Molecular Identification and Quantification of Functional Lactic Acid Bacteria and Improvement of Strain Discrimination for Staphylococcus aureus Subtyping

• Main Authors: Chieh-Hsien Lai, 賴潔賢
• Other Authors: Chi-Yue Chang
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/75671237634329209637

博士 === 大葉大學 === 生物產業科技學系 === 102 === Probiotic products such as capsules, tablets, and animal feed are advertised with lactic acid bacteria (LAB), but during the manufacturing process, transport and storage may cause LAB inactivation. Consumers are concerned with the problems of inconsistent labeling of LAB and whether it use is practical or not. Therefore, establishment of a standard identification model of probiotic products for LAB strains is very important. In this study, we used different molecular methods to identify and quantitate LAB. The results show using different typing methods and identification of target genes lead to different advantages and disadvantages. Techniques such as Real-time quantitative PCR (qPCR), Reverse transcription Real Time PCR (RT-qPCR) and Propidium monoazide combined with quantitative PCR (PMA-qPCR) are able to differentiate RNA fragments with sequences diversity. With polymerase chain reaction, we were able to quantitatively analyze the LAB in heat treatment. Colony hybridization and total plate count were rapid identification methods with detection sensitivity reaching 102 ~ 103 CFU / mL, but cannot quantitate LAB after heat treatment. However, DNA still has stability after cell death, the qPCR may cause false positive results that are not suitable for counting the number of surviving LAB. Because of the limited stability of the RNA to RT-qPCR assay when used as live LAB, the loss rate required to detect RNA of the target LAB. In addition, PMA-qPCR will prevent false positive with detection sensitivity reaching 102 ~ 103 CFU / mL which is suitable for detection of live LAB. Developed tufA and tufB gene-based PCR primers specific for the detection of E. faecium TM39 were combined Random Amplified Polymorphic DNA (RAPD) and Pulsed-field gel electrophoresis (PFGE) for the simultaneous establishment of LAB pattern profile. Furthermore, RAPD combined with other molecular techniques show great discriminate power for subtyping of S. aureus. Finally, we used PCR and API identified previously collected LAB, confirmed the correct 117 LAB strains and used RAPD method to establish LAB pattern profile. The results suggested that it has potential for developing specific primers for the detection LAB enumeration procedure by PCR.