多重引子聚合酶連鎖反應方法快速檢測致病與非致病性腸炎孤菌

致病性腸炎弧菌引起食物中毒的常見致病因子為熱穩定性溶血素(thermostable direct hemolysin; TDH)。而磷脂醯絲胺酸合成酶(phosphatidylserine synthase; PSS) 參與細胞膜上磷脂質的合成，對維持細菌細胞膜完整性十分重要。本研究利用pss基因設計了一組對腸炎弧菌有專一性的引子VP37、38 (增幅產物327 bp)，經過核酸序列同質性比對，再與可辨識致病性腸炎弧菌中溶血素基因tdh的一組探針VP21、22 (增幅產物400 bp)，以多重引子聚合酶連鎖反應技術，建立一套可同時檢測及區分致病及非致病性腸炎孤菌的方法。結果發現22株具tdh 基因的腸炎弧菌菌株，其多重引子聚合酶連鎖反應產物中均有兩個增幅產物生成(327 bp、400 bp), 55 株不具tdh 基因之腸炎弧菌中則只有一個產物產生(327 bp)，但是其他73 株非腸炎孤菌菌種則無任何增幅產物產生。本套多重引子聚合酶連鎖反應的靈敏度為10^4 cfu/mL ( 32-36 pg DNA)。

關鍵字

腸炎孤菌；多重引子聚合酶連鎖反應；磷脂醯絲胺酸合成酶；熱穩定性溶血素

並列摘要

Thermostable direct hemolysin is almost exclusively associated with clinical strains of V. parahaemolyticus and has been considered an import ant virulence factor. Moreover, the phosphatidylserine synthase involved the format ion of phospholipids in the cell membrane is found in most bacteria and yeast. We designed one set of species-specific primers, VP37 and VP38, from V. parahaemolyticus pss to identify V. parahaemolyticus. Another set of primers, VP21 and VP22, is specific for the thermostable direct hemolys in gene that is found in the virulent V. parahaemolyticus strains. These two sets of primer pairs were coamplified using a multiplex PCR technique for identification of V. parahaemolyticus and for differentiation of virulent and non-virulent V. parahaemolyticus in one step. This multiplex PCR protocol clearly coamplified pss and tdh genes and expressed two PCR products, 327 bp and 400 bp, from 22 s trains of V. parahaemolyticus carrying the tdh gene. Only one PCR product with a length of 327 bp was amplified from 55 strains of V. parahaemolyticus that were without the tdh gene. Not any PCR product was found in 73 strains of Vibrio spp. and other bacteria. The sensitivity of the multiplex PCR was 10^4 cfu/mL and 32 to 36 pg of DNA.

並列關鍵字

Vibrio parahaemolyticus ； Multiplex PCR ； Phosphatidylserine synthase ； Thermostable direct hemolysin

國際替代計量

多重引子聚合酶連鎖反應方法快速檢測致病與非致病性腸炎孤菌

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