Application of a commercial Salmonella real-time polymerase chain reaction (PCR) assay for the detection and quantitation of Salmonellaenterica in poultry ceca

2021 ◽  
Author(s):  
William Evan Chaney ◽  
April K. Englishbey ◽  
Tyler P. Stephens ◽  
Savannah F. Applegate ◽  
Marcos X. Sanchez-Plata
Keyword(s):  
Food Safety ◽  
Management Strategies ◽  
Cost Effective ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Probable Number ◽  
Pcr Screening ◽  
Standard Curve ◽  
Paired Samples ◽  
Risk Management Strategies

Foodborne Salmonellosis is commonly associated with poultry and poultry products necessitating continued development of pre- and post-harvest food safety interventions and risk management strategies. Evaluating technologies and strategies is limited by availability of cost-effective, rapid laboratory methods. The objective of this work was to evaluate a commercial, qualitative PCR assay and its novel quantitative application to detect and enumerate Salmonella in poultry ceca as an analytical matrix. Ceca were collected at harvest, contents homogenized, and paired samples evaluated with Buffered Peptone Water (BPW) and BAX® MP + Supplement (MPS) pre-enrichment broths followed by PCR screening on BAX® System Q7 (PCR) and by isolation. Additional ceca were inoculated with Salmonella to develop a standard curve for the BAX® System SalQuant™ quantitative PCR application (QA), then estimates were obtained by the QA and Most Probable Number (MPN) methods. For pre-enrichment media, PCR outcomes performed equivalently to culture isolation for detecting Salmonella in ceca with 95.65% and 87.88% sensitivity and 82.00% and 100.00% specificity (P=0.074) for BPW and MPS, respectively. However, at the sample-level, BPW performed significantly worse (47.92%) than MPS (68.75%) for overall isolation of Salmonella (P<0.0001). Post-standard curve development, the mean QA estimates obtained for the inoculated samples were 1.14 (95% CI; 0.62 - 1.66), 1.79 (95% CI; 1.50- 2.08), 2.91 (95% CI; 2.65 - 3.17) and 3.76 (95% CI; 3.26 - 4.25), respectively for each targeted inoculation of 1.0, 2.0, 3.0 and 4.0 Log10 CFU/mL and within or comparable to 95% confidence intervals of paired MPN estimates. These data demonstrate performance of MPS for the detection and isolation of Salmonella enterica from poultry ceca when screening with PCR and indicate the QA may be useful as an alternative tool to estimate Salmonella concentrations in ceca, which may support pre-harvest food safety activities.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

scholarly journals Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

2008 ◽  
Vol 98 (4) ◽  
pp. 405-412 ◽  
Author(s):  
Xinshun Qu ◽  
Leslie A. Wanner ◽  
Barbara J. Christ
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pathogenic Bacteria ◽  
Common Scab ◽  
Cost Effective ◽  
Potato Tubers ◽  
Pcr Assay ◽  
Streptomyces Species ◽  
Standard Curve ◽  
Target Dna

The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is the only known pathogenicity determinant for common scab diseases of potato and other root and tuber crops. Genes encoding thaxtomin synthetase (txtAB) are found on a pathogenicity island characteristic of genetically diverse plant pathogenic Streptomyces species. In this study, an SYBR Green quantitative real-time polymerase chain reaction (PCR) assay using primers designed to anneal to the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populations in potatoes and soil. The real-time PCR assay was specific for pathogenic Streptomyces strains. The detection limit of the assay was 10 fg of the target DNA, or one genome equivalent. Cycle threshold (Ct) values were linearly correlated with the concentration of the target DNA (correlation coefficient R2 = 0.99) and were not affected by the presence of plant DNA extracts, indicating the usefulness of the assay for quantitative analyses of the pathogenic bacteria in plant tissues. The amount of pathogenic Streptomyces DNA in total DNA extracts from 1 g asymptomatic and symptomatic tubers was quantified using the assay and ranged from 101 to 106 pg. A standard curve was established to quantify pathogenic Streptomyces in soil. Using the standard curve, numbers of pathogenic Streptomyces colony forming units were extrapolated to range from 103 to 106 per gram of soil from potato fields where common scab was found. This real-time PCR assay using primers designed from the txtAB operon allows rapid, accurate, and cost effective quantification of pathogenic Streptomyces strains in potato tubers and in soil.

scholarly journals Depuration of a Freshwater Clam (Batissa Violacea) from Rewa River in Fiji Using a Bio-Filter Set-Up in Closed and Open Water Circulatory System

2021 ◽  
pp. e824
Author(s):  
Ashneel Ajay Singh ◽  
Ravinesh Ram ◽  
Sheemal Vandhana Kumar ◽  
Sheenal Aashna ◽  
Shipaldika Verma ◽  
...  
Keyword(s):  
Bacterial Load ◽  
Circulatory System ◽  
Open Water ◽  
Cost Effective ◽  
Plate Count ◽  
Most Probable Number ◽  
Freshwater Bivalve ◽  
Probable Number ◽  
Set Up ◽  
Interior Walls

