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.

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.

scholarly journals Quantitative detection of E. coli, E. coli O157 and other shiga toxin producing E. coli in water samples using a culture method combined with real-time PCR

2006 ◽  
Vol 4 (4) ◽  
pp. 487-498 ◽  
Author(s):  
Leo Heijnen ◽  
Gertjan Medema
Keyword(s):  
Surface Water ◽  
Real Time ◽  
Shiga Toxin ◽  
Water Samples ◽  
Real Time Pcr ◽  
Matrix Effects ◽  
Quantitative Detection ◽  
Attractive Alternative ◽  
E Coli ◽  
Wastewater Samples

Recent water related outbreaks of shiga toxin producing E. coli O157 have resulted in increased attention of the water industry to this potentially deadly pathogen. Current methods to detect E. coli O157 and its virulence genes are laborious and time-consuming. Specificity, sensitivity and simple use of a real-time PCR method makes it an attractive alternative for the detection of STEC E. coli O157. This study describes the development and application of real-time PCR methods for the detection of E. coli O157, shiga toxin genes (Stx1 and Stx2) and E. coli. The specificity of the methods was confirmed by performing colony-PCR assays on characterized bacterial isolates, demonstrating the applicability of these assays as rapid tests to confirm the presence of E. coli or E. coli O157 colonies on culture plates. Sensitive culture-PCR methods were developed by combining culture enrichment with real-time PCR detection. This rapid method allowed detection of low concentrations of E. coli O157 in the presence of high concentrations of non-O157-E. coli (1:104). Culture-PCR methods were applied to 27 surface water and 4 wastewater samples. E. coli O157 and both Stx genes were detected in two wastewater samples, whereas only E. coli O157 was detected in two surface water samples. Culture-PCR methods were not influenced by matrix effects and also enabled quantitative (MPN) detection of E. coli in these samples.

Quantitative detection of selenate-reducing bacteria by real-time PCR targeting the selenate reductase gene

2016 ◽  
Vol 85 ◽  
pp. 19-24 ◽  
Author(s):  
Li-Lian Wen ◽  
Chun-Yu Lai ◽  
Qiang Yang ◽  
Jia-Xian Chen ◽  
Yin Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Reductase Gene ◽  
Reducing Bacteria ◽  
Pcr Targeting

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 Quantitative Detection of Perchlorate-Reducing Bacteria by Real-Time PCR Targeting the Perchlorate Reductase Gene

2008 ◽  
Vol 74 (6) ◽  
pp. 1941-1944 ◽  
Author(s):  
Mamie Nozawa-Inoue ◽  
Mercy Jien ◽  
Nicholas S. Hamilton ◽  
Valley Stewart ◽  
Kate M. Scow ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Soil Microbial Communities ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Gene Encoding ◽  
Soil Microbial ◽  
Reductase Gene ◽  
Reducing Bacteria ◽  
Pcr Targeting

ABSTRACT A quantitative real-time PCR assay targeting the pcrA gene, encoding the catalytic subunit of perchlorate reductase, detected pcrA genes from perchlorate-reducing bacteria in three different genera and from soil microbial communities. Partial pcrA sequences indicated differences in the composition of perchlorate-reducing bacterial communities following exposure to different electron donors.

scholarly journals Direct detection of Mycobacterium avium SUBSP. paratuberculosis in bovine milk by multiplex real-time PCR

2013 ◽  
Vol 29 (3) ◽  
pp. 513-525 ◽  
Author(s):  
A. Selim ◽  
M. El-Haig ◽  
E.S. Galila
Keyword(s):  
Real Time ◽  
Dna Extraction ◽  
Mycobacterium Avium ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Detection Sensitivity ◽  
Mycobacterium Avium Subsp ◽  
Pcr Assay ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Pcr Targeting

This study aimed to direct detection of Mycobacterium avium subsp. paratuberculosis (MAP) in milk by evaluating a multiplex real-time PCR assay targeting IS900 and ISMAV2 sequences including the amplification of PUC19-plasmid as internal control. The sensitivity of the assays was evaluated by testing MAP isolates in broad linear range of DNA (50 ng - 5 fg/?l). For the validation of the specificity, 6 MAP isolates and 22 isolates of genus Mycobacteriacea were tested. Results revealed that reproducible detection limit for real-time PCR targeting IS900 and ISMAV2 was 5 fg/?l and 50 fg/?l respectively. By targeting ISMAV2 sequence, 100% specificity was detected. However, a cross reaction with 5 ng/?l of genome of 3 M. avian subspecies avium strains was detected by targeting IS900 and negative in lower genome quantity (5pg/?l). To maximize the assay?s detection sensitivity, an efficient strategy for MAP-DNA extraction from spiked milk was assessed. Targeting of IS900 was sensitive and targeting ISMAV2 was very specific. Therefore, a multiplex real-time PCR assay targeting IS900 and ISMAV2 in combination with two commercial DNA extraction kits could be an ideal sensitive and specific protocol for routine large scale analysis of milk samples and other clinical specimens from man and animals.

scholarly journals Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

2020 ◽  
Vol 9 (1) ◽  
pp. 54
Author(s):  
Salem Belkessa ◽  
Daniel Thomas-Lopez ◽  
Karim Houali ◽  
Farida Ghalmi ◽  
Christen Rune Stensvold
Keyword(s):  
Real Time ◽  
Molecular Characterization ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Giardia Duodenalis ◽  
Specific Pcr ◽  
Stool Samples ◽  
Pcr Targeting ◽  
Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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.

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