Elsevier

Food Microbiology

Volume 25, Issue 5, August 2008, Pages 705-713
Food Microbiology

A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables

https://doi.org/10.1016/j.fm.2008.03.002Get rights and content

Abstract

In this work, a new multiplex single-tube real-time PCR approach is presented for the detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus, three of the more frequent food-borne bacterial pathogens that are usually investigated in a variety of food matrices. The study includes the design and specificity testing, of a new primer and probe specific for Salmonella spp. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the β-glucuronidase (uidA, E. coli) and Thermonulease (nuc, Sta. aureus) genes, and in the replication origin sequence (oriC, Salmonella spp.). Melting-curve analysis using a SYBR Green I RTi-PCR approach showed characteristic Tm values demonstrating the specific and efficient amplification of the three fragments. Subsequently, a TaqMan RTi-PCR approach was settled, using FAM, NED and VIC fluorescently labelled specific probes for an automated detection. It was equally sensitive than uniplex RTi-PCR reactions in Sta. aureus and E. coli O157:H7, using same amounts of purified DNA, and allowed detection of 10 genome equivalents in the presence of 102 or 104 genome equivalents of the other two pathogens. Finally, it was tested in artificially inoculated fresh, minimally processed vegetables, revealing a sensitivity of 103 CFU g−1 each of these pathogens in direct detection, following DNA extraction with DNeasy Tissue Kit (Qiagen). The multiplex RTi-PCR developed scored the sensitivity recognised for PCR in food and it allows a high-throughput and automation, thus it is promising as a rapid and cost-effective test for the food industry.

Introduction

In recent years, the growing consumption of fresh, minimally processed vegetables has lead to an increase in the number of outbreaks of food-borne disease linked to fresh produce (Anon, 2002). Agricultural irrigation with wastewater that can be raw, treated and/or partially diluted, is a common practice worldwide and constitutes the main source of pathogen contamination (Toze, 2006). Therefore, fresh or minimally processed vegetables which are often eaten raw or minimally processed, can compromise consumer health safety. The most common bacterial enteropathogens associated with fruits and vegetables are Salmonella spp. (Thunberg et al., 2002) and Escherichia coli O157:H7 (Rangel et al., 2005). They are considered among the organisms that are most likely to cause an outbreak and therefore need to be studied for produce safety (Buck et al., 2003). Salmonella is responsible for a localized, self-limiting bacterial infection of the intestinal epithelium, known as “non-typhoid salmonellosis” or “gastroenteritis” and a systemic infection known as “typhoid” or “enteric fever” with severe consequences. The incidence of typhoid salmonellosis is stable, with very few cases in developed countries, but cases of non-typhoid salmonellosis are increasing worldwide (García del Portillo, 2000). E. coli O157:H7 is the most toxigenic serotype of the genus and is an important cause of diarrhoea, hemorrhagic colitis and haemolytic-uremic syndrome worldwide, associated with high mortality rates of between 10% and 40% (Chen and Frankel, 2005). Staphylococcus aureus is the species most commonly associated with staphylococcal food poisoning. It is usually found in ready-to-eat foods since contamination frequently occurs through improper handling, and between up to 30% and 50% of the human population carries Sta. aureus as commensals (Le Loir et al., 2003). All three pathogens are among the food-borne bacteria currently observed in a wide range of food products since they are frequently reported as the causative agents in food poisoning. Furthermore, they are the human pathogens that cause the most economically important food-borne diseases throughout the world.

Rapid and specific methods to detect these pathogens are necessary, particularly in fresh vegetables, since they are highly perishable products. Besides being time-consuming, traditional culture-based methods for detection of pathogens in food are limited by their poor sensitivity and specificity, and frequently lead to uncertain identification results. In the last 10 years, rapid nucleic acid amplification and detection technologies have quickly been replacing more traditional assays; in fact, the polymerase chain reaction is increasingly applied to pathogen detection in food. In addition, PCR is a flexible detection system that allows setting up common reaction conditions to detect multiple pathogens at once (Wang et al., 1997; Bhagwat, 2003), or even simultaneous detection in a-single-tube reaction of two or more pathogens by multiplex PCR (Alarcón et al., 2004; Kim et al., 2006; Park et al., 2006). Multiplex PCR has the potential of saving time and effort in the laboratory, thus, lowering testing-related costs in the food industry (Perry et al., 2007).

