scholarly journals A Multicenter Proposal for a Fast Tool To Screen Biosecure Chicken Flocks for the Foodborne Pathogen Campylobacter

2020 ◽  
Vol 86 (20) ◽  
Author(s):  
Jeffrey Hoorfar ◽  
Ivana Koláčková ◽  
Gro S. Johannessen ◽  
Giuliano Garofolo ◽  
Francesca Marotta ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Screening Method ◽  
Foodborne Pathogen ◽  
Air Sampling ◽  
Foodborne Illness ◽  
Test Method ◽  
Air Filters ◽  
Direct Plating

ABSTRACT The present multicenter study aimed at assessing the performance of air sampling as a novel method for monitoring Campylobacter in biosecure poultry farms. We compared, using a harmonized procedure, the bacteriological isolation protocol (ISO 10272-1:2017) and a real-time PCR method used on air filter samples. Air samples and boot swabs were collected from 62 biosecure flocks from five European countries during the summer of 2019. For air filters, the frequency of PCR-positive findings was significantly higher (n = 36; 58%) than that obtained with the cultivation methods (P < 0.01; standardized residuals). The cultivation protocols (one with Bolton enrichment and one with Preston enrichment) were comparable to each other but returned fewer positive samples (0 to 8%). The association between type of sample and frequency of PCR-positive findings was statistically confirmed (P < 0.01; Fisher´s exact test), although no culture-positive air filters were detected using direct plating. For the boot swabs, the highest number of positive samples were detected after enrichment in Preston broth (n = 23; 37%), followed by direct plating after homogenization in Preston (n = 21; 34%) or Bolton broth (n = 20; 32%). It is noteworthy that the flocks in Norway, a country known to have low Campylobacter prevalence in biosecure chicken flocks, tested negative for Campylobacter by the new sensitive approach. In conclusion, air sampling combined with real-time PCR is proposed as a multipurpose, low-cost, and convenient screening method that can be up to four times faster and four times more sensitive than the current boot-swab testing scheme used for screening biosecure chicken production. IMPORTANCE Campylobacter bacteria are the cause of the vast majority of registered cases of foodborne illness in the industrialized world. In fact, the bacteria caused 246,571 registered cases of foodborne illness in 2018, which equates to 70% of all registered cases in Europe that year. An important tool to prevent campylobacters from making people sick is good data on where in the food chain the bacterium is present. The present study reports a new test method that quadruples the likelihood of identifying campylobacter-positive chicken flocks. It is important to identify campylobacter-positive flocks before they arrive at the slaughterhouse, because negative flocks can be slaughtered first in order to avoid cross-contamination along the production line.

Low-Cost Monitoring of Campylobacter in Poultry Houses by Air Sampling and Quantitative PCR

2014 ◽  
Vol 77 (2) ◽  
pp. 325-330 ◽  
Author(s):  
M. S. R. SØNDERGAARD ◽  
M. H. JOSEFSEN ◽  
C. LÖFSTRÖM ◽  
L. S. CHRISTENSEN ◽  
K. WIECZOREK ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Air Sampling ◽  
Quantitative Risk Assessment ◽  
Quantitative Real Time Pcr ◽  
Airborne Particle ◽  
Air Samples ◽  
Particle Count ◽  
Poultry Houses

The present study describes the evaluation of a method for the quantification of Campylobacter by air sampling in poultry houses. Sampling was carried out in conventional chicken houses in Poland, in addition to a preliminary sampling in Denmark. Each measurement consisted of three air samples, two standard boot swab fecal samples, and one airborne particle count. Sampling was conducted over an 8-week period in three flocks, assessing the presence and levels of Campylobacter in boot swabs and air samples using quantitative real-time PCR. The detection limit for air sampling was approximately 100 Campylobacter cell equivalents (CCE)/m3. Airborne particle counts were used to analyze the size distribution of airborne particles (0.3 to 10 μm) in the chicken houses in relation to the level of airborne Campylobacter. No correlation was found. Using air sampling, Campylobacter was detected in the flocks right away, while boot swab samples were positive after 2 weeks. All samples collected were positive for Campylobacter from week 2 through the rest of the rearing period for both sampling techniques, although levels 1- to 2-log CCE higher were found with air sampling. At week 8, the levels were approximately 104 and 105 CCE per sample for boot swabs and air, respectively. In conclusion, using air samples combined with quantitative real-time PCR, Campylobacter contamination could be detected earlier than by boot swabs and was found to be a more convenient technique for monitoring and/or to obtain enumeration data useful for quantitative risk assessment of Campylobacter.

