scholarly journals Development of a Systematic qPCR Array for Screening GM Soybeans

Foods ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 610
Author(s):  
Saet-Byul Park ◽  
Ji-Yeong Kim ◽  
Do-Geun Lee ◽  
Jae-Hwan Kim ◽  
Min-Ki Shin ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Screening Method ◽  
35S Promoter ◽  
Prediction System ◽  
Chain Reaction ◽  
Qpcr Array ◽  
Polymerase Chain ◽  
Gm Soybean

A screening method using the 35S promoter and nos terminator for genetically modified organisms (GMOs) is not sufficient to cover all GM soybean events. In this study, a real-time polymerase chain reaction (also known as quantitative polymerase chain reaction, qPCR) array targeting eight screening assays combined with a prediction system was developed for the rapid tracking of GM soybeans. Each assay’s specificity was tested and confirmed using 17 GM soybean events that have been approved in Korea. The sensitivity of each assay was determined to range from 0.01% to 0.05% using DNA mixtures with different GM ratios, and it was validated by the results of three experimenters. The applicability of this study was tested by monitoring 23 processed foods containing soybeans. It was figured out that 13 of the 23 samples included GM soybeans. The prediction system combined with screening results will be helpful to trace the absence/presence of GM soybean events. This new qPCR array and prediction system for GM soybean detection provides rapid, convenient and reliable results to users.

Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

2006 ◽  
Vol 52 (5) ◽  
pp. 451-461 ◽  
Author(s):  
S S Hynes ◽  
O Chaudhry ◽  
M A Providenti ◽  
M L Smith
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Quantitative Polymerase Chain Reaction ◽  
Pcr Primers ◽  
Chain Reaction ◽  
Environmental Persistence ◽  
Fungal Strains ◽  
Target Dna ◽  
Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

2008 ◽  
Vol 376 (2) ◽  
pp. 189-199 ◽  
Author(s):  
Katarina Cankar ◽  
Valérie Chauvensy-Ancel ◽  
Marie-Noelle Fortabat ◽  
Kristina Gruden ◽  
André Kobilinsky ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Genetically Modified Organisms ◽  
Genetically Modified ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Polymerase Chain

AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

2017 ◽  
Vol 23 (1) ◽  
Author(s):  
N.NANDHA KUMAR ◽  
K. SOURIANATHA SUNDARAM ◽  
D. SUDHAKAR ◽  
K.K. KUMAR
Keyword(s):  
Polymerase Chain Reaction ◽  
Quantitative Polymerase Chain Reaction ◽  
Proteinase K ◽  
Chain Reaction ◽  
Banana Plant ◽  
High Molecular Weight Dna ◽  
Musa Spp ◽  
Leaf Tissues ◽  
Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

scholarly journals Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

2020 ◽  
Vol 17 (1) ◽  
Author(s):  
Yang Zhang ◽  
Chunyang Dai ◽  
Huiyan Wang ◽  
Yong Gao ◽  
Tuantuan Li ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Quantitative Polymerase Chain Reaction ◽  
Clinical Samples ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Highly Sensitive ◽  
One Step ◽  
Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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