QUANTITATIVE DETECTION OF TRANSGENIC AND ENDOGENOUS DNA SEQUENCES IN SEEDS AFTER AUTOMATED DNA PREPARATION

2004 ◽  
Vol 16 (01) ◽  
pp. 1-6 ◽  
Author(s):  
CHRISTOPH PETERHÄNSEL ◽  
SILKE HAHNEN ◽  
RAINER KALAMAJKA ◽  
SASCHA OFFERMANN ◽  
BRIGITTE MIEDL ◽  
...  
Keyword(s):  
High Throughput ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Genetically Modified Organisms ◽  
Quantitative Detection ◽  
Specific Detection ◽  
Large Populations ◽  
Single Seeds ◽  
Hybridisation Probe ◽  
Multiple Pcr

In applied transgenic research as well as in agriculture there is an increasing need for high-throughput analyses of plants for genotypic selection or to identify the purity of seed stocks, e.g. for transgenic contaminations or the identification of pathogens. We developed and optimised conditions for the isolation of DNA from single seeds using an automated high-throughput protocol. Our results show that the system provided is capable of isolating DNA from any tested seed source. Furthermore, seeds remain capable of germinating during the homogenisation procedure. Quantification of endogenous and transgenic sequences by Real-Time PCR revealed that the prepared DNA is suitable in quality and quantity for multiple PCR analyses with both SYBR Green and hybridisation probe detection. The described method will be a useful tool for routine analyses like screening of large populations or the specific detection of genetically modified organisms (GMO).

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.

High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups

2009 ◽  
Vol 45 (4) ◽  
pp. 304-310 ◽  
Author(s):  
Elisa Leo ◽  
Simona Venturoli ◽  
Monica Cricca ◽  
Monica Musiani ◽  
Marialuisa Zerbini
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Risk Groups ◽  
Quantitative Detection ◽  
Pcr Assay

scholarly journals Bovine ticks harbour a diverse array of microorganisms in Pakistan

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Abdul Ghafar ◽  
Alejandro Cabezas-Cruz ◽  
Clemence Galon ◽  
Dasiel Obregon ◽  
Robin B. Gasser ◽  
...  
Keyword(s):  
Real Time ◽  
High Throughput ◽  
Dna Sequences ◽  
Real Time Pcr ◽  
Distribution Patterns ◽  
Diagnostic Methods ◽  
Mixed Infections ◽  
Ecological Zones ◽  
Wide Range ◽  
Relationship Of

Abstract Background Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique. Methods Ticks were collected from clinically healthy cattle (n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein. Results PCR detected DNA of at least one microorganism in each of 221 ticks tested (94.4%, 221/234). DNA-based detection inferred that single pathogens/endosymbionts were the most common (43.4%, 96/221) followed by double (38.9%, 86/221), triple (14.5%, 32/221), quadruple (2.3%, 5/221) and quintuple (0.9%, 2/221) mixed infections. Piroplasms (Babesia/Theileria spp.) were the most prevalent (31.6%, 74/234), followed by Ehrlichia spp. (20%, 47/234) and Anaplasma marginale (7.7%, 18/234). Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like (91.5%, 214/234) and Coxiella-like (1.3%, 3/234) organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms (41.2%, 54/131; P < 0.05) than those from buffaloes (19.4%, 20/103). However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs. Conclusions To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan.

scholarly journals Real-Time Detection of Genetically Modified Soya Using Lightcycler and ABI 7700 Platforms with TaqMan, Scorpion, and SYBR Green I Chemistries

2002 ◽  
Vol 85 (4) ◽  
pp. 938-944 ◽  
Author(s):  
Catherine F Terry ◽  
Della J Shanahan ◽  
Lydia D Ballam ◽  
Neil Harris ◽  
David G McDowell ◽  
...  
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Sybr Green ◽  
Sybr Green I ◽  
Quantitative Detection ◽  
Roundup Ready ◽  
Detection Systems ◽  
Copy Numbers

Abstract A comparative cross platform evaluation of real-time polymerase chain reaction detection of DNA sequences present in Roundup Ready soya was undertaken using the ABI 7700 and Roche Lightcycler detection systems in combination with 3 different detection chemistries: TaqMan, Scorpion primers, and SYBR Green I fluorescent dye. Various copy numbers of a plasmid containing the soya lectin sequence were used to determine the sensitivity and reproducibility of the different technology combinations and to examine both inter and intra machine variability. To examine the relative accuracy of each technology, the genetically modified soya content of baked products containing known amounts of Roundup Ready soya was determined by detection of lectin and the EPSPS transgene. It was determined that the combination of TaqMan detection chemistry and the ABI 7700 platform represented the best method for quantitative detection of genetically modified organisms in terms of both precision and accuracy.

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.

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