Development of Multitarget Real-Time PCR for the Rapid, Specific, and Sensitive Detection of Yersinia pestis in Milk and Ground Beef

Real-time PCR has been used previously to detect Yersinia pestis; this study applies this rapid, specific, and sensitive nucleic acid–based method to the detection and quantitation of Y. pestis specifically in food. Five sets of primers and corresponding TaqMan dual-labelled fluorogenic hybridization probes for Y. pestis were designed and optimized for specificity testing using genomic DNA from 71 bacterial strains. Four Y. pestis–specific primer and probe sets were developed, based on the virulence plasmid targets, and used to distinguish this bacterium from the various Yersinia and other bacterial species tested. An additional primer and probe set, based on a chromosomal gene target, distinguished Y. pestis and Yersinia pseudotuberculosis from the various Yersinia and other bacterial species tested. With optimized conditions, the quantitative detection limit of the probes for Y. pestis pure cultures ranged from 13 to 220 CFU. Standard curves were generated for the probes and used to determine the amplification efficiencies. The primers and probes demonstrated high amplification efficiencies, and their performance was evaluated using spiked milk and ground beef samples. The quantitative detection limit was 101 to 103 CFU/ml in milk and 102 to 105 CFU/g in ground beef without any preenrichment step. Testing the hybridization probes on food samples demonstrated the detection of Y. pestis in a foodborne application; this is the first such report, to our knowledge.

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Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation†

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1366 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1366-1372 ◽

Cited By ~ 104

Author(s):

LUXIN WANG ◽

YONG LI ◽

AZLIN MUSTAPHA

Keyword(s):

Escherichia Coli ◽

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Quantitative Detection ◽

Escherichia Coli O157 ◽

The Real ◽

E Coli

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 103 CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli O157:H7, Salmonella, and Shigella in food.

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Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1373 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1373-1378 ◽

Cited By ~ 30

Author(s):

ANNA-CLARA RÖNNER ◽

HANS LINDMARK

Keyword(s):

Detection Limit ◽

Real Time ◽

Dna Extraction ◽

Campylobacter Jejuni ◽

Real Time Pcr ◽

Close Correlation ◽

Quantitative Detection ◽

Quantitative Real Time Pcr ◽

Direct Plating ◽

Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

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Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

Clinical Chemistry ◽

10.1093/clinchem/45.11.1932 ◽

1999 ◽

Vol 45 (11) ◽

pp. 1932-1937 ◽

Cited By ~ 65

Author(s):

Andreas Nitsche ◽

Nina Steuer ◽

Christian Andreas Schmidt ◽

Olfert Landt ◽

Wolfgang Siegert

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Detection System ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Hybridization Probe ◽

Sequence Detection ◽

Hybridization Probes ◽

Assay Time

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

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Identification of New PCR Targets and its Validation for Development of Nucleic Acid-based Detection Assay for Melioidosis

Defence Life Science Journal ◽

10.14429/dlsj.1.10056 ◽

2016 ◽

Vol 1 (1) ◽

pp. 18

Author(s):

Sonia Arora ◽

Duraipandian Thavaselvam ◽

Archna Prakash ◽

Ashu Kumar ◽

Anita Barua ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Bacterial Species ◽

Cost Effective ◽

Sybr Green ◽

Specific Gene ◽

Pcr Assay ◽

Gene Target ◽

Geographical Regions ◽

Species Specific

Burkholderia pseudomallei the gram negative, soil saprophyte is the causative agent of melioidosis in human and animals. Development of rapid, sensitive, species specific and cost effective molecular assays are needed for detection of B. pseudomallei from clinical and environmental samples and to differentiate it from other closely related bacterial species. In this study, insilico approach was used to identify new species specific gene targets for molecular diagnosis of B. pseudomallei. The identified targets were then analyzed by SYBR Green real time PCR assay for their specificity, sensitivity and presence across different Indian clinical and soil isolates of B. pseudomallei. Out of the three targets studied SYBR Green real time PCR assay targeting bpss0091 gene of B. pseudomallei was found 100% specific, having detection limit of 12.3fg/µl DNA. The bpss0091 gene target was present in all clinical and soil isolates of B. pseudomallei tested thus suggesting bpss0091 gene based SYBR Green real time PCR assay will be useful for detection of B. pseudomallei in different geographical regions.

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Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

Applied and Environmental Microbiology ◽

10.1128/aem.02706-05 ◽

2006 ◽

Vol 72 (6) ◽

pp. 4256-4263 ◽

Cited By ~ 35

Author(s):

David Rodrï¿½guez-Lï¿½zaro ◽

Deborah A. Lewis ◽

Alain A. Ocampo-Sosa ◽

Ursula Fogarty ◽

Lï¿½szlï¿½ Makrai ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Dynamic Range ◽

Rhodococcus Equi ◽

Plasmid Copy Number ◽

Quantitative Detection ◽

Virulence Plasmid ◽

Plasmid Copy ◽

Pcr Method ◽

Species Specific

ABSTRACT We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA + and vapA-negative (including vapB +) plasmid genotypes. The vapA + frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to ≈10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA +) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA + “horse-pathogenic” genotype determination.

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Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3911-3916.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3911-3916 ◽

Cited By ~ 71

Author(s):

Mark G. Wise ◽

Gregory R. Siragusa

Keyword(s):

Gastrointestinal Tract ◽

Real Time ◽

Clostridium Perfringens ◽

Real Time Pcr ◽

Limit Of Detection ◽

Quantitative Detection ◽

Necrotic Enteritis ◽

Analytical Sensitivity ◽

Quantitative Real Time Pcr ◽

Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

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Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

Journal of Food Protection ◽

10.4315/0362-028x-68.6.1217 ◽

2005 ◽

Vol 68 (6) ◽

pp. 1217-1221 ◽

Cited By ~ 27

Author(s):

PAVEL KRCMAR ◽

EVA RENCOVA

Keyword(s):

Mitochondrial Dna ◽

Real Time ◽

Dna Sequences ◽

Real Time Pcr ◽

Single Species ◽

Absolute Quantification ◽

Quantitative Detection ◽

Vertebrate Species ◽

Target Species ◽

Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

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