A Quantitative Real-Time PCR Approach for Assessing Campylobacter jejuni and Campylobacter coli Colonization in Broiler Herds

2017 ◽  
Vol 80 (4) ◽  
pp. 604-608 ◽  
Author(s):  
Katrin Haas ◽  
Gudrun Overesch ◽  
Peter Kuhnert
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Developed Countries ◽  
Human Infection ◽  
Health Concern ◽  
Campylobacter Coli ◽  
Quantitative Real Time Pcr ◽  
Simple Method

ABSTRACT Human campylobacteriosis is a major public health concern in developed countries, with Campylobacter jejuni and Campylobacter coli from poultry recognized as the main source of human infection. Identification of Campylobacter-positive broiler herds before slaughter is essential for implementing measures to avoid carryover of pathogens via the slaughter process into the food chain. However, appropriate methods that have been validated for testing poultry flocks antemortem are lacking for Campylobacter. A quantitative real-time PCR (qPCR) that allows simultaneous detection and quantification of C. jejuni and C. coli was adapted and optimized to be applied on boot socks. The adjusted qPCR serves as an easy, sensitive, and quantitative method for Campylobacter detection in poultry flocks antemortem by analysis of boot socks. An adequate correlation was found between qPCR and culture, as well as between boot socks and cecal samples, which are regarded as the “gold standard.” Therefore, boot sock sampling followed by qPCR analysis provides a reliable and simple method for assessing Campylobacter load within a flock prior to slaughter. The approach allows categorization of broiler herds into negative, low, moderate, or high Campylobacter colonization. Based on the results of this new approach, risk assessment models, such as evaluating the possible effect of sorting flocks before slaughter, can be easily implemented. Similarly, targeted identification of highly colonized flocks for improvement of biosecurity measures at the farm level will become feasible, presenting an opportunity to increase food safety.

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 Establishment of a simpler method for measuring HDL-microRNAs

2018 ◽  
Vol 56 (1) ◽  
pp. 49-55 ◽  
Author(s):  
Hiroaki Ishikawa ◽  
Hiroya Yamada ◽  
Kanako Kondo ◽  
Takeru Ota ◽  
Mirai Yamazaki ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Phosphotungstic Acid ◽  
Density Lipoprotein ◽  
Clinical Applications ◽  
Processing Capacity ◽  
Quantitative Real Time Pcr ◽  
Simple Method ◽  
Standard Curve ◽  
Purification Methods

Background MicroRNAs are present not only in exosomes but also in high-density lipoprotein (HDL) and have the potential as biomarkers for various diseases. Various purification methods have been developed to quantify HDL-miRNAs; however, they are unsuitable for clinical applications. Therefore, we aimed to establish a simpler analytical method to quantify HDL-miRNAs for clinical applications. Methods We purified HDL fraction from pooled plasma using a three-step protocol consisting of ultracentrifugation, phosphotungstic acid/MgCl2 precipitation and desalting/buffer exchange followed by the quantification of HDL-miRNAs by quantitative real-time PCR. In order to establish a method to quantify HDL-miRNAs by quantitative real-time PCR, we prepared standard curves for miR-223 and miR-92. The HDL-miRNAs of 10 volunteers were assessed. Results Exosomes and LDL were not detected in the purified HDL fraction. Furthermore, we confirmed that only HDL was purified and that the HDL recovery rate of our method was at least approximately 50%. The threshold cycle values of miR-223, miR-92, miR-146a and miR-150 in the same subject were 32.11 ± 0.58, 32.50 ± 0.35, 34.30 ± 0.70 and 34.91 ± 0.77, respectively ( n = 10). The coefficient of variation values for these miRNAs were 1.08–2.21%. In addition, the standard curve for the quantitative analysis of miRNAs showed high linearity (30–30,000 copies/ μL) with a correlation coefficient of >0.99. The concentrations of HDL-miR-223 and HDL-miR-92 in the plasma of 10 subjects were 1.98 ± 0.32 and 0.90 ± 0.14 copies/mL (×104). Conclusions We established a simple method for quantifying HDL-miRNAs and improved the sample processing capacity compared with conventional methods.

Simultaneous Detection and Differentiation of Campylobacter jejuni, C. coli, and C. lari in Chickens Using a Multiplex Real-Time PCR Assay

2010 ◽  
Vol 3 (4) ◽  
pp. 321-329 ◽  
Author(s):  
Yiping He ◽  
Xiaomin Yao ◽  
Nereus W. Gunther ◽  
Yanping Xie ◽  
Shu-I Tu ◽  
...  
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay

scholarly journals Development and evaluation of internal amplification controls for use in a real-time duplex PCR assay for detection of Campylobacter coli and Campylobacter jejuni

2010 ◽  
Vol 59 (2) ◽  
pp. 172-178 ◽  
Author(s):  
Luke Randall ◽  
Fabrizio Lemma ◽  
John Rodgers ◽  
Ana Vidal ◽  
Felicity Clifton-Hadley
Keyword(s):  
Real Time ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Dna Amplification ◽  
Pcr Primers ◽  
Campylobacter Coli ◽  
Coding Region ◽  
Pcr Assay ◽  
Viral Haemorrhagic Septicaemia Virus ◽  
Duplex Pcr Assay

A common problem of both conventional and real-time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate internal amplification controls (IACs) for use with an existing duplex real-time PCR assay for Campylobacter coli and Campylobacter jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the fish viral haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilized an extra set of universal 16S rDNA primers. Both IAC-PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable for detecting inhibition which could lead to false-negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR assay. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6 %, specificity 80.1 %); however, it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.

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