Detection and quantification of Salmonella in pure cultures using 5′-nuclease polymerase chain reaction

Abstract

Advances in detection and quantification assays based on nucleic acids conceivably will revolutionize the ability to quickly and specifically detect and quantify microorganisms in foods. Among these assays, the polymerase chain reaction (PCR) assay and the TaqMan^™ PCR Detection System (Perkin-Elmer) probably are among the most promising. Since a 5′-nuclease PCR renders possible the automated and direct detection and quantification of PCR products (Holland et al., 1991. Proc. Natl. Acad. Sci. USA 88, 7276–7280), microorganisms in foods can be detected and quantified indirectly within a few hours through analysis of the microbial DNA or RNA sequences present. In the present report we have adapted a 5′-nuclease-based kit for the quantification of Salmonella.

Introduction

Foodbourne salmonellosis is caused by the ingestion of food that contains a critical number of cells of the nonhost-specific species or serotypes of the genus Salmonella. Generally, 10⁷–10⁹ cells are necessary for salmonellosis, but outbreaks from significantly lower numbers of cells have been noted (D’Aoust and Pivnick, 1972, Jay, 1996). From the time of ingestion, symptoms usually develop within 12–14 h. The average mortality rate is 4.1% (Jay, 1996).

Results from kits based on immunological detection of Salmonella usually can be obtained after approximately 48 h. However, some kits still require the positive results to be confirmed serologically and biochemically, thus leading to another 48 h delay before a final conclusion can be drawn (Guttorm, 1998). DNA-based methods such as polymerase chain reaction (PCR) therefore have been increasingly used for rapid, sensitive and specific nonquantitative detection of foodborne pathogens, including Salmonella (Olsen et al., 1995, Wang et al., 1997).

Among the various PCR strategies available, those based on the 5′-nuclease chemistry (Holland et al., 1991) probably are the most promising. A 5′-nuclease -based PCR differs from a standard PCR in the incorporation of a fluorogenic DNA probe into the PCR, and in the exploitation of the 5′–3′ nuclease activity of the Taq DNA polymerase to hydrolyze this probe during the DNA polymerization step. The probe is labeled with two different fluorogenic dyes, a reporter dye and a quencher dye, and when both dyes are attached to the same molecule, the quencher dye will absorb the “excited state” energy of the reporter dye. But as the probe anneals to a target sequence between the two PCR primers, and subsequently is cleaved by the 5′–3′ nuclease activity of the DNA polymerase, the quenching effect comes to an end as the two dyes are separated from each other. Accordingly, there will be an increase in reporter dye emission during amplification that is proportional to the amount of amplified DNA produced (Holland et al., 1991). Based on these characteristics, formats for employing direct and automated detection of the amplified DNA, i.e., “end-point” or “plate-read” detection (Livak et al., 1995), as well as formats for automated and continuous monitoring of the PCR, i.e., “real-time” detection (Heid et al., 1996), have been developed. While the former can be used for nonquantitative purposes only, the latter can be applied both qualitatively and quantitatively. In addition, due to a requirement for 100% homology between probe and template, 5′-nuclease-based amplifications frequently are more specific than standard PCR amplifications.

Perkin-Elmer Applied Biosystems (PE ABI; Foster City, CA, US) reports that the TaqMan^®Salmonella PCR Amplification/Detection Kit tested positive for 99.7% of 300 Salmonella strains comprising at least 121 different serotypes. No false positives were recorded among 72 strains of 48 non-Salmonella organisms, including Escherichia coli, Shigella, Citrobacter, and Listeria.

The aim of this work was to evaluate if this kit, designed for nonquantitative purposes, also could be used for quantification. Pure cultures of Salmonella were used in the attempt to demonstrate the possibilities for a quantitative assay. In the present report we show that this 5′-nuclease-based kit also can be adapted for the quantification of Salmonella, when applied to pure cultures.