Nucleic acid sequence based amplification for the rapid and sensitive detection of Salmonella enterica from foods

Abstract

Aims: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. Methods and Results: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens Ò Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 10 1 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. Conclusions: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 10 2 and 10 1 CFU per 25 g food sample.Significance and Impact of the Study: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.

Loop‐Mediated Isothermal Amplification (LAMP) for the Rapid and Sensitive Detection of Salmonella Typhimurium from Pork

“…Simpkins and others (2000) successfully developed the mRNA‐based NASBA to detect Salmonella enterica targeting dnaK gene without the interference of DNA. D'Souza and Jaykus (2003) further applied this assay to detect Salmonella in food samples. The detection sensitivities at 10 2 to 10 1 CFU/25 g from fresh meats, poultry, fish, ready‐to‐eat salads, and bakery products were obtained after 18‐h enrichment using the same assay.…”

Enrichment cultivation in detection of food-borne Salmonella

“…Real-time PCR together with enrichment cultivation has been applied for the detection of Salmonella in various food matrices, such as eggs Malorny, Bunge, & Helmuth, 2007), tomatoes (Warren, Yuk, & Schneider, 2007), and different meats (McGuinness et al, 2009;O'Regan et al, 2008). Alternative amplification-based nucleic acid detection methods have also been developed for food-borne Salmonella, including isothermal nucleic acid sequencebased amplification (NASBA) (D'Souza & Jaykus, 2003), and loopmediated isothermal amplification (LAMP) (Ye et al, 2011).…”

Detection of Salmonella in Chicken Meat by Insulated Isothermal PCR

“…Compared with the LAMP assay, iiPCR methods for bacterial detection are easier to design because the primer system is simpler. In the NASBA assay described by D'Souza and Jaykus (12), primer annealing at 65°C was required before isothermal amplification at 41°C, and the enzyme mixture was added after primer annealing, which could be inconvenient. In the iiPCR assay, the experimental procedures are simpler than those of the NASBA assay.…”