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標題: 不同方法偵測家禽樣本中沙門氏菌之比較
Investigation of different methods in detecting Salmonella from porltry samples
作者: Cheng-Wen Wang
關鍵字: 無;no
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沙門氏菌為世界上最主要的食媒性病原之一,而沙門氏菌感染禽類後,不僅可能 會出現臨床症狀,人類更有機會因食用感染沙門氏菌之禽類相關產品造成中毒。 而以往傳統檢驗樣品中是否含有沙門氏菌病原至少需要 5 天以上的時間,不僅費 時,且過程繁複。因此,本研究旨在找出符合快速檢測、高特異性以及操作方便, 以期快速準確監測上游現場沙門氏菌的病原有無 確保下游家禽產品的食物安全,。 本次試驗之樣本為現場 3 項樣本:雞蛋(分蛋內與蛋外)、飼糧以及糞便。樣本取 材後,首先以 ISO 6579:2002 確認樣品中無沙門氏菌。而後以人工接種 4 株標準 菌株:Salmonella Enteritidis、Salmonella Typhimurium(此二株是為 Motile strains; MT)、Salmonella Pullorum 以及 Salmonella Gallinarum(此二株是為 Non-motile strains; NMT),並將已定量之菌液分別加入。以 PCR、LAMP 以及 Kit 3 種分子生 物學的檢驗方法,並以傳統檢驗法為黃金準則(gold standard),比較 3 種方法之正 確度(AC)、敏感度(SE)、特異度(SP)、陽性預測值(PPV)以及陰性預測值(NPV), 最後以比較兩方法間一致性之 kappa 值比較各方法間的一致性。結果顯示,PCR 在偵測 MT 時,與傳統培養法之 AC、SP、PPV 與 NPV 皆顯著高於 LAMP 與 Kit (p<0.05)。另一方面,在偵測 NMT 的糞便樣本時,PCR 則無法檢出陽性樣本,且 傳統培養法的敏感度降低;而 LAMP 與 Kit 仍保持高度特異性以及敏感性,不受 糞便內微生物或雜質影響。但 LAMP 與 Kit 在使用煮沸法萃取之蛋內沙門氏菌核 酸時,受到蛋黃內物質之抑制,無陽性樣本。另 kappa 值在 MT 的部分,PCR 與 傳統培養法為 0.86,為幾乎完全吻合之一致性;而在 NMT 的部分,則 kappa 值 僅 0.53,為中等吻合。LAMP 與 kit 則不論在 MT 或 NMT,兩者之 kappa 值皆為 幾乎完全吻合。綜上所述,PCR 實為檢測現場樣品時,與傳統培養法一致性最高 之方法,但其耗時相對比 LAMP 與 Kit 要長;而 LAMP 與 Kit 在檢測蛋內沙門氏 菌核酸時,必須使用非煮沸法之核酸萃取方式,另 LAMP 與 Kit 在檢測 PCR 與傳 統培養法檢出之陽性樣本較少的 NMT 糞便樣本時,仍保有其高度的敏感度與特 異度 不受糞便樣本雜質干擾 故現場之 MT 非糞便樣本 可考慮以 PCR 監測之, 而 NMT 之糞便樣本,則以 LAMP 與 Kit 來監控效果較佳。

Salmonella spp. is one of the major foodborne pathogens in the world. When Salmonella infected poultry, it not only occurred the clinical symptoms, but also transmit to the product of poultry. The traditional culture method for detecting the samples with Salmonella which spend at least 5 days. It costs much time and the process is complex. Thus, the aim of my study is to find the best method in the field included the characters of rapid detection, high specificity and easy to operation. The samples are all take from the fields, and it will be tested by ISO6579:2002 to make sure samples without Salmonella spp. Then inoculate 4 standard Salmonella strains: Salmonella Enteritidis, Salmonella Typhimurium (Motile strains; MT), Salmonella Pullorum and Salmonella Gallinarum (Non-motile strains; NMT) to each sample. Using the traditional culture method as the gold standard, PCR, LAMP and Kit will be calculated the accuracy (AC), sensitivity (SE), specificity (SP), positive predictive value (PPV) and negative predictive value (NPV). Finally, calculate the kappa coefficient to compare the consistency of each method. The results show that when PCR detected MT, AC, SP, PPV and NPV has a significant for LAMP and Kit (p<0.05). Otherwise, when PCR detect the fecal sample with NMT, it's unable to show positive of samples. Though the traditional culture method detected the NMT would decreases the sensitivity, LAMP and Kit don't be effect by microorganism in fecal samples. It still shows high specificity and sensitivity for detection the nucleic acid of Salmonella. But using the boiling method to extract the nucleic acid in egg yolk, LAMP and Kit can't show positive expectedly. It was inhibited by some material in egg yolk. The kappa coefficient at the part of MT, PCR/traditional culture method is 0.86, shows almost perfect coincide. In the part of NMT is 0.53, shows moderate coincide. LAMP and Kit also show almost perfect coincide of each other for kappa coefficient in MT and NMT. In summary, PCR is more consistency with traditional culture method than LAMP and Kit, but it costs more time than these two methods. When LAMP and kit detect the sample of egg yolk, it needs another way to extract the nucleic acid of Salmonella. These two methods that detect the fecal samples of NMT are better than PCR, because it would not be inhibited by the microorganism in fecal sample. In conclusion, PCR is suitable to monitor and detect the MT samples besides fecal of the field. LAMP and Kit is suitable to monitor and detect NMT samples in fecal sample of the field.
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