Development of rDNA Molecular Diagnosis for Lactobacillus Species

• Main Authors: Chin-Gin Chao, 趙縉君
• Other Authors: Hau-Yang Tsen
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/28159402330704472507

碩士 === 國立中興大學 === 食品科學系 === 92 === Lactobacillus strains could be found in the intestines of human and animals. Some Lactobacillus spp. strains are commonly used in fermented foods, meat processing and feed products. Classical methods for Lactobacillus spp. identification are mostly based on the biochemical tests, which are time consuming and labor cost. Recently, molecular diagnostic techniques were used. For example, rRNA genes have become one of the useful targets for bacteria identification and differentiation due to their sequence conservation in evolution and sequence variation between species. In this study, we developed the molecular diagnostic techniques including PCR, oligonucleotiede array and colony hybridization methods for the detection of common Lactobacillus spp. Based on the sequences of ITS and 23S rRNA genes, in this study, we designed 16 sets of PCR primers while could be used for the differentiation of Lb. jensenii, Lb. saliverius, Lb. crispatus, Lb. gasseri, Lb. acidophilus, Lb. heleveticus, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. reuteri. Also, based on the sequences of 23S genes, an olignucleotides array specific for the identification of Lb. jensenii, Lb. saliverius, Lb. crispatus, Lb. gasseri, Lb. delbrueckii and Lb. acidophiluswas designed. This array allowed us to detect several different Lactobacillus species. Finally, in this study, specific documented PCR primers were biotin labeled and successfully used for the direct detection and enumeration of Lactobacillus cells in feed supplements. These Lactobacillus cells include cells of Lb. plantarum, Lb. acidophilus, Lb. reuteri, Lb. bulgaricus, Lb. paracasei and Lb. fermentum. In conclusion, the PCR methods and oligonucleotide array designed from our laboratory could be used for the rapid detection of several Lactobacillus species. Also, specific probes developed could be used for the simultaneous identification and enumeration of different Lactobacillus species.