Development of a quantitative molecular method for enumeration of Salmonella enterica | Priprava kvantitativne molekularno biološke metode za ugotavljanje števila celic vrste Salmonella enterica

English. 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.

Slovenian. Pripravili smo molekularno biološko metodo za hitro štetje bakterijskih celic iz vrste Salmonella enterica, ki temelji na kompetitivni različici verižne reakcije s polimerazo (c-PCR). Uporabili smo začetna oligonukleotida MINF in MINR-Hex, ki sta zagotavljala specifičnost metode. Notranji standard DNK smo pripravili z encimskim razrezom in lepljenjem stranskih fragmentov pomnožka gena za 16S rRNK seva S. Agona (U92197). Notranji standard DNK je imel isti prepoznavni mesti za začetna oligonukleotida kot tarčna DNK, bil pa je za 27 bp krajši. Pomnožke c-PCR smo ločevali s kapilarno elektroforezo in rezultate obdelali z regresijsko analizo. Z opisano metodo in 5 × 10–5 redčenim notranjim standardom smo lahko učinkovito ugotavljali število celic vrste S. enterica v bujonski kulturi v območju med 1,2 × 104 in 6 × 105 celic na reakcijsko mešanico. Z metodo smo v preliminarnem poskusu ugotavljali število celic seva S. Livingstone v bujonski kulturi in rezultate primerjali z rezultati indirektne števne metode. Rezultati opisane metode c-PCR so bili 2,9 krat višji od rezultatov indirektnega štetja kolonij, vendar je glede na številne prednosti opisana metoda kljub temu uporabna za hitro, specifično in dovolj natančno ugotavljanje števila celic vrste S.enterica v naravnih mikrobioloških vzorcih.