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研究生:黃淳維
研究生(外文):Huang, Chuen-Uei
論文名稱:蜂產品中美洲幼蟲病原孢子檢測技術的建立與應用
論文名稱(外文):The applications and establishment of detection methods for American foulbrood spores in bee products
指導教授:陳素瓊陳裕文陳裕文引用關係
指導教授(外文):Chen, Su-Chiung Ph. D.Chen, Yue-Wen Ph. D.
學位類別:碩士
校院名稱:國立宜蘭大學
系所名稱:園藝學系碩士班
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
語文別:中文
論文頁數:75
中文關鍵詞:西洋蜂美洲幼蟲病幼蟲芽孢桿菌疾病防治蜂產品聚合酶鏈鎖反應標準檢測流程
外文關鍵詞:Apis mellifera L.American foulbroodPaenibacillus larvaedisease controlbee productspolymerase chain reactionstandard operation procedures
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美洲幼蟲病(American foulbrood)是西洋蜂(Apis mellifera L.)最嚴重的細菌性病害,病原為幼蟲芽孢桿菌(Paenibacillus larvae)。蜂群感病初期不易察覺病徵,因此檢測病原孢子在疾病防治上極為重要。本研究收集來自台灣各地的蜂蜜及泰國進口蜂蜜,透過平板培養法(plate counting methods)檢測蜂蜜中美洲幼蟲病之病原孢子,並計算其CFU(colony forming units)。根據研究結果顯示,2005年,台灣蜂蜜樣本的孢子檢出率顯著高於泰國進口蜜(18.8% vs. 9.8%, p < 0.05),而該年台灣蜂蜜的分類樣本中,蜂場採收蜜檢出率21.1%為最高;2006年台灣蜂蜜樣本的孢子檢出率亦顯著高於泰國進口蜜(34.8 % vs. 20.0%, p < 0.05),且該年台灣蜂蜜的分類樣本中,蜂場採收蜜檢出率亦為最高(44.2%),表示近年來美洲幼蟲病仍普遍發生於台灣蜂群。此外,為鑑定平板培養法檢測之準確性,並將檢測技術運用在不同蜂產品,本研究以常見的分子檢測技術-PCR,並設計一對nest PCR引子,用以檢測蜂產品中是否含有病原孢子;結果顯示,將孢子分別加入成蜂、蜂蜜、蜂王乳及蜂花粉中,均可以nest PCR檢出病原孢子DNA片段,經過計算得知,三種蜂產品最低的檢出濃度皆為5 CFU/g,而在第一次PCR中的DNA模板數量為0.125 CFU,即可經nest PCR測得。然而,將分子檢測應用於0及1 ~ 10 CFU/15 g的田間蜂蜜樣本(n=20)時,檢出率為80%(n=15),顯示即使蜂蜜中僅含少量孢子亦可成功檢出孢子;此外,應用分子檢測於田間蜂花粉樣本(n=36)時,檢出率則為97%(n=35),顯示亦可成功檢出蜂花粉中的病原孢子。最後,本研究應用分子檢測技術於田間蜂群的監控上,根據檢測結果顯示,蜂王乳最適合做為蜂群感染美洲幼蟲病的初步指標,蜂蜜次之。因此,本研究證實分子檢測技術可充分應用於各種蜂產品中,並建立了一套完整的標準檢測流程,如能落實美洲幼蟲病原孢子監測制度,並結合蜂群管理的模式,即可有效預警並防治美洲幼蟲病的發生,以避免使用抗生素可能造成的殘留問題。
American foulbrood (AFB) is a serious bacterial disease of honeybee (Apis mellifera L.) caused by the spores of Paenibacillus larvae. Since the disease signs of the early infection stage were difficult to find by naked eyes in bee colony, it is very important to detect the P. larvae spores for disease controls. This study collected honey samples from Taiwan and Thailand in 2005-2006 and detected P. larvae spores by plate counting methods. Results showed, in 2005, the rate of spore-positive samples from Taiwan is significant higher than from Thailand (18.8% vs. 9.8%, p < 0.05), and the highest (21.1%) was the apiary honey from Taiwan. Also in 2006 samples, the spore-positive rate from Taiwan is significant higher than Thailand (34.8% vs. 20.0%, p < 0.05), and apiary honey from Taiwan was up to 44.2%. These showed the AFB spores presented commonly in Taiwan.
