豬霍亂沙門氏菌Std 線毛的分析及 stdA 基因在檢測沙門氏菌的應 用Analysis of the Std fimbriae in Salmonella enterica serotype Choleraesuis and the application of the stdA gene in detecting Salmonella
中文摘要
豬霍亂沙門氏菌 ( Salmonella enterica serovar Choleraesuis ) 具有宿主 適應性，會造成豬隻副傷寒病症，另外，它會感染人類，並造成敗血症的發生。
線毛（fimbriae）是一種位於大多數革蘭氏陰性菌外膜上的一種毛髮狀蛋白質 結構物，豬霍亂沙門氏菌具有std 線毛基因組，其可轉譯出相似於線毛的結構 物。
本實驗室藉由PCR 方式，增幅 7.4 kb 的 std 線毛基因組，將其選殖至 pCR- XL-TOPO 質體 DNA，委外定序後，分析 std 線毛基因組之開放性讀框
( ORF )，進而將此重組 DNA 送入大腸桿菌 HB101，並藉由 RT-PCR 及西方點 墨試驗觀察到S. Choleraesuis 在靜置培養液中可以表現 stdA 線毛基因，進 而有StdA 蛋白質的產生，而震盪培養液及固態培養基均無法觀察到 stdA 線毛 基因表現或是StdA 蛋白質的產生。Std 線毛的主要線毛次單位（major
fimbrial subunit）為 StdA，而其 N 端的胺基酸序列僅與沙門氏菌屬有同源性，
在其他非沙門氏菌屬中則無同源性。因此若能以stdA 當作偵測沙門氏菌的目標 基因，在S. Choleraesuis 之 stdA 基因上設計一對引子，藉由 PCR 方式，測 試45 種沙門氏菌血清型及 15 種非沙門氏菌菌株，用以做為偵測沙門氏菌的方 法。在經PCR 反應後，可觀察到這 45 種沙門氏菌血清型，共 268 株，均可產 生518-bp 大小的 PCR 產物，而 15 種非沙門氏菌菌株則無任何的 PCR 產物。
而PCR 的偵測敏感度為菌體 DNA 濃度 3.4 × 10-1 pg，使用南方點墨試驗再 次偵測，其敏感度提高為3.4 × 10-2 pg。並且進行接種試驗，屠體液中非沙 門氏菌細菌數量為4 × 107 CFU/ml，當 4.4 × 100 CFU/ml 的沙門氏菌加上 屠體液於培養液中增殖後，可以使用stdA 成對引子藉由 PCR 方式看到 stdA 的產物，表示stdA 成對引子在 PCR 反應液中存在沙門氏菌及其他菌種時，仍 然可以順利進行反應，而不會受到影響。藉由這些實驗，我們了解到針對stdA 所設計的成對引子確實具有專一性及高敏感度，能夠確實偵測出沙門氏菌。
英文摘要
Salmonella enterica serovar Choleraesuis is host adapted and causes swine paratyphoid. In addition, S. Choleraesuis is invasive in human and usually causes septicemia without involvement of gastrointestinal tract. Fimbriae are proteinous appendages present on the most of the Gram negative bacteria including
Salmonella. The genomic DNA of S. Choleraesuis possesses the std fimbrial gene cluster that has the potential to encode fimbrial appendages. The 7.4 kb DNA
fragment containing the S. Choleraesuis std fimbrial gene cluster was PCR
amplified and cloned into the pCR-XL-TOPO vector. The open reading frames of the std fimbrial gene cluster were analyzed. The resulting recombinant DNA was transformed into Escherichia coli HB101 strain. RT-PCR and Western blot analysis demonstrated that stdA expression and StdA protein were detected only when S.
Choleraesuis strains were grown in static broth, but not on solid agar or shaking broth culture conditions. The amino acid sequence of the N-terminal portion of the StdA, the major fimbrial subunit of Std fimbriae, reveals no homology to that of the non-Salmonella strains and is conserved within Salmonella strains. To test the feasibility if stdA could be used as a target to detect Salmonella, primers internal to stdA in S. Choleraesuis were designed to amplify specific PCR products from the genomic DNA of 45 Salmonella serovars and 15 non-Salmonella strains. The 518- bp stdA derived PCR products were present in 268 strains composing of 45 Salmonella serovars. Non-Salmonella strains yielded no specific DNA products.
The limit of detection by PCR was 3.4 × 10-1 pg using the genomic DNA from S.
Choleraesuis while Southern hybridization detected to the level of 3.4 × 10-2 pg.
The sensitivity of the stdA-specific primer set was demonstrated on a Salmonella- free swab sample from a pork carcass surface, which was then artificially
contaminated with different concentrations of S. Typhimurium. Inoculation studies showed that stdA-specific primer set detected Salmonella in samples having initial 4.4 × 100 CFU/ml of Salmonella inoculum prior to the enrichment step in the presence of 4 × 107 CFU/ml of non-Salmonella florae. Results in this study demonstrate that stdA is unique to Salmonella species and is an appropriate PCR target for detecting these microorganisms.
