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J Hoorfar

Publications and source records attributed to J Hoorfar.

At least 19 recordsLinked to original sources
PubMed2006

Comparative, collaborative, and on-site validation of a TaqMan PCR method as a tool for certified production of fresh, campylobacter-free chickens.

Certified Campylobacter-free poultry products have been produced in Denmark since 2002, the first example of fresh (unprocessed and nonfrozen) chickens labeled "Campylobacter free." This success occurred partly through use of a 4-hour gel-based PCR testing scheme on fecal swabs. In this study, a faster, real-time PCR approach was validated in comparative and collaborative trials, based on recommendations from the Nordic system for validation of alternative microbiological methods (NordVal). The comparative real-time PCR trial was performed in comparison to two reference culture protocols on naturally contaminated samples (99 shoe covers, 101 cloacal swabs, 102 neck skins from abattoirs, and 100 retail neck skins). Culturing included enrichment in both Bolton and Preston broths followed by isolation on Preston agar and mCCDA. In one or both culture protocols, 169 samples were identified as positive. The comparative trial resulted in relative accuracy, sensitivity, and specificity of 98%, 95%, and 97%, respectively. The collaborative trial included nine laboratories testing neck skin, cloacal swab, and shoe cover samples, spiked with low, medium, and high concentrations of Campylobacter jejuni. Valid results were obtained from six of the participating laboratories. Accuracy for high levels was 100% for neck skin and cloacal swab samples. For low levels, accuracy was 100% and 92% for neck skin and cloacal swab samples, respectively; however, detection in shoe cover samples failed. A second collaborative trial, with an optimized DNA extraction procedure, gave 100% accuracy results for all three spiking levels. Finally, on-site validation at the abattoir on a flock basis was performed on 400 samples. Real-time PCR correctly identified 10 of 20 flocks as positive; thus, the method fulfilled the NordVal validation criteria and has since been implemented at a major abattoir.

Animals↗
PubMed2005

Evaluation of probe chemistries and platforms to improve the detection limit of real-time PCR.

A validated PCR-based Salmonella method targeting a 94-bp sequence of the ttr gene was used as a model to compare six different combinations of reporter and quencher dyes of a TaqMan probe, on three different instruments, to improve the detection limit in a real-time PCR assay with the aim of a same-day analysis. The use of locked nucleic acids (LNA) and Scorpion probes were also tested. The combination FAM-BHQ1 or Cy5-BHQ3, both dark quenchers, gave the best results (Cycle threshold (Ct) of 25.42+/-0.65 and 24.47+/-0.18 at 10(3) DNA copies). When comparing different probe technologies, the LNA probe (FAM-BHQ1) was the most sensitive with the strongest fluorescence signal (dR last 48066), resulting in 0.6 to 1.1 lower Ct values than a DNA TaqMan probe, and 1.9 to 4.0 lower Ct than the Scorpion system (FAM-BHQ1). The RotorGene real-time PCR instrument gave 0.4-1.0 lower Ct values (more sensitive) than the Mx3005p, and 1.5-3.0 lower than the ABI 7700. Using the LNA in a RotorGene instrument, we detected the following Salmonella DNA copies in 1-ml pre-enriched samples: fishmeal (100 copies), chicken rinse (100 copies) and pig feces (10 copies). The detection probability of the final assay on inoculated fecal samples was 100% at 2x10(4) copies per ml. In conclusion, the LNA probe with annealing temperature of 65 degrees C could be useful for more sensitive detection limits.

Animals↗
PubMed2005

A real-time PCR assay for the specific identification of serotype O:9 of Yersinia enterocolitica.

A real-time PCR assay was developed based on a 181-bp fragment of the recently cloned per gene, including an internal amplification control (124 bp), for the detection of Yersinia enterocolitica O:9 (Ye O:9). The validation included 48 Ye O:9, 33 Y. enterocolitica non-O:9 and 35 other closely-related bacterial strains, containing per gene homologies. The assay was specific for the Ye O:9 tested, the detection limit was 1-10 genome copies of purified DNA and amplification efficiency was between 90.5-103%, indicating a linear regression throughout the detection window.

Animals↗
PubMed2005

Novel approach for assessing performance of PCR cyclers used for diagnostic testing.

As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 microl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was +/-0.3 degrees C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer's specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5 degrees C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20 degrees C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.

DNA, Bacterial↗
PubMed2004

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples.

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).

Animals↗
PubMed2004

Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters.

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10(2) to 10(7) CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 x 10(3) and 2 x 10(3) CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 10(1) to 1 x 10(7) (R(2) = 1.00) for the RotorGene and 10(3) to 10(7) (R(2) = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R(2)) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.

Animals↗
PubMed2004

Validation of a PCR-based method for detection of food-borne thermotolerant campylobacters in a multicenter collaborative trial.

A PCR-based method for rapid detection of food-borne thermotolerant campylobacters was evaluated through a collaborative trial with 12 laboratories testing spiked carcass rinse samples. The method showed an interlaboratory diagnostic sensitivity of 96.7% and a diagnostic specificity of 100% for chicken samples, while these values were 94.2 and 83.3%, respectively, for pig samples.

Animals↗
PubMed2004

Validated 5' nuclease PCR assay for rapid identification of the genus Brucella.

A real-time, genus-specific 5' nuclease PCR assay for amplification of a 322-bp fragment of the per gene was developed for rapid (<2 h) identification of Brucella spp. from agar plates. The assay, including an internal amplification control (116 bp), identified Brucella strains (n = 23) and did not detect non-Brucella strains (n = 174), indicating its usefulness, particularly for laboratories with stringent quality assurance programs.

