以多套式聚合酶鏈反應同時檢測具腸毒素基因之Staphylococcus aureus   全文下載

Multiplex PCR for the Simultaneous Detection of the SEA, SEB, SEC, SED and SEE Genes of Enterotoxigenic Staphylococcus aureus

腸毒素型金黃色葡萄球菌（Staphylococcus aureus） （含A, B, C, D及E腸毒素），常引起食物中毒。傳統分析檢測費時、費力，且易有偽結果發生。本研究設計七段合成的寡核?酸引子（primer），以多套式聚合?鏈反應（multiplex PCR）探討同時檢測腸毒素A, B, C, D及E的可能性。這七段寡核?酸引子中包括二段共同引子（U1、U2）及五段具特異性的毒素基因引子（A2、B2、C2、D2及E2）。結果顯示，組合成的五組引子（A2／U2、B2／U1、C2／U1、D2／U2 及E2／U2）中，雖然含U1、U2兩個共同引子，但仍可產生五段具特異性的PCR產物，大小分別為582 bp、732 bp、403 bp、251 bp及474 bp。將已知的各類腸毒素型金黃色葡萄球菌混合培養後，亦可利用此多套式PCR法成功的將各類毒素型檢測出來。進一步應用於食品微生物的檢驗時，我們發現食品均質液中若按入含SEA-SEE Staphylococcus aureus 102-103 cells／mL經6-8小時培養後，即可利用此法檢測出正確的腸毒素型。"

The enterotoxins of Staphylococcus aureus (A, B, C, D and E) are known agents of food poisoning. Classical identification for these enterotoxigenic S. aurreus strains is laborious, time consuming and sometimes gives erronneous results. In this work, seven synthetic oligonucleotide primers were used in a multiplex PCR protocol to detect the genes encoding the Staphylococcal enterotoxins A to E simultaneously. The primers include two universal primers, which encode consensus sequences, and five primers, which encode unique sequences for toxin genes. None of the specific primer pairs (U2/A2, U1/B2, U1/C2, U2/D2 and U2/E2) cross-reacted with each other. Each primer was specific for the detection of its corresponding toxin gene, even though U2 and U1 are universal primers. The sizes of the amplified PCR products were 582 bp, 732 bp, 403 bp, 251 bp and 474 bp for enterotoxin genes, A, B,C,D and E, respectively. Unequivocal discrimination of the toxin genes was obtained by PCR using DNA extracted from strains of S. aureus whose toxigenicity had been previously established biologically and immunologically. When these primers were used to detect S. aureus in spiked foods containing102 to 103 cells per ml of food homogenate, all five types of genes could be identified after six to eight hours of preincubation. 腸毒素型金黃色葡萄球菌（Staphylococcus aureus） （含A, B, C, D及E腸毒素），常引起食物中毒。傳統分析檢測費時、費力，且易有偽結果發生。本研究設計七段合成的寡核苷酸引子（primer），以多套式聚合酶鏈反應（multiplex PCR）探討同時檢測腸毒素A, B, C, D及E的可能性。這七段寡核苷酸引子中包括二段共同引子（U1、U2）及五段具特異性的毒素基因引子（A2、B2、C2、D2及E2）。結果顯示，組合成的五組引子（A2／U2、B2／U1、C2／U1、D2／U2 及E2／U2）中，雖然含U1、U2兩個共同引子，但仍可產生五段具特異性的PCR產物，大小分別為582 bp、732 bp、403 bp、251 bp及474 bp。將已知的各類腸毒素型金黃色葡萄球菌混合培養後，亦可利用此多套式PCR法成功的將各類毒素型檢測出來。進一步應用於食品微生物的檢驗時，我們發現食品均質液中若按入含SEA-SEE Staphylococcus aureus 102-103 cells／mL經6-8小時培養後，即可利用此法檢測出正確的腸毒素型。