Quantitative Detection of Species-Specific DNA in Feedstuffs and Fish Meals

2005 ◽  
Vol 68 (6) ◽  
pp. 1217-1221 ◽  
Author(s):  
PAVEL KRCMAR ◽  
EVA RENCOVA
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Single Species ◽  
Absolute Quantification ◽  
Quantitative Detection ◽  
Vertebrate Species ◽  
Target Species ◽  
Species Specific

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species–derived material in concentrate feed mixtures and fish meals.

Highly Sensitive Matrix-Independent Quantification of Major Food Allergens Peanut and Soy by Competitive Real-Time PCR Targeting Mitochondrial DNA

2018 ◽  
Vol 101 (1) ◽  
pp. 170-184 ◽  
Author(s):  
Eva-Maria Ladenburger ◽  
Markus Dehmer ◽  
Ruben Grünberg ◽  
Hans-Ulrich Waiblinger ◽  
Dieter Stoll ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Matrix Effects ◽  
Detection Sensitivity ◽  
Quantitative Detection ◽  
Food Allergens ◽  
Major Food ◽  
Pcr Targeting

Abstract The development of two competitive real-time PCR assays for the quantitative detection of trace amounts of two major food allergens, peanut and soybean, is reported. In order to achieve very low detection levels for both allergens, we established PCR primers and probes targeting mitochondrial DNA sequences. We were able to demonstrate that this approach led to an increase in detection sensitivity in the range of at least 1 order of magnitude compared with published assays targeting nuclear DNA. Furthermore, we generated corresponding competitor molecules, which were used as internal standards to compete with matrix effects that are evident during DNA extraction and PCR amplification in heterogeneous analytical matrixes like food. According to the recently described competitive quantitative PCR method published by Holzhauser et al. (2014), we performed threshold calibration against milk powder spiked with 10 ppm peanut and soy. Matrix-independent quantitative determination of peanut and soy could be demonstrated for three different calibrated food matrix standards in a range between 1 and 100 ppm. The data presented indicate that both assay concepts are powerful analytical tools for the quantitative detection of trace amounts of peanut and soy in commercial food products.

Application of Quantitative Real-Time PCR in the Detection of Prion-Protein Gene Species-Specific DNA Sequences in Animal Meals and Feedstuffs

2006 ◽  
Vol 69 (4) ◽  
pp. 891-896 ◽  
Author(s):  
FEDERICA BELLAGAMBA ◽  
SERGIO COMINCINI ◽  
LUCA FERRETTI ◽  
FRANCO VALFRÈ ◽  
VITTORIO M. MORETTI
Keyword(s):  
Real Time ◽  
Prion Protein ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Animal Feed ◽  
Quantitative Estimation ◽  
Taqman Assay ◽  
Quantitative Real Time Pcr ◽  
The Real ◽  
Species Specific

This study describes a method for quantitative and species-specific detection of animal DNA from different species (cattle, sheep, goat, swine, and chicken) in animal feed and feed ingredients, including fish meals. A quantitative real-time PCR approach was carried out to characterize species-specific sequences based on the amplification of prion-protein sequence. Prion-protein species-specific primers and TaqMan probes were designed, and amplification protocols were optimized in order to discriminate the different species with short PCR amplicons. The real-time quantitative PCR approach was also compared to conventional species-specific PCR assays. The real-time quantitative assay allowed the detection of 10 pg of ruminant, swine, and poultry DNA extracted from meat samples processed at 130°C for 40 min, 200 kPa. The origin of analyzed animal meals was characterized by the quantitative estimation of ruminant, swine, and poultry DNA. The TaqMan assay was used to quantify ruminant DNA in feedstuffs with 0.1% of meat and bone meal. In conclusion, the proposed molecular approach allowed the detection of species-specific DNA in animal meals and feedstuffs.

