Real-Time Polymerase Chain Reaction Method for Detecting Contamination of Beef by Material from Genetically Engineered Cattle

Nakajima, Osamu; Akiyama, Hiroshi; Teshima, Reiko

doi:10.1248/bpb.32.1313

Cited by 4 publications

(3 citation statements)

References 11 publications

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“…Probe #5 (Roche Diagnostics, Basel, Switzerland) from the Universal Probe Library, conjugated with FAM, was used to detect amplification of CO CYBB . The probe structures are described for PURO 13 and RPP30. 14 VCN was determined with the QX200 droplet digital PCR system (Bio-Rad, Hercules, CA) as per the manufacturer's instructions and as follows.…”

Section: Methodsmentioning

confidence: 99%

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Lin

Okumura

Yatsuda³

et al. 2016

Human Gene Therapy Methods

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Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

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Section: Methodsmentioning

confidence: 99%

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Lin

Okumura

Yatsuda³

et al. 2016

Human Gene Therapy Methods

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“…The existence of viable non-cultivable microorganisms, or the long time that certain microorganisms may have to multiply in the environment, ends up being an obstacle to some researches [ 14 , 25 , 26 , 27 ]. On the other hand, molecular methods are essentially based on the amplification of target genes (i.e., PCR, real-time PCR (qPCR), multiplex PCR, random amplified polymorphic DNA (RAPD), PCR combined with restriction fragment length polymorphism (PCR–RFLP)), whole-genome sequencing and metagenomics [ 28 , 29 , 30 ]. Although more expensive than traditional cultivation, they are essential tools for the study of multiple microbiomes.…”

Section: Introductionmentioning

confidence: 99%

Antimicrobial Resistance Gene Detection Methods for Bacteria in Animal-Based Foods: A Brief Review of Highlights and Advantages

et al. 2021

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Antimicrobial resistance is a major public health problem and is mainly due to the indiscriminate use of antimicrobials in human and veterinary medicine. The consumption of animal-based foods can contribute to the transfer of these genes between animal and human bacteria. Resistant and multi-resistant bacteria such as Salmonella spp. and Campylobacter spp. have been detected both in animal-based foods and in production environments such as farms, industries and slaughterhouses. This review aims to compile the techniques for detecting antimicrobial resistance using traditional and molecular methods, highlighting their advantages and disadvantages as well as the effectiveness and confidence of their results.

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“…Few papers describe the use of an internal plasmid reference standard for the detection of hidden ingredients in food, such as allergens , or GMOs − . Oguchi and collaborators reported the production and use of a linearized modified plasmid (pSCM) in a duplex real-time PCR calibration to detect genetically modified maize.…”

Section: Introductionmentioning

confidence: 99%

Development and Validation of a SYBR-Green I Real-Time PCR Protocol To Detect Hazelnut (Corylus avellana L.) in Foods through Calibration via Plasmid Reference Standard

D’Andrea¹,

Coı̈sson²,

Travaglia³

et al. 2009

J. Agric. Food Chem.

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Many tree nuts are considered to be a serious problem in food safety, because of the presence of causative factors in IgE-mediated food allergies. Among these, hazelnut (Corylus avellana L.) seeds are largely used in a range of confectionery products and contain many well-characterized allergens. DNA-based methods and ELISA tests may prove to be useful to assess the presence of hidden ingredients in foods. The aim of this work was the development and validation of a species-specific SYBR Green I real-time PCR protocol for the detection of hazelnut in foods. A novel efficient primer pair on the Cor a 8 genomic coding region was designed by preparing a plasmid vector-based internal reference standard to calibrate the PCR. A good sensitivity, down to 20 (genomic) and 15 (plasmid) DNA copies, was established. All of the commercial samples considered in our study (containing hazelnut as ingredient or as a potential trace cross-contamination) were effectively amplified by PCR, showing a perfect correspondence with an ELISA commercial test, employed as a reference standard method.

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Real-Time Polymerase Chain Reaction Method for Detecting Contamination of Beef by Material from Genetically Engineered Cattle

Cited by 4 publications

References 11 publications

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Antimicrobial Resistance Gene Detection Methods for Bacteria in Animal-Based Foods: A Brief Review of Highlights and Advantages

Development and Validation of a SYBR-Green I Real-Time PCR Protocol To Detect Hazelnut (Corylus avellana L.) in Foods through Calibration via Plasmid Reference Standard

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