Next, in order to confirm its accuracy of the plate counting method and to detect the other bee products, this study designed a nest PCR primer pair to detect P. larvae spores from bee products by polymerase chain reaction (PCR). Results showed a specific DNA fragment could be detected from infection adult bees, honey, bee pollen, and royal jelly by nest PCR. The detection limit is 0.125 CFU of the DNA template in primary PCR that equilibrated to 5 CFU per gram of bee products. Using this method, 80% (n = 15) of honey samples containing 0 ~ 10 CFU/15 g (n = 20) counted by plate counting methods were spore-positive. When tested the bee pollen samples (n = 36), 97% (n = 35) of them were spore-positive. These show that the proposed methods can succeed detecting the specific DNA fragment of P. larvae spores from bee pollen and honey at a lower spore density. When using this detection method to monitor the diseased levels of bee colony, royal jelly may be the suitable subject than honey or bee pollen.This study has demonstrated that the proposed nest PCR method can succeed detecting P. larvae spores from bee products. In addition, there are standard operation procedures for different bee products have been proposed to monitor AFB levels. Combing the well hive management, this system may pre-warn or prevent the emergence of American foulbrood without using antibiotics in bee colony.
目錄
壹、前言 1
貳、往昔研究 4
一、美洲幼蟲病背景資料 4
二、病原菌的特性 5
三、檢測美洲幼蟲病原孢子的意義與方法 6
四、以分子技術檢測美洲幼蟲病的方法 8
參、材料與方法 11
一、美洲幼蟲病原孢子源的製備 11
二、國產與進口蜂蜜中美洲幼蟲病原孢子的檢測 12
(一)蜂蜜樣品的來源 12
(二)平板培養基檢測法 12
1. 培養基試驗 12
2. Nalidixic acid的劑量試驗 13
3. 分離條件對病原孢子殘留量影響的試驗 13
4. 檢測流程的建立 14
三、分子技術檢測方法的建立 14
(一)蜂產品來源 14
(二)引子與PCR條件 15
1. 引子試驗 15
2. PCR反應條件試驗 16
3. PCR產物膠體電泳分析 17
(三)DNA萃取方法試驗 17
1. 物理加熱檢測方法 17
2. GTC檢測方法 18
3. CTAB檢測方法 18
(四)蜜蜂與蜂產品檢測法的建立 19
1. 蜜蜂成蜂的檢測法 20
2. 蜂蜜的檢測法 20
3. 蜂花粉的檢測法 20
4. 蜂王乳的檢測法 21
四、分子檢測法應用於田間蜂群的監測 21
(一)蜂群管理與蜂產品樣本的取得 22
(二)田間蜂群中蜂產品的監測 22
肆、結果 24
一、美洲幼蟲病徵觀察及病原孢子取得的結果 24
二、國產與進口蜂蜜中美洲幼蟲病原孢子檢測 24
(一)平板培養基檢測法 24
1. 培養基及的nalidixic acid的劑量試驗結果 24
3. 分離條件對病原孢子殘留量影響的試驗結果 25
4. 平板培養基檢測結果 25
三、分子技術檢測方法 26
(一)引子與PCR條件試驗結果 26
1. 引子試驗結果 26
2. PCR反應條件試驗結果 26
(二)DNA萃取方法試驗結果 26
(三)蜜蜂與蜂產品的檢測結果 28
1. 人為汙染蜜蜂與蜂產品檢測結果 28
2. 田間蜂蜜的檢測結果 29
3. 田間蜂花粉的檢測結果 29
四、分子檢測法應用於田間蜂群的監測 29
(一)試驗蜂群的臨床症狀 29
(二)蜂產品的監測結果 30
伍、討論 32
一、平板培養基檢測病原孢子的探討 32
(一)平板培養基檢測技術建立的探討 32
(二)平板培養基檢測蜂蜜中病原孢子的探討 33
二、分子檢測技術建立的探討 35
(一)nest PCR成效的探討 35
(二)DNA萃取法與檢測敏感度的探討 36
三、田間蜂產品監測之探討 38
(一)以田間蜂蜜及蜂花粉實測檢測技術成效的探討 38
(二)透過蜂產品監測田間蜂群的探討 39
四、標準檢驗流程 42
(一)平板培養檢測法 42
(二)分子技術檢測法 42
陸、引用文獻 64
柒、附錄 71
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