Quantitative determination of Streptococcus mutans by using competitive polymerase chain reaction

Abstract

Mutans streptococci are among the range of pathogens strongly related to human dental caries. The determination of total amounts of these pathogens as well as their proportion in relation to other oral bacteria is of interest for the assessment of the risk that a patient runs of developing dental caries. This paper presents a competitive polymerase chain reaction (PCR) method for the specific quantitative determination of Streptococcus mutans which uses a homologous DNA for internal standardisation. For quantification of these bacteria, calibration curves were obtained by coamplification of known amounts of S. mutans DNA in the presence of different known amounts of the competitor DNA. The same procedure was performed with known amounts of cultured S. mutans cells. In a clinical study, the reliability of the newly developed quantitative PCR method was assessed by comparing its results with those obtained in parallel with a standard chair side culture method. The described method enables a rapid and exact determination of unknown amounts of S. mutans and could provide an efficient tool for evaluating the caries risk in a patient and to monitor the efficiency of preventive and therapeutic measures.

Peroxidase Reaction as a Parameter for Discrimination of <i>Streptococcus mutans</i> and <i>Streptococcus sobrinus</i>

“…The developed methods should be tested by means of reference methods for possible cross-reactions with closely related species. The present investigation examined the specificity of the S. mutans and S. sobrinus PCRs by comparison with human and plant pathogenic bacteria, archaea, fungi and mammalian cells [Rupf et al, 1999]. MS and other oral streptococci were also included.…”

“…The sequences of the primers were: S. mutans -forward primer: 5*-GGTCAGGAAAGTCTGGAGTAAAAGGCTA-3*, S. mutans -reverse primer: 5*-GCGTTAGCTCCGGCACTAAGCC-3* (length of the PCR product: 282 bp); S. sobrinus -forward primer: 5*-CGGAC-TTGCTCCAGTGTTACTAA-3*, S. sobrinus -reverse primer: 5*-GC-CTTTAACTTCAGACTTAC-3* (length of the PCR product: 546 bp). The specificities of the respective PCRs were tested using 34 bacterial reference strains, archaea, fungi and mammalian cells [Rupf et al, 1999] as well as 17 further reference strains of mutans and other oral streptococci (table 2). The bacterial sediments (cultivation section) were resuspended in 500 µl of distilled water.…”

Chromosomal Insertions and Deletions in <i>Streptococcus mutans</i>

“…The confirmation of strains as S. mutans was done using primers for 16S ribosomal RNA genes described by Rupf et al [1999]. Primers for amplification of regions of chromosomal DNA were designed with the aid of the Primer3 programme of Rozen and Skaletsky [1998] available at http://www-genome.wi.mit.edu/genome-software/other/primer3.html using published sequences or data from the genome of S. mutans UA159, available from http://www.genome.ou.edu/smutans.html.…”

Real-time PCR for quantification of Streptococcus mutans

“…One of the reasons may be the di⁄culty in isolating DNA from mutans streptococci, owing to the hard outer envelope of these Gram-positive bacteria. In general, rapid methods of DNA isolation, such as those based on boiling and hot phenol, have merits for quick detection by conventional PCR, but the recovery rate and/ or quality of the DNA seems to be insu⁄cient for direct quanti¢cation by real-time PCR [20,21]. In this study, we have established two quantitative real-time PCR assays targeting gtf genes of mutans streptococci, one for S. mutans and the other for S. sobrinus and S. downei, by adapting the glass beads method of DNA isolation.…”