Species-specific Polymerase Chain Reaction Primers for Detecting Prevotella nigrescens

A previous study reported the cloning of a putative Prevotella nigrescens-specific DNA probe, Pn23, using random shotgun method. The present study evaluated the species-specificity of Pn23 for P. nigrescens using the clinical strains of Prevotella intermedia and P. nigrescens to develop Prevotella nigrescens-specific polymerase chain reaction (PCR) primers. The specificity was tested against 10 clinical isolates of P. nigrescens, 6 clinical isolates of P. intermedia and 20 type strains of oral bacteria. The sensitivity of PCR primers was determined by testing serial dilutions of the purified genomic DNA of P. nigrescens ATCC 33563 as a standard strand. Southern blot analysis showed that the DNA probe, Pn23, detected only the genomic DNA of P. nigrescens strains. PCR showed that the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, had species-specificity for P. nigrescens. The detection limits of the four primer sets were 40 pg or 4 pg of the purified genomic DNA of P. nigrescens ATCC 33563. These results suggest that the DNA probe, Pn23, and the two sets of PCR primers, Pn23-F1/Pn23-R1 and Pn23-F2/Pn23-R2, can be useful for the detection of P. nigrescens in the molecular epidemiological studies of oral infectious diseases.