scholarly journals In-House Validation and Comparison of Two Wheat (Triticum aestivum) Taxon-Specific Real-Time PCR Methods for GMO Quantification Supported by Droplet Digital PCR

2017 ◽  
Vol 11 (5) ◽  
pp. 1281-1290 ◽  
Author(s):  
Annalisa Paternò ◽  
Daniela Verginelli ◽  
Pamela Bonini ◽  
Marisa Misto ◽  
Cinzia Quarchioni ◽  
...  
Keyword(s):  
Triticum Aestivum ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gmo Quantification

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽  
2019 ◽  
Vol 98 ◽  
pp. 380-388 ◽  
Author(s):  
Xiaofu Wang ◽  
Ting Tang ◽  
Qingmei Miao ◽  
Shilong Xie ◽  
Xiaoyun Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Processed Foods ◽  
Conventional Pcr ◽  
Rice Line ◽  
Transgenic Rice Line

42P Method optimization for the detection of chimerism by real-time PCR and droplet digital PCR

2021 ◽  
Vol 32 ◽  
pp. S1358
Author(s):  
I.M. Lambrescu ◽  
V.S. Ionescu ◽  
G. Gaina ◽  
A. Popa ◽  
C. Niculite ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Method Optimization

Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real‐time PCR for serum HBV RNA quantification

2020 ◽  
Vol 92 (12) ◽  
pp. 3365-3372 ◽  
Author(s):  
Umaporn Limothai ◽  
Natthaya Chuaypen ◽  
Kittiyod Poovorawan ◽  
Watcharasak Chotiyaputta ◽  
Tawesak Tanwandee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Rna Quantification

scholarly journals Absolute quantification by droplet digital PCR versus analog real-time PCR

2013 ◽  
Vol 10 (10) ◽  
pp. 1003-1005 ◽  
Author(s):  
Christopher M Hindson ◽  
John R Chevillet ◽  
Hilary A Briggs ◽  
Emily N Gallichotte ◽  
Ingrid K Ruf ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Absolute Quantification ◽  
Droplet Digital Pcr

scholarly journals Comparison of real-time PCR and droplet digital PCR for the detection of Xylella fastidiosa in plants

2019 ◽  
Vol 162 ◽  
pp. 86-95 ◽  
Author(s):  
Enora Dupas ◽  
Bruno Legendre ◽  
Valérie Olivier ◽  
Françoise Poliakoff ◽  
Charles Manceau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Xylella Fastidiosa ◽  
Digital Pcr ◽  
Droplet Digital Pcr

scholarly journals Quantification of BK Virus Standards by Quantitative Real-Time PCR and Droplet Digital PCR Is Confounded by Multiple Virus Populations in the WHO BKV International Standard

2017 ◽  
Vol 63 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Allen C Bateman ◽  
Alexander L Greninger ◽  
Ederlyn E Atienza ◽  
Ajit P Limaye ◽  
Keith R Jerome ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Bk Virus ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
International Standard ◽  
Multiple Virus ◽  
Generation Sequencing

Abstract BACKGROUND The WHO recently released a BK virus (BKV) international standard. This study evaluated the WHO international standard and commercially available BKV standards by quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). METHODS WHO, Exact Diagnostics, Acrometrix, and Zeptometrix BKV standards were tested by qPCR and ddPCR. Two preparations of NIST BKV clones were also tested. Nucleic acid was extracted with the Roche MP96 and MPLC, followed by quantification in duplicate. To resolve discrepancies, we sequenced the WHO and NIST materials. RESULTS Manufacturers' expected copies/mL were close to WHO IU/mL: linear regression of qPCR data revealed 1.12 Exact copies/IU, 0.76 Acrometrix copies/IU, and 0.70 Zeptometrix copies/IU. For ddPCR, similar concentrations were measured when either the VP1 region or the T region was targeted, and concentrations were almost 2-fold higher when both regions were targeted simultaneously. ddPCR results for the VP1 and T regions were similar for all commercial standards, but targeting the T region of the WHO standard led to a 4-fold lower result than the VP1 region. Next-generation sequencing revealed no primer or probe mismatches. However, large differences in coverage across the WHO standard and junctional reads were observed, indicating subpopulations of the WHO standard with deletions in the T region. CONCLUSIONS BKV standards showed concordance among providers, but the WHO standard contains subpopulations of viruses with various deletions in the T region. PCR results will vary depending on which region of the WHO standard is targeted.

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