The effectiveness of the freshwater bivalve Batissa violacea depuration was tested in closed and open water circulatory system over a 48 h period. The closed circulatory system included a sand biofilter. Microbial levels were assessed every 4 h using Total Aerobic Plate Count (TPC) for heterotrophs and Most Probable Number (MPN) for coliforms. TPC and coliform loads in bivalve tissue reduced rapidly to low and undetectable levels in a closed circulatory system while open system showed a slower reduction. Both TPC and coliform loads remained above detectable levels throughout the depuration period. Closed system showed similar patterns of logarithmic reduction of TPC and coliforms in all cases with R2>0.95 and p<0.001. Similar results were observed for tank water however, reduction of TPC and coliforms were slower. Biofilm formation was observed in the interior walls of the aquarium tanks over 48 h in all cases. Physicochemical parameters did not show any significant change. The reduction in TPC and coliform load in B. violacea suggests that biofilter in a closed water circulatory system is a simple, cost-effective, water conserving and effective way to significantly reduce the spoilage and coliform bacterial load that is accumulated in the clams.

Miniaturized Most Probable Number and Enrichment Serology Technique for the Enumeration of Salmonella spp. on Poultry Carcasses

1997 ◽  
Vol 60 (11) ◽  
pp. 1306-1311 ◽  
Author(s):  
FLORENCE S. HUMBERT ◽  
GILLES SALVAT ◽  
FRANÇOISE LALANDE ◽  
PIERRE COLIN
Keyword(s):  
Analytical Approach ◽  
Analytical Procedure ◽  
Cost Effective ◽  
Test Material ◽  
Most Probable Number ◽  
Salmonella Spp ◽  
Probable Number ◽  
Chicken Skin ◽  
Peptone Water ◽  
Skin Samples

The ability of two 8-tube most probable number (MPN) techniques to quantitatively recover Salmonella spp. from 26 fresh, naturally contaminated chicken skin samples was compared. Individual macerated skin samples were tested in parallel using a traditional (tMPN) and a miniaturized (mMPN) analytical procedure. In the tMPN assay, replicate aqueous portions from each macerated sample were preenriched individually in buffered peptone water, selectively enriched in Müller-Kauffmann tetrathionate brilliant green broth (MKTBG), and plated on Rambach agar. Each MKTBG was also postenriched in M Broth, and the resulting postenrichment culture screened for the presence of Salmonella cells by enrichment serology (ES). Although a similar analytical approach was used in the mMPN assay, it differed from the tMPN in the use of smaller test volumes dispensed in microplates, and on a sedimented portion of skin macerate as test material. Of the 26 Salmonella-contaminated samples examined in this study, the tMPN coupled to Rambach agar or ES identified 23 and 24 positive samples, respectively. Under homologous conditions, the mMPN detected all 26 positive Salmonella contaminated samples. The most probable numbers in 100-g skin samples analyzed by the tMPN ranged from 18/100 g to 9,530,000/100 g with a median value of 570/100 g. Levels of contamination by the mMPN procedure ranged from 90/100 g to 556,000/100 g with a median value of 1,200/100 g. Statistical analysis of experimental data underlined the equivalence of the tMPN and the mMPN procedures and nonequivalence of the Rambach plating and ES conditions. It is suggested that the microplate mMPN coupled to ES offers a reliable and more cost-effective analytical approach for the quantitative recovery of Salmonella on broiler carcasses.

scholarly journals Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

2001 ◽  
Vol 67 (4) ◽  
pp. 1613-1618 ◽  
Author(s):  
Line Fredslund ◽  
Flemming Ekelund ◽  
Carsten Suhr Jacobsen ◽  
Kaare Johnsen
Keyword(s):  
Dna Sequences ◽  
Sequence Data ◽  
Small Subunit ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Probable Number ◽  
Soil Protozoa

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

Detection of Escherichia albertii in retail oysters

2021 ◽  
Author(s):  
Sakura Arai ◽  
Satoko Yamaya ◽  
Kayoko Ohtsuka ◽  
Noriko Konishi ◽  
Hiromi Obata ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pacific Oyster ◽  
Genetic Characterization ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Macconkey Agar ◽  
Pcr Assay ◽  
Probable Number ◽  
Pacific Oysters ◽  
Rock Oyster

Escherichia albertii  is an emerging foodborne pathogen. Owing to its distribution in river water,  it is important to determine the presence of  E. albertii  in aquaculture-related foods. In this study, we investigated the distribution of  E. albertii  in retail oyster samples.  A total of  427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in  modified Escherichia coli  broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for  E. albertii -specific nested PCR assay, as well as for  E. albertii  isolation using  deoxycholate hydrogen sulfide lactose agar  (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of  E. albertii  in nested PCR-positive samples was  determined using the  most probable number  (MPN) method.  E. albertii  isolates were subjected to biochemical and genetic characterization.  E. albertii   was detected in 5 of 315 (1.6%) Pacific oyster samples  (one piece each), 2 of 70 (2.9 %)  Pacific oyster samples  (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples  procured from four geographically distant regions. A total of 64  E. albertii  strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and  the MPN value was under the detection limit (&lt; 3 MPN/10 g).  A specific season or month for detecting  E. albertii  was not observed in this study, suggesting that the pathogen is present in seawater.   All the  E. albertii  isolates, except one, were positive for the virulence factor  eae,  indicating that these isolates have  the potential to infect humans.

scholarly journals Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

2000 ◽  
Vol 66 (4) ◽  
pp. 1453-1459 ◽  
Author(s):  
Young-Rok Kim ◽  
John Czajka ◽  
Carl A. Batt
Keyword(s):  
Bacillus Cereus ◽  
Positive Sequence ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Probable Number ◽  
Fluorogenic Probe ◽  
Hemolysin Gene ◽  
Dry Milk ◽  
Fluorogenic Probes ◽  
Nonfat Dry Milk

ABSTRACT A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereusstrains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negativeB. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated withB. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.

