The devolopment of duplex real-time PCR based on SYBR Green florescence for rapid ıdentification of ruminant and poultry origins in foodstuff

Şakalar, Ergün; Abasıyanık, M. Fatih

doi:10.1016/j.foodchem.2011.07.130

Cited by 25 publications

(14 citation statements)

References 26 publications

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“…The specificity of oligonucleotides is one of most important points; the optimal primer should hybridize only with the concrete target sequence. The specificity of the primers in this work has been tested for livestock species in previous studies (Dalmasso and others 2004;Sakalar and Abasıyanık 2012). It was determined that primers specific to the species of ruminant, pork, and poultry showed no cross-reaction with any of the nontarget species.…”

Section: Resultsmentioning

confidence: 96%

Effect of Heat Processing on DNA Quantification of Meat Species

Şakalar¹,

Abasıyanık²,

Bektik³

et al. 2012

Journal of Food Science

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In this study, real-time polymerase chain reaction (PCR) was used for identifying the effects of different temperatures and times of heat treatment on the DNA of meat products. For this purpose, beef, pork, and chicken were baked at 200 °C for 10, 20, 30, 40, 50 min, and for 30 min at 30, 60, 90, 120, 150, 180, 210 °C and also cooked by boiling at 99 °C for 10, 30, 60, 90, 120, 150, 180, 210, and 240 min. The DNA was then extracted from all samples after the heat treatment. Further, a region of 374, 290, and 183-bp of mitochondrial DNA of beef, pork, and chicken, respectively, was amplified by real-time PCR. It was found that baking and boiling of the beef, pork, and chicken resulted in decreases in the detectable copy numbers of specific genes, which varied with the heating time and degree. The results indicated that species determination and quantification using real-time PCR are affected by the temperature, duration of the heat treatment, and size of the DNA fragment to be amplified.

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Section: Resultsmentioning

confidence: 96%

Effect of Heat Processing on DNA Quantification of Meat Species

Şakalar¹,

Abasıyanık²,

Bektik³

et al. 2012

Journal of Food Science

Self Cite

View full text Add to dashboard Cite

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“…In addition, it is difficult to design and optimize a probe ( Varga and James, 2005 ). For these reasons, melting curve analysis after SYBR green-based real-time PCR has been used to identify ruminant (cattle) and poultry (turkey) in foodstuffs ( Şakalar and Abas yan k, 2012 ), discrimination of plum pox virus isolates of strain D and M ( Varga and James, 2005 ), and discrimination of deer and six common domestic species ( You et al ., 2014 ).…”

Section: Resultsmentioning

confidence: 99%

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

Cho¹,

Dong²,

Cho³

2014

Korean Journal for Food Science of Animal Resources

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Meat source fraud and adulteration scandals have led to consumer demands for accurate meat identification methods. Nucleotide amplification assays have been proposed as an alternative method to protein-based assays for meat identification. In this study, we designed Loop-mediated isothermal amplification (LAMP) assays targeting species-specific mitochondrial DNA to identify and discriminate eight meat species; cattle, pig, horse, goat, sheep, chicken, duck, and turkey. The LAMP primer sets were designed and the target genes were discriminated according to their unique annealing temperature generated by annealing curve analysis. Their unique annealing temperatures were found to be 85.56±0.07℃ for cattle, 84.96±0.08℃ for pig, and 85.99±0.05℃ for horse in the BSE-LAMP set (Bos taurus, Sus scrofa domesticus and Equus caballus); 84.91±0.11℃ for goat and 83.90±0.11℃ for sheep in the CO-LAMP set (Capra hircus and Ovis aries); and 86.31±0.23℃ for chicken, 88.66±0.12℃ for duck, and 84.49±0.08℃ for turkey in the GAM-LAMP set (Gallus gallus, Anas platyrhynchos and Meleagris gallopavo). No cross-reactivity was observed in each set. The limits of detection (LODs) of the LAMP assays in raw and cooked meat were determined from 10 pg/μL to 100 fg/μL levels, and LODs in raw and cooked meat admixtures were determined from 0.01% to 0.0001% levels. The assays were performed within 30 min and showed greater sensitivity than that of the PCR assays. These novel LAMP assays provide a simple, rapid, accurate, and sensitive technology for discrimination of eight meat species.

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“…The uniformity of each species-specific amplicon in conjunction with fluorophor-specific probes would make these assays amenable to multi-color multiplex detection, whereas fluorescent dye based detection would not ( Elızaquıvel and Aznar, 2008 , Walker et al , 2004 ). In fact, recent research shows that the use of fluorescent dye may result in a high multiplexing index in less time, in a single reaction ( Fukushıma et al , 2005 ; Pafundo et al , 2009 ; Şakalar and Abasıyanık 2012 ) as shown by schematic determination of porcine and horse meats by EvaGreen based duplex real time PCR ( Fig. 1 ).…”

Section: Introductionmentioning

confidence: 99%

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

Şakalar¹,

Ergün²,

Akar³

2015

Korean Journal for Food Science of Animal Resources

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A duplex real-time polymerase chain reaction (PCR) based assay for the detection of porcine and horse meat in sausages was designed by using EvaGreen fluorescent dye. Primers were selected from mitochondrial 12S rRNA and 16S rRNA genes which are powerful regions for identification of horse and porcine meat. DNA from reference samples and industrial products was successfully extracted using the GIDAGEN® Multi-Fast DNA Isolation Kit. Genomes were identified based on their specific melting peaks (Mp) which are 82.5℃ and 78℃ for horse and porcine, respectively. The assay used in this study allowed the detection of as little as 0.0001% level of horse meat and 0.001% level of porcine meat in the experimental admixtures. These findings indicate that EvaGreen based duplex real-time PCR is a potentially sensitive, reliable, rapid and accurate assay for the detection of meat species adulterated with porcine and horse meats.

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The devolopment of duplex real-time PCR based on SYBR Green florescence for rapid ıdentification of ruminant and poultry origins in foodstuff

Cited by 25 publications

References 26 publications

Effect of Heat Processing on DNA Quantification of Meat Species

Effect of Heat Processing on DNA Quantification of Meat Species

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

A Simultaneous Analytical Method for Duplex Identification of Porcine and Horse in the Meat Products by EvaGreen based Real-time PCR

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