Animals↗
PubMed2004

A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model--towards an international standard.

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

Animals↗
PubMed2003

Preston and Park-Sanders protocols adapted for semi-quantitative isolation of thermotolerant Campylobacter from chicken rinse.

Human campylobacteriosis has become the major cause of foodborne gastrointestinal diseases in several European countries. In order to implement effective control measures in the primary production, and as a tool in risk assessment studies, it is necessary to have sensitive and quantitative detection methods.Thus, semi-quantitative detection of thermophilic Campylobacter spp. in 20 naturally contaminated chicken rinse samples was carried out using the two most common standard protocols: Preston and Park-Sanders, as proposed by Nordic Committee on Food Analysis (NMKL) and International Standard Organization (ISO), respectively. For both protocols, the chicken rinse samples were prepared in 500 ml buffered peptone water, as recommended in the ISO protocol no. 6887-2. The results indicated that the Preston protocol was superior to the Park-Sanders protocol in supporting growth of Campylobacter spp. In conclusion, the established semi-quantitative assessment using Preston broth could be useful in monitoring the outcome of control programs or quantitative risk assessments.

Animals↗
PubMed2003

Toward an international standard for PCR-based detection of Escherichia coli O157. Part 1. Assay development and multi-center validation.

As part of a major European research project, a diagnostic PCR assay, including an internal amplification control, was developed and validated in a collaborative trial for the detection of Escherichia coli O157. The assay is based on amplification of sequences of the rfbE O157 gene. The collaborative trial, including 12 international laboratories, was carried out in two phases: phase (a) was performed with identical PCR reagents, including the internal control, provided by the sending laboratory; phase (b) was performed on the same samples and internal control but using in-house PCR reagents of own choice. Phase (a) showed an inclusivity (detection of target strains) of 96.8% and the exclusivity (negative response from nontarget strains) was 100%. The overall performance resulted of phase (a) in an accordance of 98.8, concordance of 98.6, and a concordance odds ratio of 1.11. Phase (b) results showed an accuracy of 100% with all partners and by using different polymerase types and thermocycler models. This indicates that the assay, under consideration as an international standard, was just as reproducible between laboratories, as repeatable within a laboratory. The assay is taken further for validation on carcass-rinse samples.

Animals↗
PubMed2003

Addition of Novobiocin in pre-enrichment step can improve Salmonella culture protocol of modified semisolid Rappaport-Vassiliadis.

The aim was to investigate the effect of addition of Novobiocin to the non-selective buffered peptone water (BPW) for pre-enrichment of Salmonella in connection with plating on modified semisolid Rappaport-Vassiliadis (MSRV). In a semi-quantitative study, the level of Salmonella following pre-enrichment of 32 presumably naturally contaminated swine fecal samples were assessed for BPW with and without addition of Novobiocin (22 microg/ml). In another experiment, a total of 400 swine fecal samples were screened for the presence of Salmonella spp., in order to compare the performance of the non-selective pre-enrichment broth with BPW made semi-selective by addition of Novobiocin. The semi-quantitative assessment of the Salmonella level showed that addition of Novobiocin in the pre-enrichment step on average increased the level of Salmonella 1.2 log dilution steps. When growth was scored at five levels, 90 samples opposed to 50 yielded a strong positive reading (+++) when Novobiocin was applied. Growth was on average 0.3 scores higher when pre-enriched with Novobiocin. The difference in growth score medians of the two methods was highly significant (Sign test; p<0.001). Despite the increased sensitivity, 13 culture-positive samples were missed when using the Novobiocin-containing BPW. In conclusion, a simple addition of Novobiocin in the BPW pre-enrichment step of fecal samples may facilitate reading and thereby detection of Salmonella on MSRV. The increase of Salmonella in the semi-quantitative study may be caused by a reduction in the number of competitive microorganisms.

Animals↗
PubMed2003

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: assay development and analytical validation.

As part of a European research project (FOOD-PCR), we developed a standardized and robust PCR detection assay specific for the three most frequently reported food-borne pathogenic Campylobacter species, C. jejuni, C. coli, and C. lari. Fifteen published and unpublished PCR primers targeting the 16S rRNA gene were tested in all possible pairwise combinations, as well as two published primers targeting the 23S rRNA gene. A panel of 150 strains including target and nontarget strains was used in an in-house validation. Only one primer pair, OT1559 plus 18-1, was found to be selective. The inclusivity and exclusivity were 100 and 97%, respectively. In an attempt to find a thermostable DNA polymerase more resistant than Taq to PCR inhibitors present in chicken samples, three DNA polymerases were evaluated. The DNA polymerase Tth was not inhibited at a concentration of 2% (vol/vol) chicken carcass rinse, unlike both Taq DNA polymerase and DyNAzyme. Based on these results, Tth was selected as the most suitable enzyme for the assay. The standardized PCR test described shows potential for use in large-scale screening programs for food-borne Campylobacter species under the assay conditions specified.

Base Sequence↗
PubMed2003

Toward an international standard for PCR-based detection of food-borne thermotolerant Campylobacters: validation in a multicenter collaborative trial.

As part of a European research project, the performance of a PCR assay to detect food-borne thermotolerant campylobacters (Campylobacter jejuni, C. coli, and C. lari) was evaluated through an international collaborative trial involving 12 participating laboratories. DNA from 10 target and 8 nontarget strains was tested, and the results were reported as the presence of a positive signal after gel electrophoresis. The overall inclusivity (sensitivity) was 93.7%, and the exclusivity (specificity) was 100%. The results indicate that the assay can become an international standard and can be confidently applied in microbiological laboratories.

Arcobacter↗