Microbiological Food Safety Status of Commercially Produced Tomatoes from Production to Marketing

2016 ◽  
Vol 79 (3) ◽  
pp. 392-406 ◽  
Author(s):  
BRIGITTE N. van DYK ◽  
WILLEKE de BRUIN ◽  
ERIKA M. du PLESSIS ◽  
LISE KORSTEN
Keyword(s):  
Food Safety ◽  
Salmonella Typhimurium ◽  
Contact Surface ◽  
Personal Hygiene ◽  
Most Probable Number ◽  
Source Water ◽  
Probable Number ◽  
E Coli ◽  
Processing Facility ◽  
The Impact

ABSTRACT Tomatoes have been implicated in various microbial disease outbreaks and are considered a potential vehicle for foodborne pathogens. Traceback studies mostly implicate contamination during production and/or processing. The microbiological quality of commercially produced tomatoes was thus investigated from the farm to market, focusing on the impact of contaminated irrigation and washing water, facility sanitation, and personal hygiene. A total of 905 samples were collected from three large-scale commercial farms from 2012 through 2014. The farms differed in water sources used (surface versus well) and production methods (open field versus tunnel). Levels of total coliforms and Escherichia coli and prevalence of E. coli O157:H7 and Salmonella Typhimurium were determined. Dominant coliforms were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. No pathogens or E. coli were detected on any of the tomatoes tested throughout the study despite the high levels of coliforms (4.2 to 6.2 log CFU/g) present on the tomatoes at the market. The dominant species associated with tomatoes belonged to the genera Enterobacter, Klebsiella, and Citrobacter. Water used on the farm for irrigation considered not fit for purpose according to national agricultural irrigation standards, with high E. coli levels resulting from either a highly contaminated source water (river water at 3.19 log most probable number [MPN]/100 ml) or improper storage of source water (stored well water at 1.72 log MPN/100 ml). Salmonella Typhimurium was detected on two occasions on a contact surface in the processing facility of the first farm in 2012. Contact surface coliform counts were 2.9 to 4.8 log CFU/cm2. Risk areas identified in this study were water used for irrigation and poor sanitation practices in the processing facility. Implementation of effective food safety management systems in the fresh produce industry is of the utmost importance to ensure product safety for consumers.

Quantitative Effect of Refrigerated Storage Time on the Enumeration of Campylobacter, Listeria, and Salmonella on Artificially Inoculated Raw Chicken Meat

2007 ◽  
Vol 70 (3) ◽  
pp. 739-743 ◽  
Author(s):  
KATARINA PINTAR ◽  
ANGELA COOK ◽  
FRANK POLLARI ◽  
ANDRÉ RAVEL ◽  
SUSAN LEE ◽  
...  
Keyword(s):  
Cost Effective ◽  
Bacterial Count ◽  
Developed Countries ◽  
Most Probable Number ◽  
Surveillance Program ◽  
Refrigerated Storage ◽  
Two Stage ◽  
Active Monitoring ◽  
Probable Number ◽  
Retail Food

Active monitoring of pathogens on retail foods has been recommended and implemented in a number of developed countries. Because only a portion of retail food is contaminated with pathogens, a cost-effective and informative surveillance program at the retail level often involves a two-stage approach of initial presence-absence analysis and subsequent pathogen enumeration in any positive samples. Most-probable-number (MPN) methods are more resource intensive and therefore used only for samples considered positive by presence-absence methods. Interpretation of the results assumes that the initial bacterial count remains relatively stable between the initiation of the presence-absence analysis and the enumeration analysis. The objective of this study was to quantify the influence of 4°C storage for 5 and 8 days on pathogen counts on raw chicken. The three pathogens examined were Salmonella Typhimurium, Campylobacter jejuni, and Listeria monocytogenes. No significant differences were found between treatments for Salmonella and Campylobacter. However, significant differences were observed for Listeria; counts at day 0 were lower than counts after 5 or 8 days of refrigerated storage (the maximum mean difference was less than 0.6 log units). These findings suggest that a two-stage approach could overestimate the number of Listeria cells on chicken at the time of purchase. By using an MPN analysis on the presumptive positive samples after 5 days of refrigerated storage, this difference will be reduced. These findings support the decision to reduce surveillance costs by performing a two-stage analysis for Salmonella and Campylobacter on retail chicken. This study provides direction for future sampling or surveillance programs that include enumeration of Listeria on retail food.

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