing for S. aureus. Overall, results of the present study indicate that the m-PCR is a potential technique for the rapid detection of foodborne bacteria for routine monitoring and risk assessment of food.