Complement C3 Genotyping of Slow and Fast Variants by Real Time PCR-High Resolution Melting

2012 ◽  
Vol 10 (3) ◽  
pp. 329-334 ◽  
Author(s):  
D.M. Valero-Hervás ◽  
P. Morales ◽  
M.J. Castro ◽  
P. Varela ◽  
M. Castillo-Rama ◽  
...  
Keyword(s):  
High Resolution ◽  
Real Time ◽  
Real Time Pcr ◽  
High Resolution Melting ◽  
Low Cost ◽  
Complement C3 ◽  
Amplification Refractory Mutation System ◽  
Rt Pcr ◽  
Dna Amount ◽  
Mutation System

“Slow” and “Fast” C3 complement variants (C3S and C3F) result from a g.304C>G polymorphism that changes arginine to glycine at position 102. C3 variants are associated with complement-mediated diseases and outcome in transplantation. In this work C3 genotyping is achieved by a Real Time PCR - High Resolution Melting (RT-PCR-HRM) optimized method. In an analysis of 49 subjects, 10.2% were C3FF, 36.7% were C3SF and 53.1% were C3SS. Allelic frequencies (70% for C3S and 30% for C3F) were in Hardy-Weinberg equilibrium and similar to those published previously. When comparing RT-PCR-HRM with the currently used Tetraprimer-Amplification Refractory Mutation System PCR (T-ARMS-PCR), coincidence was 93.8%. The procedure shown here includes a single primer pair and low DNA amount per reaction. Detection of C3 variants by RT-PCR-HRM is accurate, easy, fast and low cost, and it may be the method of choice for C3 genotyping.

Ultrafast real-time PCR with integrated melting curve analysis and duplex capacities using a low-cost polymer lab-on-a-chip system

2015 ◽  
Author(s):  
Rainer Gransee ◽  
Tristan Schneider ◽  
Deniz Elyorgun ◽  
Xenia Strobach ◽  
Tobias Schunck ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Low Cost ◽  
Melting Curve ◽  
Lab On A Chip ◽  
Curve Analysis ◽  
Melting Curve Analysis

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

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
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.

scholarly journals Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 757
Author(s):  
Sara Franco Ortega ◽  
Ilenia Siciliano ◽  
Simona Prencipe ◽  
Maria Lodovica Gullino ◽  
Davide Spadaro
Keyword(s):  
Food Safety ◽  
Real Time ◽  
Aspergillus Flavus ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Screening Method ◽  
Lamp Assay ◽  
High Sensitivity ◽  
Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

scholarly journals Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

2020 ◽  
Author(s):  
Dennis Back Holmgaard ◽  
Celine Barnadas ◽  
Seyed Hossein Mirbarati ◽  
Lee O’Brien Andersen ◽  
Henrik Vedel Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Relevance ◽  
Bacterial Species ◽  
Screening Method ◽  
Infectious Keratitis ◽  
Next Generation ◽  
Specificity And Sensitivity ◽  
Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

scholarly journals Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

2011 ◽  
Vol 77 (22) ◽  
pp. 8193-8196 ◽  
Author(s):  
Lucja M. Jarosz ◽  
Bastiaan P. Krom
Keyword(s):  
Candida Albicans ◽  
Real Time ◽  
Real Time Pcr ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Rapid Screening ◽  
Content Type ◽  
Wide Range ◽  
Hyphal Formation ◽  
Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

scholarly journals Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory

2009 ◽  
Vol 107 (5) ◽  
pp. 1433-1439 ◽  
Author(s):  
K.L. Leung ◽  
C.W. Yip ◽  
W.F. Cheung ◽  
A.C.T. Lo ◽  
W.M. Ko ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Low Cost ◽  
Pcr Method
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