Development of cPCR technique for detection and enumeration of Prevotella bryantii

Abstract

Competitive PCR (cPCR) system for the detection and enumeration of Prevotella bryantii cells in rumen samples was developed. PBB₁4 primer, specific for P. bryantii was used as the reverse PCR primer and EUB 338 bacterial primer as the forward primer. An internal DNA control, containing primer specific sequences at 5’ and 3’ ends, but lacking 41 bp at the middle of the sequence, was constructed. C-PCR products were quantified by capillary electrophoresis and the results were calculated with regression equation (Reilly and Attwood, 1998). By coamplification of P.bryantii B₁4 genomic DNA extracted from known numbers of cells and internal control, standard curve was constructed which enables quantification of P.bryantii cells in the range of 2.15 x 10³ to 4.3 x 10⁴ cells.