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Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR
SIMPLE SUMMARY: Digital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantificatio...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869681/ https://www.ncbi.nlm.nih.gov/pubmed/35205068 http://dx.doi.org/10.3390/biology11020201 |
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author | Demeke, Tigst Lee, Sung-Jong Eng, Monika |
author_facet | Demeke, Tigst Lee, Sung-Jong Eng, Monika |
author_sort | Demeke, Tigst |
collection | PubMed |
description | SIMPLE SUMMARY: Digital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantification of two, three, and four GM canola and soybean events at the same time using the QX200 Droplet Digital PCR (ddPCR) system. Adjustments of the probe concentrations and labels for some of the assays were needed for successful multiplex ddPCR. Absolute quantification of GM canola and soybean events was achieved for duplex, triplex, and tetraplex ddPCR at 0.1%, 1%, and 5% concentrations. ABSTRACT: The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection. |
format | Online Article Text |
id | pubmed-8869681 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-88696812022-02-25 Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR Demeke, Tigst Lee, Sung-Jong Eng, Monika Biology (Basel) Article SIMPLE SUMMARY: Digital PCR (dPCR) technology has been used for absolute quantification of genetically modified (GM) events. Duplex dPCR consisting of a target gene and a reference gene is mostly used for absolute quantification of GM events. We investigated the feasibility of absolute quantification of two, three, and four GM canola and soybean events at the same time using the QX200 Droplet Digital PCR (ddPCR) system. Adjustments of the probe concentrations and labels for some of the assays were needed for successful multiplex ddPCR. Absolute quantification of GM canola and soybean events was achieved for duplex, triplex, and tetraplex ddPCR at 0.1%, 1%, and 5% concentrations. ABSTRACT: The number of genetically modified (GM) events for canola, maize, and soybean has been steadily increasing. Real-time PCR is widely used for the detection and quantification of individual GM events. Digital PCR (dPCR) has also been used for absolute quantification of GM events. A duplex dPCR assay consisting of one reference gene and one GM event has been carried out in most cases. The detection of more than one GM event in a single assay will increase the efficiency of dPCR. The feasibility of detection and quantification of two, three, and four GM canola and soybean events at the same time was investigated at 0.1%, 1%, and 5% levels using the QX200 Droplet Digital PCR (ddPCR) system. The reference gene assay was carried out on the same plate but in different wells. For some of the assays, optimization of the probe concentrations and labels was needed for successful ddPCR. Results close to the expected result were achieved for duplex, triplex, and tetraplex ddPCR assays for GM canola events. Similar ddPCR results were also achieved for some GM soybean events with some exceptions. Overall, absolute quantification of up to four GM events at the same time improves the efficiency of GM detection. MDPI 2022-01-27 /pmc/articles/PMC8869681/ /pubmed/35205068 http://dx.doi.org/10.3390/biology11020201 Text en © 2022 Crown Copyright. https://creativecommons.org/licenses/by/4.0/This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Demeke, Tigst Lee, Sung-Jong Eng, Monika Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title | Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title_full | Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title_fullStr | Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title_full_unstemmed | Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title_short | Increasing the Efficiency of Canola and Soybean GMO Detection and Quantification Using Multiplex Droplet Digital PCR |
title_sort | increasing the efficiency of canola and soybean gmo detection and quantification using multiplex droplet digital pcr |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8869681/ https://www.ncbi.nlm.nih.gov/pubmed/35205068 http://dx.doi.org/10.3390/biology11020201 |
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