Interlaboratory Validation for a Real-Time PCR Salmonella Detection Method Using the ABI 7500 FAST Real-Time PCR System

Abstract

Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U. S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods.

Protein particulate retention and microorganism recovery for rapid detection of Salmonella

“…Hence, the industry is seeking rapid sample preparation techniques that will bring microbial pathogens to levels that are compatible with rapid detection methods for food‐borne pathogens . While methods that facilitate bacteria quantitation in hours are available, monitoring is subject to enrichment steps preceding microbial detection that add a day or more to the process …”

“…5 While methods that facilitate bacteria quantitation in hours are available, monitoring is subject to enrichment steps preceding microbial detection that add a day or more to the process. [6][7][8][9] Here we report detection of Salmonella cells in ground turkey burger, using hollow fiber microfiltration to increase the concentration of these microorganisms from undetectable levels to concentrations of several hundred microorganisms/mL, in about 8 h. 10 The method is based on recently reported concentration of 1 CFU/g (less than 1 CFU/mL) Salmonella enterica in egg whites in 8 h. 10 Unlike egg whites, extracts of turkey burger contain microparticulates capable of blocking the inlets to the 0.5 mm diameter hollow fibers through which microfiltration is carried out. [10][11][12] Prefiltration is needed to remove these particulates, but undesirable loss of microorganisms during prefiltration may also occur ( Table 1).…”

Recent and emerging innovations inSalmonelladetection: a food and environmental perspective

“…The largest advance towards faster detection of Salmonella has been in the realm of molecular biology, where PCR and real-time, quantitative PCR (qPCR) are predominantly being applied as the methods of choice for the detection stage of this process. Many different protocols targeting different genes or gene regions specific to Salmonella have been published (Cohen et al, 1996;Guo et al, 2000;Malorny et al, 2003Malorny et al, , 2004Chen et al, 2010;Postollec et al, 2011;Gonz alez-Escalona et al, 2012;Cheng et al, 2015). Additionally, many targets have been investigated for the specific and sensitive detection of all salmonellae in food and environmental samples.…”

Comparison data of a two-target real-time PCR assay with and without an internal control in detecting Salmonella enterica from cattle lymph nodes

“…The bead suspension was then transferred into 3 mL RV broth and incubated at 42°C for 18-20 h. One hundred microliters of enriched homogenate was streaked onto HE agar plates and incubated at 37°C for 24 h. Six dark-colored colonies with morphology consistent with Salmonella were re-streaked onto BAPs and incubated at 37°C for 18-20 h. The resulting cultures was used for DNA extraction by boiling 1 ml of culture for 10 min and centrifuging at 9300 g for 5 min; the supernatant was used as template for the qPCR reactions with the duplex qPCR assay using both invA and pagC genes as molecular targets. Endogenous housekeeping genes [2,3], or irrelevant exogenous gene [4,5] have been widely used as internal controls to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. We have selected an endogenous housekeepig gene, 18S rRNA gene, as internal control in this study.…”

“…A real-time PCR (qPCR) assay targeting on invA and pagC genes was developed and validated for the detection and quantification of Salmonella enterica strains (Bai et al, 2018) [1]. A host gene, normally an endogenous housekeeping gene (Beer-Davidson et al, 2018; Poon et al, 2004) [2,3], or an irrelevant exogenous gene (Cheng et al, 2015;Sedlak et al, 2014) [4,5] has been widely used as an internal control to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. An endogenous internal control designed based on the 18S rRNA gene was used in the above-mentioned qPCR assay.…”