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標題: 應用環形恆溫核酸增幅法及巢式聚合酶鏈鎖反應-酵素免疫分析法偵測鳥型分枝桿菌副結核桿菌亞種
Detection of Mycobacterium avium subsp. paratuberculosis by LAMP (Loop-mediated Isothermal Amplification) and nested PCR-ELISA
作者: 陳怡如
Chen, Yi-Ju
關鍵字: 環形恆溫核酸增幅法
LAMP (Loop-mediated Isothermal Amplification)
nested PCR-ELISA
Mycobacterium avium subsp. paratuberculosis
出版社: 獸醫學系暨研究所
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摘要: 鳥型分枝桿菌副結核桿菌亞種(Mycobacterium avium subsp. paratuberculosis; MAP)是一個人畜共通並在全世界散佈且造成經濟損失的一種重要的病原,在反芻獸造成Johne''s disease,而每年Johne''s disease在美國會造成15億美元的經濟損失,近幾年來,MAP在台灣及美國的盛行率有逐年升高的趨勢。目前有許多種檢測方法來區分動物是否感染MAP,如PCR及ELISA等,但PCR及ELISA都有敏感度及特異度不夠高的問題,所以臨床上需要發展新的檢測技術。本實驗的目的為以環形恆溫核酸增幅法及nested PCR-ELISA來檢測糞便中的MAP,並以糞便細菌培養為gold standard作比較。檢驗樣本分成兩個部分,第一部分為21個經淡水家衛所以糞便細菌培養檢測證實的樣本,第二部分為11個臨床上疑似感染MAP牛隻的樣本,並經由ELISA檢測結果均為陽性,所有的樣本皆分別以細菌培養、PCR、nested PCR、LAMP及nested PCR-ELISA檢測作比較。結果顯示,PCR檢測的敏感度為100 pg/μl的DNA,約為3,000個MAP的量,而nested PCR與LAMP敏感度相同可以檢測到1 pg/μl的DNA,約為30個MAP的量,而nested PCR-ELISA 的敏感度為0.1 pg/μl的DNA,約為3個MAP的量,在特異度方面,無論是PCR、nested PCR、LAMP及nested PCR-ELISA都不會檢測到E. coli、Staphylococcus sp.、Enterococcus sp.的DNA。在檢測21個實驗室確認的樣本部分,LAMP及nested PCR-ELISA的檢測結果皆與細菌培養相符合(16個陽性及9個陰性),其敏感度及特異度皆為100%。在檢測臨床疑似病例樣本的部分,有9個樣本PCR、nested PCR、LAMP及nested PCR-ELISA的結果為陽性,但細菌培養結果為陰性,顯示檢測結果差異較大,推測可能是因為採集糞便樣本後的保存方式不當影響到細菌分離培養的結果。本實驗之結果顯示,以LAMP及nested PCR-ELISA檢測糞便樣本的MAP的準確度相當高。
Mycobacterium avium subsp. paratuberculosis (MAP) is one of the most widespread and economically important zoonotic pathogens of ruminants and human, which may cause Johne''s disease in ruminants. The cost of Johne''s disease to the cattle industry is staggering with an estimated $1.5 billion loss every year in the USA. In recent years, the prevalence of MAP in the USA and Taiwan had increased remarkably. Accurate diagnostic tests for MAP such as ELISA and PCR are routinely used to identified the disease-free animals, however, the sensitivity and specificity of ELISA and PCR are unsatisfied. The purpose of this study was to detect the presence of MAP in fecal samples of dairy cows by Loop-mediated Isothermal Amplification (LAMP) system and nested PCR-ELISA, which were compared by using bacteriological culture as the gold standard. The samples were collected from two sources, 21 laboratory-confirmed samples and 11 field samples of suspected cases with positive result of ELISA assay. All 32 faecal samples were detected by bacteriological culture, PCR, nested PCR, LAMP and nested PCR-ELISA. The results indicated that PCR can detect 100 pg/μl of MAP, while LAMP and nested PCR can detect 1 pg/μl of MAP. However, nested PCR-ELISA can detect 0.1 pg/μl of MAP. None of the negative controls including E. coli, Staphylococcus sp. and Enterococcus sp. was detected by PCR, nested PCR, LAMP and nested PCR-ELISA. In 16 positive and 9 negative laboratory-confirmed samples, the results from LAMP and nested PCR-ELISA were identical to those from bacteriological culture, which yielded a sensitivity of 100% and specificity of 100%. However, all of the 9 field samples detected by PCR, nested PCR, LAMP and nested PCR-ELISA showed positive results, which were inconsistent with those by bacteriological culture. It is inferred that the storage condition of faecal samples may affect the results of bacteriological culture. In conclusion, the accuracies for detection of MAP in faecal samples by LAMP and nested PCR-ELISA are relatively high as compared to the bacteriological culture.
其他識別: U0005-2307201313470600
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