In the last few years, real-time PCR (RTi-PCR) has merged with a variety of chemical and detection instruments to become a high-sample-throughput technology. RTi-PCR technology is based on the detection of a fluorescent signal coming either from dsDNA specific dyes (SYBR Green I), or from specific probes which include hydrolysis probes (TaqMan or TaqMan-MGB) and hybridization probes (Molecular beacons or Scorpion primers). Such methods allow the automated detection of amplicons without post-PCR manipulation, thus reducing the risk of cross-contamination (Mackay, 2004; McKillip and Drake, 2004). RTi-PCR protocols are available for the specific detection of many food-borne bacterial pathogens in single reactions; however, multiplex RTi-PCR approaches are still very scarce. Multiplex RTi-PCR has mainly been approached using the TaqMan system since it allows various target organisms to be detected in the same reaction by using spectrally distinct dye-labelled probes. Moreover, it has been successfully applied in clinical microbiology to the detection of meningitis pathogens (Corless et al., 2001), mastitis pathogens (Gillespie and Oliver, 2005) or pneumonia pathogens (Welti et al., 2003), as well as to enterotoxin detection and genotyping (Ibekwe et al., 2002; Jothikumar and Griffiths, 2002; Grant et al., 2006; Perelle et al., 2007). Regarding food-borne pathogens, Sharma and Carlson (2000) described a fluorigenic PCR assay for the simultaneous detection of Salmonella and E. coli O157:H7, after enrichment in artificially inoculated meat and faeces. To our knowledge, the application of multiplex RTi-PCR in food has only been approached with SYBR Green I for simultaneous detection of Salmonella serovars and Listeria monocytogenes after enrichment in water and milk spiked samples (Jothikumar et al., 2003) and raw sausage meat (Wang et al., 2004).

The aim of this study was to develop a multiplex real-time PCR assay for the simultaneous detection of Salmonella spp., Sta. aureus and E. coli O157:H7 using both SYBR Green I and TaqMan assays. To our knowledge, this is the first study to deal with the simultaneous detection of these three pathogens by RTi-PCR using both, SYBR Green I and TaqMan assays.

Section snippets

Bacterial strains, culture media and growth conditions

A total of 114 Salmonella strains (including 22 reference strains and 92 food isolates), and 84 non-Salmonella strains were used in this study. For detailed information on the strains, see Table 1. All reference strains were supplied by the Spanish Type Culture Collection (CECT). They were all grown on TSB (Trypticasein Soy Broth) at 37 °C for 24 h except lactic acid bacteria (Carnobacterium, Leuconostoc, Lactobacillus and Pediococcus) that were grown in MRS broth (Man Rogosa Sharpe, Oxoid) at 30 

Specificity of the RTi-PCR assays

A new primer P3 (antisense primer) and a probe were designed to target the replication origin of Salmonella spp. Primer P3, used in combination with sense primer P1 previously described by (Widjojoatmodjo et al., 1991), delimit a 100 bp fragment. Primer and probe specificity were tested: (i) in silico using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (NCBI) nucleotide public database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) which showed

Discussion

A novel triplex real-time PCR assay has been developed that utilizes specific PCR primers and TaqMan probes to detect Salmonella spp., E. coli O157:H7 and Sta. aureus. Its ability to detect them in different proportions has been assessed in fresh vegetables, following DNA extraction by DNeasy Tissue Kit (Qiagen), which successfully removed PCR inhibitors. Previous studies on simultaneous detection of food-borne pathogens by multiplex fluorigenic PCR or RTi-PCR have been published focusing on

Acknowledgements

This work has been supported by “Comisión Interministerial de Ciencia y Tecnología” (CICYT) grant PTR95-0773-OPCT and by “Consellería de Sanitat de la Generalitat Valenciana” grants EVES 050/2005 and EVES 001/2006.

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