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Studies on vibrio parahaemolyticus by PCR during the four seasons in the south coast of taiwan

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  1. Studies on vibrio parahaemolyticus byPCR during the four seasons in the south coast of taiwan 指導老師:張福林副教授 研究生:陳念群

  2. Introduction

  3. 致病三大弧菌 V.vulnificus V.cholerae V. parahaemolyticus • 霍 亂 弧 菌 與 腸 炎 弧 菌 是 食 品 界 及 防 疫 單 位 最 重 視 的 食 品 中 毒 菌 (資料來源:行政院衛生署)

  4. TCBS V. parahaemolyticus :直徑1~2mm、有光澤、綠色 V. cholerae :直徑2~4mm 、平滑、黃色、中心不透明、周圍透明 • 嗜鹽性試驗(0% 、1% 、6% 、8% 、10%) V. parahaemolyticus :6% 、8%生長良好 V. cholerae :0% 、 1%生長良好

  5. Vibrio parahaemolyticus is one of the most important food-borne pathogens in Taiwan, Japan and other coastal countries. • Vibrio parahaemolyticus is a gram-negative, halophilic bacterium that occurs naturally in estuarine environments world-wide. (Environmental Microbiology 5(8),706-710 ,2003)

  6. 致病因子 • Thermostable direct hemolysin (TDH) • TDH/ • TDH-related haemolysin (TRH) • Urease positive • Lethal toxin • 附著因子

  7. Pathogenic V.parahaemolyticus generally produces a thermostable direct hemolysin (TDH). • More than 90% of clinical V.parahaemolyticus isolates, but fewer than 1% of food or environmental strains produce TDH. (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2003)

  8. Aim 橫跨四季有系統地蒐集﹑保存與研究台灣南部海域中的腸炎弧菌,並且進行一些基本特性分析,進而由分析結果來評估腸炎弧菌在台灣南部海域的分佈情形,而最後再來加以更深入的探討。

  9. 研究架構 海水、有機樣本收集 (水質測量) 利用PCR偵測樣本中 致病性腸炎弧菌(tdh/ trh)及分析KP和 血清型 傳統方法培養 腸炎弧菌 利用腸炎弧菌ToxR基因專一性 引子進行最確數-聚合酵素連鎖 反應(MPN-PCR),偵測樣本中全 部的腸炎弧菌密度 數據整理與分析

  10. 採樣地點 安平漁港 興達漁港 前鎮漁港

  11. 採樣方法 • 水樣:利用採樣筒丟入海水中採得水樣後放入無菌塑膠試管中 • 有機物質:刮取岸邊岩石上的藻類、貝類或水中魚類後放入無菌塑膠試管中 • 保存於室溫3小時內帶回實驗室分析

  12. 水質測量 • PH (METTLER TOLEDO) • 比重 • 鹽度 • 溫度 (VWR 1551-004) • 透視度 • 濁度 (HACH 46500-00) • 綜合水質DO、TDS、 EC (DR/2500 Prot HACH5900) • COD (HACH COD Reactor) 、(HACH ODYSSEY)

  13. Sample collection (3水樣3有機樣本) APW 每管APW各取1loop接種於TCBS 抽取DNA挑選典型綠色菌落分別各接種於TSB-2及TSA-2 PCR(toxR)由TSB-2 抽取DNA PCR (toxR) toxR(+): toxR(-): PCR (tdh) PCR (16S rRNA)toxR(+) PCR (trh) 致病性分析 生化鑑定 菌體抗原 PCR (tdh) KP試驗 PCR (trh)

  14. toxR • 對腸炎弧菌的專一性非常強 • L-GTCTTCTGACGCAATCGTTG R-ATACGAGTGGTTGCTGTCATG • (JOURNAL OF CLINICAL MICROBIOLOGY,Apr. 1999, p. 1173–1177)

  15. TDH & TRH • 通常致病性腸炎弧菌都會具有的致病因子 • TDH: L-GGTACTAAATGGCTGACATC R-CCACTACCACTCTCATATGC • TRH: L-GGCTCAAAATGGTTAAGCG R-CATTTCCGCTCTCATATGC (Applied and Environmental Microbiology, Nov. 2004, P.6401-6406)

  16. 16S rRNA • 所有的菌都具有16s rRNA ,於是選擇保守的序列來做為primer ,這樣就可知道樣本中的DNA是否有毀損. • L-ACGGCGCAGACTCCTACGGGAGGC R-GGGTTGCGCTCGTTGCGGCACTTA (Journal of Clinical Microbiology, Aug. 1994, P.1911-1917)

