Development of a quantitative molecular method for enumeration of Salmonella enterica

Abstract

A competitive polymerase chain reaction (c-PCR) was developed for rapid enumeration of Salmonella enterica cells in broth culture. The primers MINF and MINR-Hex ensured the methods specificity. An internal DNA control was prepared by enzymatic restriction and subsequent ligation of the flanking regions of the amplified 16S rRNA gene of S. Agona (U92197) strain. The prepared internal control had the same primer specific sequences as the target DNA but was 27 bp shorter. The c-PCR products were quantified by capillary electrophoresis and the results were obtained by regression analysis. Using the described system and 5 × 10^–5 times diluted internal control, a quantification of S. enterica cells in broth culture in the range between 1.2 × 10⁴ and 6 × 10⁵ per reaction mixture was made possible.   The preliminary test showed that the results of the c-PCR quantification of the salmonella cells in broth culture are 2.9 times higher in comparison with the results of the indirect counting method. The authors are still convinced, however, that the method offers a rapid, specific and sufficiently accurate counting of S. enterica cells in natural microbiological samples.