Direct Quantitation and Detection of Salmonellae in Biological Samples without Enrichment, Using Two-Step Filtration and Real-Time PCR

Abstract

A new two-step filtration protocol followed by a real-time PCR assay based on SYBR green I detection was developed to directly quantitate salmonellae in two types of biological samples: i.e., chicken rinse and spent irrigation water. Four prefiltration filters, one type of final filter, and six protocols for recovery of salmonellae from the final filter were evaluated to identify an effective filtration protocol. This method was then combined with a real-time PCR assay based on detection of the invA gene. The best results were obtained by subsequent filtration of 100 ml of chicken rinse or 100 ml of spent irrigation water through filters with pore diameters of >40 m to remove large particles and of 0.22 m to recover the Salmonella cells. After this, the Salmonella cells were removed from the filter by vortexing in 1 ml of physiological saline, and this sample was then subjected to real-time quantitative PCR. The whole procedure could be completed within 3 h from sampling to quantitation, and cell numbers as low as 7.5 ؋ 10 2 CFU per 100-ml sample could be quantified. Below this limit, qualitative detection of concentrations as low as 2.2 CFU/100 ml sample was possible on occasion. This study has contributed to the development of a simple, rapid, and reliable method for quantitation of salmonellae in food without the need for sample enrichment or DNA extraction.Salmonella is one of the most common causes of food-borne disease (27), with 40,000 reported annual cases of salmonellosis and an even higher number of estimated cases in the United States (data available at www.cdc.gov). In order to minimize risks for the consumer, microbial auditing of food is increasingly being applied. For this reason, the number of rapid test methods for Salmonella has grown rapidly in the last decade. PCR and real-time PCR have become powerful tools for the detection of pathogens in food. Many different PCR assays have been developed for Salmonella, all with different specificities, accuracies, and detection limits (13,15,35). The most recent assays, with detection in 12 to 20 h, have drastically improved the speed of the detection process compared to that of culture-based methods (3, 10). However, due to the potential for very low levels of salmonellae in foods and standards requiring detection of 1 CFU of salmonellae in food, all these methods include a significant enrichment time that limits the ability for same-day analysis. Also, even though real-time PCR allows for quantitation of targets, after enrichment the number of cells present has generally been changed in an unpredictable manner, making quantitation of the initial amount of target difficult. For these reasons, it would be considered an improvement to be able to detect and, if possible, quantitate (low) levels of salmonellae in foods without the need for enrichment.Aside from concentrating the target, sample treatments are performed prior to PCR in order to remove PCR inhibitors or to improve the homogeneity of samples (19). There are currently several methods for detect...

Detection of viable Salmonella Typhi by reverse transcription-multiplex polymerase chain reaction

“…This result is consistent with other previous reports mentioning that the primer pair was Salmonella specific. Many previous works have used the invA gene to specifically detect Salmonella (Amini et al, 2015, Chiu andOu, 1996;Malorny et al, 2003;Krascsenicsova et al, 2008;Paiao et al, 2013;Rahn et al, 1992;Wolffs et al, 2006;Zhai et al, 2014). Apart from the invA gene, other Salmonella genes have been used for the specific detection of Salmonella such as the fimA (Cohen et al, 1996), fimY (Yeh et al, 2002), stdA (Chuang et al, 2008) and ompC (Ngan et al, 2010) (Levy et al, 2008, Lim andThong, 2009).…”

Genetic characterization of <i>Salmonella</i> and <i>Shigella</i> spp. isolates recovered from water and riverbed sediment of the Apies River, South Africa

“…Two 0.45 µm filters were used; one was placed onto xylose lysine deoxycholate agar (XLD) (Biorad, South Africa) and/ or Salmonella-Shigella agar (SS) (Merck, South Africa) and incubated at a temperature of 37°C for 24 h under aerobic conditions, and the other was preserved in 1 mL of 15% glycerol at 0°C for genetic analysis. Since the desired counts required were of the actual water and sediment samples, no enrichment step was needed to enhance and multiply the initial bacterial counts, as also performed by Wolffs et al (2006) and Marathe et al (2012). The abundance of cultured Salmonella spp.…”

Rapid Filtration Separation-Based Sample Preparation Method for Bacillus Spores in Powdery and Environmental Matrices

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