  17. 生化鑑定

  18. KP 使用 Wagatsuma agar 加以培養,若有 β溶血 ( 在培養基上形成透明圈 ) 現象則稱為 Kanagawa phenomenon ( KP 陽性 ),很可能就是致病菌株.

  19. 命 名 系 統 • (一)方式: 西元年/月/地點/樣本編號/菌株編號 • (二)說明: 西元年2005 →取後二碼 05 月份:01、02、03、04、05、06、07、08、09、10、11、12 地點:安平港→A 興達港→B 前鎮港→C • 樣本編號 : • 菌株編號:自01開始編號 • 例如 : 0508A1302

  20. RESULTS

  21. Table 1.安平港水質結果

  22. Table 2.興達港水質結果

  23. Table 3.前鎮港水質結果

  24. Fig1.Amplification of the toxR gene by PCR 1 2 3 4 5 6 The primers amplified a 368bp fragment Lane1…….100bp marker Lane2…….CCRC 10806 Lane3…….negative control Lane4…….(+)sample Lane5…….(-)sample Lane6…….100bp marker 368bp

  25. 原液 10倍 100倍 1000倍 10ml原液 90ml緩衝液 90ml緩衝液 90ml緩衝液 每稀釋檢液各接種3支(稱三階三支),並於35~37℃,培養16~18小時

  26. Table 4.判定為腸炎弧菌陽性者,利用最確數表(如附表),推算出腸炎弧菌之最確數(MPN/g或MPN/ml )。

  27. Table 5. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0508Awater1 32 7.62 < 3.6 < 3.6 0508Awater2 30 8.38 15 93 0508Awater3 32 7.58 < 3.6 < 3.6 0508Aorganics1 32 7.62 160 > 1100 0508Aorganics2 30 8.38 35 > 1100 0508Aorganics3 32 7.58 290 > 1100 0508Bwater1 28 6.89 3.6 93 0508Bwater2 28 7.41 < 3.6 240 0508Bwater3 29 7.08 6.2 9.4 0508Borganics1 28 6.89 < 3.6 > 1100 0508Borganics2 28 7.41 3.0 > 1100 0508Borganics3 29 7.08 28 > 1100 0508Cwater1 29 8.27 < 3.6 3 0508Cwater2 29 8.27 < 3.6 < 3.6 0508Cwater3 29 8.27 < 3.6 < 3.6 0508Corganics1 29 8.27 3.6 460 0508Corganics2 29 8.27 < 3.6 > 1100 0508Corganics3 29 8.27 < 3.6 64

  28. Table 6. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0509Awater1 29 7.93 15 9.2 0509Awater2 29 7.93 7.4 9.2 0509Awater3 29 7.94 3.6 23 0509Aorganics1 29 7.93 7.2 > 1100 0509Aorganics2 29 7.93 < 3.6 > 1100 0509Aorganics3 29 7.94 < 3.6 > 1100 0509Bwater1 30 7.84 3.0 3.0 0509Bwater2 30 7.91 3.6 43 0509Bwater3 30 7.89 15 > 1100 0509Borganics1 30 7.84 3.0 1100 0509Borganics2 30 7.91 15 290 0509Borganics3 30 7.89 6.1 > 1100 0509Cwater1 29 8.11 < 3.6 3.6 0509Cwater2 29 8.14 < 3.6 < 3.6 0509Cwater3 29 8.16 < 3.6 < 3.6 0509Corganics1 29 8.11 < 3.6 3.6 0509Corganics2 29 8.14 3.6 23 0509Corganics3 29 8.16 < 3.6 23

  29. Table 7. Vibrio parabaemolyficus densities in different samples from three areas Data Temp. pH MPN (MPN/ml或g) MPN-PCR(toxR) (MPN/ml或g) 0510Awater1 30.1 7.12 3 93 0510Awater2 30.1 7.76 3 >1100 0510Awater3 30.1 7.85 9.2 36 0510Aorganics1 30.1 7.12 7.4 >1100 0510Aorganics2 30.1 7.76 <3.6 9.2 0510Aorganics3 30.1 7.85 <3.6 >1100 0510Bwater1 31.1 8.02 9.2 93 0510Bwater2 30.8 7.83 6.1 >1100 0510Bwater3 30.8 7.94 9.2 43 0510Borganics1 31.1 8.02 3.0 >1100 0510Borganics2 30.8 7.83 15 93 0510Borganics3 30.8 7.94 6.2 >1100 0510Cwater1 30.2 8.18 <3.6 <3.6 0510Cwater2 30.2 8.23 3.0 75 0510Cwater3 30.2 8.17 <3.6 3.6 0510Corganics1 30.2 8.17 <3.6 <3.6 0510Corganics2 30.2 8.23 <3.6 23 0510Corganics3 30.2 8.17 <3.6 23

