Multiplex PCR with 16S rRNA gene-targeted primers of bifidobacterium spp. to identify sources of fecal pollution

Abstract

Bifidobacteria are one of the most common bacterial types found in the intestines of humans and other animals and may be used as indicators of human fecal pollution. The presence of nine human-related Bifidobacterium species was analyzed in human and animal wastewater samples of different origins by using species-specific primers based on 16S rRNA sequences. Only B. adolescentis and B. dentium were found exclusively in human sewage. A multiplex PCR approach with strain-specific primers was developed. The method showed a sensitivity threshold of 10 cells/ml. This new molecular method could provide useful information for the characterization of fecal pollution sources.

FIG. 1.

Agarose gel electrophoresis patterns of the ADO-DEN multiplex PCR products obtained from 10-fold dilutions of mixtures of pure cultures of B. adolescentis DSM 20083^T and B. dentium DSM 20084^T to determine the threshold of the method. Lanes: M, DNA marker (φX174); A to G, 10⁶, 10⁵, 10⁴, 10³, 10², 10¹, and 10⁰ cells/ml, respectively; H and I, negative and positive controls, respectively.

FIG. 2.

Agarose gel electrophoresis patterns of the ADO-DEN multiplex PCR products obtained from experimental mixtures of wastewater samples to evaluate the lowest proportion of human pollution that can be detected. Lanes: M, DNA marker (φX174); A to H, mixtures of human (H) and animal (A) wastewater (vol/vol), 100H:0A, 90H:10A, 10H:90A, 7.5H:92.5A, 5H:95A, 2.5H:97.5A, 1H:99A, and 0H:100A, respectively; I, negative control; J, positive control.