Detection of Pathogenic Mycobacteria Nucleic Acid in Biofilm of Dairy Farm Drinking Trough
Date Issued
2012
Date
2012
Author(s)
Yang, En-Cheng
Abstract
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.
Subjects
dairy farm
drinking troughs
biofilm
SDGs
Type
thesis
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