Application of mitochondrial DNA analysis for microbial source tracking purposes in shellfish harvesting waters

2010 ◽  
Vol 61 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Craig Baker-Austin ◽  
Rachel Rangdale ◽  
James Lowther ◽  
David N. Lees
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Pcr Amplification ◽  
Source Tracking ◽  
Pcr Analysis ◽  
Taqman Probes ◽  
Faecal Matter ◽  
Species Specific

We present a method for the reliable detection and source characterisation of faecal pollution in water and shellfish matrices, utilising real-time PCR analysis of mitochondrial DNA targets. In this study we designed real-time PCR (TaqMan) probes to target human, bovine, ovine and swine mtDNA. PCR amplification using species-specific TaqMan probes on faecal matter and mixed effluent slurries revealed no cross-reactions between species of interest and other vertebrate faecal matter. Performed as a single blind experiment we were able to correctly identify faecal material in 17/20 effluents (85% correct). mtDNA degrades relatively quickly in faecally-spiked water samples (∼2 weeks), a similar timeframe of environmental persistence to several bacterial faecal indictors, highlighting its applicability. The procedure described here is specific, rapid (<5 hours) and sensitive. These results confirm the suitability of using species-specific mtDNA as an indicator in source tracking studies in surface waters, shellfish harvesting areas and shellfish matrices.

scholarly journals Identification of tuna species Thunnus albacares and Katsuwonus pelamis in canned products by real-time PCR method

2019 ◽  
Vol 88 (3) ◽  
pp. 323-328 ◽  
Author(s):  
Pavel Krčmář ◽  
Zora Piskatá ◽  
Eliška Servusová
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Yellowfin Tuna ◽  
Close Relative ◽  
Thunnus Albacares ◽  
Skipjack Tuna ◽  
Katsuwonus Pelamis ◽  
Variable Regions

Tuna species are a popular food among consumers. They are mostly sold as heat-processed canned products on the market. Different quality and price of tuna species can lead the producer to the adulteration of food products. The main difficulties in developing a method for species identification in these fish is the high similarity of DNA sequences among close relative fish species. All complete mitochondrial DNA sequences of skipjack tuna (Katsuwonus pelamis) and yellowfin tuna (Thunnus albacares) were compared to all other mitochondrial DNA sequences of tuna fish deposited in the GenBank. The most variable regions within species were determined and primers and probes were designed in this region for the species-specific DNA amplification of skipjack tuna and yellowfin tuna. Moreover, to check the content of amplifiable DNA of fish (namely tuna) in the sample, primers and a probe of mitochondrial 12S rRNA gene in the region of conservative sequence were designed. Real time PCR methods were verified by investigating 51 samples of canned tuna with the declared content of tuna species from the market; the species was confirmed in all tested samples. This method was designed to be suitable for the determination of DNA sequences especially in highly heat treated products.

scholarly journals Internally Controlled Real-Time PCR Method for Quantitative Species-Specific Detection and vapA Genotyping of Rhodococcus equi

2006 ◽  
Vol 72 (6) ◽  
pp. 4256-4263 ◽  
Author(s):  
David Rodr�guez-L�zaro ◽  
Deborah A. Lewis ◽  
Alain A. Ocampo-Sosa ◽  
Ursula Fogarty ◽  
L�szl� Makrai ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Rhodococcus Equi ◽  
Plasmid Copy Number ◽  
Quantitative Detection ◽  
Virulence Plasmid ◽  
Plasmid Copy ◽  
Pcr Method ◽  
Species Specific

ABSTRACT We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA + and vapA-negative (including vapB +) plasmid genotypes. The vapA + frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to ≈10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA +) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA + “horse-pathogenic” genotype determination.

Species-specific real-time PCR cell number quantification of the bloom-forming cyanobacterium Planktothrix agardhii

2012 ◽  
Vol 194 (9) ◽  
pp. 749-757 ◽  
Author(s):  
Catarina Churro ◽  
Paulo Pereira ◽  
Vitor Vasconcelos ◽  
Elisabete Valério
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Cell Number ◽  
Planktothrix Agardhii ◽  
Species Specific

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

A novel method to quantify deleted mitochondrial DNA in a real time PCR

2006 ◽  
Vol 1288 ◽  
pp. 756-758 ◽  
Author(s):  
T. Schwark ◽  
C. Fisch-Kohl ◽  
N. von Wurmb-Schwark
Keyword(s):  
Mitochondrial Dna ◽  
Real Time ◽  
Real Time Pcr ◽  
Novel Method
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