A Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Detection ofSalmonellaspp.,Escherichia coliO157, andListeria monocytogenesin Meat Products

2010 ◽  
Vol 7 (6) ◽  
pp. 619-628 ◽  
Author(s):  
Biao Suo ◽  
Yiping He ◽  
Shu-I Tu ◽  
Xianming Shi
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Simultaneous Detection ◽  
Meat Products ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Quantification of Venturia inaequalis Growth in Malus × domestica with Quantitative Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2012 ◽  
Vol 96 (12) ◽  
pp. 1791-1797 ◽  
Author(s):  
Michele Gusberti ◽  
Andrea Patocchi ◽  
Cesare Gessler ◽  
Giovanni A. L. Broggini
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Malus Domestica ◽  
Venturia Inaequalis ◽  
Simultaneous Detection ◽  
Scab Resistance ◽  
Infected Leaf ◽  
Chain Reaction ◽  
Limit Of Quantification ◽  
Polymerase Chain

A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus × domestica. The method is based on dual-labeled hybridization probes, allowing simultaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible ‘Gala’ was 118 times higher than in resistant ‘Florina’ and ‘Discovery’ while intermediate values were found with the intermediate susceptible ‘Milwa’. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.

A Duplex SYBR Green I-based Real-time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

2021 ◽  
Author(s):  
Yang Pan ◽  
Jing Chen ◽  
Junhuang Wu ◽  
Yongxia Wang ◽  
Junwei Zou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Rapid Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Canine Kobuvirus

Abstract Background: Canine Kobuvirus (CaKoV) and Canine Circovirus (CaCV) are viruses that infect dogs causing diarrheal symptoms that are very similar. However, there is no clinical method to detect a co-infection of these two viruses.Results: In this study, a duplex SYBR Green I-based quantitative real-time polymerase chain reaction (PCR) assay for the rapid and simultaneous detection of CaKoV and CaCV was established. CaKoV and CaCV were distinguished by their different melting temperature which was 86℃ for CaKoV and 78℃ for CaCV. The assay was highly specific, with no cross-reactivity with other common canine viruses and demonstrated high sensitivity. The detection limits of CaKoV and CaCV were 8.924 × 101 copies/μL and 3.841 × 101 copies/μL, respectively. The highest intra- and inter-assay Ct value variation coefficients (CV) of CaKoV were 0.40% and 0.96%, respectively. For CaCV, the highest intra- and inter-assay Ct value variation coefficients were 0.26% and 0.70%, respectively. In 57 clinical samples, positive detection rates of CaKoV and CaCV were 8.77% (7/57) and 15.79% (9/57), respectively. The co-infection rate was 7.02% (4/57). Conclusions: The duplex SYBR Green I-based real-time PCR assay established in this study is a fast, efficient, and sensitive method for the simultaneous detection of the two viruses and provides a powerful tool for the rapid detection of CaKoV and CaCV in clinical practice.

scholarly journals Collaborative Trial for Validation of a Real-Time Reverse Transcriptase-Polymerase Chain Reaction Assay for Detection of Central Nervous System Tissues as Bovine Spongiform Encephalopathy RiskMaterial: Part 1

2006 ◽  
Vol 89 (5) ◽  
pp. 1335-1340
Author(s):  
Amir Abdulmawjood ◽  
Holger Schnenbrcher ◽  
Michael BÜlte
Keyword(s):  
Central Nervous System ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Products ◽  
Spongiform Encephalopathy ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Heat Treated ◽  
Polymerase Chain

Abstract A collaborative trial was conducted to evaluate a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detection of central nervous system (CNS) tissues in meat products (e.g., sausages). The method is based on the detection of ruminant glial fibrillary acidic protein (GFAP) mRNA by applying real-time RT-PCR. The assay was evaluated through a multicenter trial involving 12 participating laboratories that received coded cDNA obtained from 3 different types of sausages. The participants used 5 different real-time detection systems. The results obtained in this validation revealed that this real-time RT-PCR assay performed well in the different laboratories with a detection limit of at least 0.1% CNS in those test materials that contained strongly heat-treated samples (sausages cooked at 120C) and the medium heat-treated samples (sausages cooked at 80C). The detection limit of liver sausages was determined to be 0.2% of CNS. Neither the samples with no CNS additive nor the bovine DNA and the negative control containing 100% swine brain gave any positive signals. The presented results indicate that the real-time RT-PCR assay was just as reproducible between laboratories, as repeatable within a laboratory, could reliably be used for detection of bovine spongiform encephalopathy risk material in meat and meat products, and signify that it may be used with confidence in any laboratory.

