Identification and quantification of lactic acid bacteria by real-time PCR

Objetive. To establish a PCR-TR assay to assess the population size and composition of total bacteria, total lactic acid bacteria, and particularly bacteria of the genus Lactobacillus and Bifidobacterium in samples of the gastrointestinal tract of chickens. Materials and methods. The specificity of primers was verified using conventional PCR technique. Dilutions, 101 to 10-4 ng/mu l, of bacterial DNA prepared from each microbial culture were used in the calibration curves. Melting temperature and efficiency of each RT-PCR reaction were determined using iQCycler software 3.1 (BioRad). Results. The primers sets used were found to be specific for each bacterial group with no detectable cross reaction. Efficiencies of RT-PCR reactions for total bacteria, lactic acid bacteria, Lactobacillus and Bifidobacterium were 104.4%, 98.1%, 113.3% and 103.3%, respectively. Conclusions. As specific RT-PCR reactions were obtained and efficiencies of RT-PCR reactions were about 100%, the total bacteria, lactic acid bacteria, Lactobacillus and Bifidobacterium could be quantified with high specificity. Therefore, based on the results found in this study, the RT-PCR technique can be used to monitoring bacterial population shifts in environments such as the gastrointestinal tract of broiler chickens, where lactic acid bacteria, Lactobacillus and Bifidobacterium are common habitants.