乳牛場飲水槽生物膜病原性分枝桿菌核酸偵測

Abstract

威脅牛隻健康的病原性分枝桿菌包括造成牛結核病的Mycobacterium bovis (M. bovis)、伺機病原性的M. avium及引起副結核病的M. avium subsp. Paratuberculosis (MAP)。分枝桿菌細胞壁富含分枝酸，對環境中的物化因子具有抵抗性，目前已知腐生性的非結核分枝桿菌 (Nontuberculosis mycobacterium, NTM) 廣泛的分布於各種自然及人造的環境，而對絕對胞內寄生病原性的M. bovis在活體外存活情形有不同看法。分枝桿菌細胞壁的厭水屬性使其易在環境中形成生物膜，然M. bovis於乳牛場飲水槽生物膜媒介則尚無相關研究。本論文即設計分子生物學偵測技術以便探討病原性分枝桿菌於乳牛場飲水槽生物膜的存在。研究分為四個部份： (i) 設計Nested PCR合併限制酶切割作為偵測病原核酸的定性分析； (ii) 建立病原核酸定量法； (iii) 評估生物膜M. bovis、M. avium及MAP核酸的回收； (iv) 驗證檢測技術可行性。結果顯示： (i) Nested PCR可以區分M. bovis、M. avium、MAP及其他生物膜內常見的細菌，M. bovis的偵測極限為3 ×10-4 ng核酸； (ii) 經Melting curve analysis 確定Real time PCR-SYBR Green的特異性，M. bovis使用SYBR Green及TaqMan分析之偵測極限分別為1.95 ×10-6 ng及1.30 ×10-7 ng核酸； (iii) B/T Genomic DNA Extraction Miniprep System對生物膜內M. bovis、M. avium及MAP的核酸萃取回收率分別為 (53.7 ±32.4)%、 (28.7 ±21.3)%及 (26.7 ±25.7)%； (iv) 乳牛場飲水槽195個生物膜樣本以Nested PCR搭配限制酶切割分析，M. bovis核酸陽性率為28.72%，核酸濃度介於106-8 gene copies，最後一次採樣升至1011 gene copies。PCR偵測生物膜樣本NTM、M. avium及MAP核酸陽性率分別為54.87%、10.77%及3.59%；(v) 比較不同飲水槽材質，M. bovis陽性率於塑膠製飲水槽生物膜最低 (29.23%)，而NTM陽性率於塑膠製飲水槽生物膜最高 (72.31%)。總結本研究設計出生物膜樣本病原性分枝桿菌核酸快速檢測技術，並推論絕對胞內寄生病原M. bovis核酸可以普遍、持續存在於乳牛場飲水槽生物膜中。

Pathogenic mycobacteria pose threats to cattle health- including bovine tuberculosis from Mycobacterium bovis (M. bovis), opportunist pathogenic M. avium and paratuberculosis from M. avium subsp. Paratuberculosis (MAP). Mycobacteria are more likely to resist to environmental attacks because of the abundant mycolic acid in cell wall. Non-tuberculosis mycobacterium (NTM) distributes widely in most natural and artificial environment. However, different opinions are raised in academic regarding whether absolutely existence of the pathogenic M. bovis in living animals or not. Mycobacteria can form biofilm that provides protection and also adaption easily to environment. The existence of pathogenic mycobacteria via biofilm formation in drinking trough of dairy farm is not reported and worth investigation. This study aims to design feasible molecular biology detection techniques for the investigation of pathogenic mycobacteria in the biofilm of the drinking trough of dairy farms. The approach methods include: (i) To design nested PCR and restriction enzymes digestion protocol for the analyzing nucleic acid of bacterial pathogens. (ii) To establish reliable pathogenic nucleic acid quantitative method. (iii) To evaluate nucleic acid recovery efficiencies for the extraction of M. bovis, M. avium and MAP in biofilm. (iv) To verify the feasibility of applying the detection techniques in field sampling. Results showed that: (i) Nested PCR can distinguish M. bovis, M. avium and MAP from each other and also from other commensal bacteria in the biofilm. Method detection limit of M. bovis is 3×10-4 ng nucleic acid. (ii) By applying the melting curve analysis, the real time PCR-SYBR Green can determine detection specificity. The detection limit of SYBR Green and TaqMan analysis of M. bovis are 1.95 ×10-6 ng and 1.30 ×10-7 ng nucleic acid. (iii) The recoveries of the nucleic acid through B/T Genomic DNA Extraction Miniprep System for M. bovis, M. avium and MAP in biofilm are (53.7 ±32.4)%, (28.7 ±21.3)% and (26.7 ±25.7)%, respectively. (iv) Positive rate of M. bovis nucleic acid in the biofilm of 195 dairy farm drinking trough samples with nested PCR and restriction enzymes digestion analysis is 28.72%, and the nucleic acid concentration is stability between 106 and 108 gene copies; the last collection sample rises to 1011 gene copies. PCR positive rate of NTM, M. avium and MAP are 54.87%, 10.77% and 3.59%, respectively. (v) The highest and lowest positive rates found in the biofilm of different drinking trough materials were 72.31% of NTM in plastic tank and 29.23% of M. bovis in plastic tank. In conclusion, this study provides feasible techniques to screen pathogenic mycobacterium nucleic acid quickly in biofilm samples, and time-series study infers that the nucleic acid of M. bovis can exist continuously in the biofilm of drinking trough of the dairy farms.