Combination of Immunomagnetic Separation and Polymerase Chain Reaction for the Simultaneous Detection of Listeria monocytogenes and Salmonella spp. in Food Samples

2001 ◽  
Vol 64 (11) ◽  
pp. 1744-1750 ◽  
Author(s):  
HSIEN-YEE HSIH ◽  
HAU-YANG TSEN
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Simultaneous Detection ◽  
Meat Products ◽  
Immunomagnetic Separation ◽  
Food Samples ◽  
Chain Reaction ◽  
Salmonella Spp ◽  
Pcr Method ◽  
Polymerase Chain

A method that combined the immunomagnetic separation (IMS) technique and the multiplex polymerase chain reaction (PCR) method (i.e., the IMS-mPCR method) was developed for simultaneous detection of Listeria monocytogenes and Salmonella spp. in food samples. When only the multiplex PCR method was used, it was found that if cell numbers of each of the two target organisms (L. monocytogenes and Salmonella spp.) were above the detection limit, but differed by more than 2 logs—e.g., n × 107 to n × 104 or n × 106 to n × 103—the organism presenting the lower numbers might go undetected. Following the enrichment step with universal preenrichment (UP) broth, if an IMS method using equal quantities of anti-Listeria and anti-Salmonella immunomagnetic beads was performed prior to PCR, both pathogens could be detected unambiguously. Such results could be obtained for target organisms in food samples, such as milk, dairy, and meat products, if similar enrichment and IMS steps were performed prior to PCR.

scholarly journals REAL TIME POLYMERASE CHAIN REACTION (RT-PCR) FOR DNA PIG CONTAMINATION IDENTIFICATION SAUSAGE IN SIX DISTRICT PANDEGLANG BANTEN

2021 ◽  
Vol 1 (1) ◽  
pp. 81-88
Author(s):  
Hadi Susilo
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Meat Product ◽  
Meat Products ◽  
Pcr Analysis ◽  
Processed Meat ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Dna Purity ◽  
Polymerase Chain

Sausage is a meat product processed that is popular food especially in Pandeglang, Banten Province. The importance of halal certificates or the existence of the MUI (Indonesian Ulama Council) halal logo for processed meat products makes Muslim people confident to consume them. The aim this research was to identify pig DNA contamination in sausage products in six  districts in Pandeglang without the MUI halal labels using RT-PCR (Real Time-Polymerase Chain Reaction). RT PCR that can calculate to pig to fill these sample free from pig contamination. This research was divided into two stage, the first stage is extracted or carried out DNA and the second stage is RT PCR analysis. The results of the DNA purity test on sausage samples had DNA purity values ​​of 1.84-1.9 (A260 / A280) and resulted in sample concentrations ranging from 37.8 to 102.5 ng / µl.  The only amplification on the FAM curve was in the positive control pig.  the Cq value ranges from 30 - 31.29. The results of RT PCR on sausage samples in the district area in Pandeglang Banten did not detect the presence of pig DNA.

scholarly journals Designing a time-effective TaqMan probe-based real-time polymerase chain reaction protocol for the identification of Yersinia enterocolitica in raw pork meat

2017 ◽  
Vol 86 (4) ◽  
pp. 317-323
Author(s):  
Milena Alicja Stachelska
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Yersinia Enterocolitica ◽  
Meat Products ◽  
Taqman Probe ◽  
Pork Meat ◽  
Chain Reaction ◽  
Microbial Risk ◽  
Polymerase Chain

The aim of this study was to design a time-effective method comprising a short pre-enrichment step in a non-selective broth in combination with the TaqMan probe applied in the real-time polymerase chain reaction to detectYersinia enterocoliticastrains in raw pork meat. The method enabled to detect 1 colony forming unit per 25 mg ofYersinia enterocoliticain pork meat. The specificity and reliability of the method was not diminished by the company of microflora naturally present in meat. The method was found successful to detect pathogenicYersinia enterocoliticastrains in pork meat. It is advised to be used for assessing the microbial risk and for controlling the microbial quality of meat and meat products.

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