  30. Table 8. Detection of Vibrio parahaemolyticus from water and organic samples V. parahaemolyticus detected by Sample (n) Culture method toxR-PCR Water (n= 27) 14 (51.9) 20 (74.1) Organics (n = 27) 18 (66.7) 26 (96.3) Total (n= 54) 32 (59.3) 46 (85.2)

  31. Fig2.Amplification of the 16S rRNA gene by PCR 1 2 3 4 5 6 7 8 9 10 11 12 763bp The primers amplified a 763bp fragment Lane1…..marker Lane2…..0508C32 Lane3…..0508C33 Lane4…..0508C34 Lane5…..0508C35 Lane6…..0508C36 Lane7…..0508C45 Lane8…..0508C47 Lane9…..0508C48 Lane10….. negative control Lane11…no sample Lane12…...no sample

  32. Fig 3. tdhFig 4. trh 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 251bp 250bp The primers amplified a 251bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A42 Lane4……0508A43 Lane5……0508A44 Lane6……0508A45 Lane7……0508A46 Lane8……0508A47 Lane9……0508A48 Lane10….0508A49 Lane11……vp1 Lane12…negative control The primers amplified a 250bp fragment Lane1……marker Lane2……0508A41 Lane3…… 0508A42 Lane4……0508A43 Lane5……0508A44 Lane6……0508A45 Lane7……0508A46 Lane8……0508A47 Lane9……0508A48 Lane10….0508A49 Lane11……vp2 Lane12…negative control

  33. Fig 5. Amplification of the tdh gene by PCR The primers amplified a 251bp fragment Lane1…..marker Lane2…..0508B44 Lane3…..0508B45 Lane4…..0508B46 Lane5…..0508B47 Lane6…..0508B48 Lane7…..0508B49 Lane8…..0508B50 Lane9…..0508B51 Lane10…..0508B52 Lane11…..positive control Lane12…..negative control 1 2 3 4 5 6 7 8 9 10 11 12 300bp 200bp

  34. Fig 6. Amplification of the trh gene by PCR The primers amplified a 250bp fragment. Lane1…..marker Lane2…..0508B53 Lane3…..0508B54 Lane4…..0508B55 Lane5…..0508B56 Lane6…..0508B57 Lane7…..0508B58 Lane8…..0508B59 Lane9…..0508B60 Lane10…0508B61 Lane11…..positive control Lane12…..negative control 1 2 3 4 5 6 7 8 9 10 11 12 300bp 200bp

  35. Table 9. Detection of haemolysin genes (tdh) from water and organic samples tdh detected by Sample (n) Culture method tdh-PCR Water (n= 27) 0 (0) 0 (0) Organics (n = 27) 0 (0) 4 (14.8) Total (n= 54) 0 (0) 4 (7.4)

  36. Table 10. Detection of haemolysin genes (trh) from water and organic samples trh detected by Sample (n) Culture method trh-PCR Water (n= 27) 0 (0) 0 (0) Organics (n = 27) 1 (3.7) 5 (27.8) Total (n= 54) 1 (1.9) 5 (13.9)

  37. Table 11. Serotyping of the 101 strains isolated O1………………………………………………21 O2………………………………………………20 O3………………………………………………16 O4………………………………………………5 O5………………………………………………8 O6………………………………………………0 O7………………………………………………3 O8………………………………………………3 O9………………………………………………1 O10……………………………………………23 O11……………………………………………..1 血清型 數量

  38. Fig 7. percentage of serotyping from three areas % serotyping

  39. Conclusion • 研究中所發現的tdh(7.4)及trh(13.9)大多是藉由MPN-PCR所發現(MPN只發現1.9%trh). • 在MPN與MPN-PCR兩者間比較的話,可以發現藉由MPN-PCR所偵測到的腸炎弧菌數是較多的. • 在三個採樣點中可以發現,其中前鎮港所分離出的菌量都是很少量的,其次是安平港.

  40. END Thank for your attention