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Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods
Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to es...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10572612/ https://www.ncbi.nlm.nih.gov/pubmed/37835336 http://dx.doi.org/10.3390/foods12193681 |
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author | Ding, Lin Xu, Xiaoli Wang, Xiaofu Chen, Xiaoyun Lu, Yuwen Xu, Junfeng Peng, Cheng |
author_facet | Ding, Lin Xu, Xiaoli Wang, Xiaofu Chen, Xiaoyun Lu, Yuwen Xu, Junfeng Peng, Cheng |
author_sort | Ding, Lin |
collection | PubMed |
description | Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the Cas gene. In the present study, the primers and probes were designed and screened for Cas12a (Cpf1), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting Cpf1 in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing Cpf1 DNA, while the detection rate of other samples without Cpf1 was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for Cpf1 are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future. |
format | Online Article Text |
id | pubmed-10572612 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-105726122023-10-14 Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods Ding, Lin Xu, Xiaoli Wang, Xiaofu Chen, Xiaoyun Lu, Yuwen Xu, Junfeng Peng, Cheng Foods Article Effective regulation of gene-edited products and resolution of public concerns are the prerequisites for the industrialization of gene-edited crops and their derived foods. CRISPR-associated protein, the core element of the CRISPR system, requires to be regulated. Thus, there is an urgent need to establish qualitative and quantitative detection methods for the Cas gene. In the present study, the primers and probes were designed and screened for Cas12a (Cpf1), which is the most commonly used target site in gene editing; we performed PCR system optimization, determined the optimal primer concentration and annealing temperature, and established qualitative PCR and quantitative PCR (qPCR) assays for detecting Cpf1 in gene editing by specificity and sensitivity tests. In specificity testing, qualitative PCR and qPCR methods could 100% detect samples containing Cpf1 DNA, while the detection rate of other samples without Cpf1 was 0%. In the assay sensitivity test, the limit of detection of qualitative PCR was 0.1% (approximately 44 copies), and the limit of detection of the qPCR method was 14 copies. In the stability test, both the qualitative PCR and qPCR methods were repeated 60 times at their corresponding lowest detection limit concentrations, and the results were positive. Thus, the qualitative and quantitative assays for Cpf1 are specific, sensitive, and stable. The method provides technical support for the effective monitoring of gene-edited products and their derived foods in the future. MDPI 2023-10-07 /pmc/articles/PMC10572612/ /pubmed/37835336 http://dx.doi.org/10.3390/foods12193681 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. 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 Ding, Lin Xu, Xiaoli Wang, Xiaofu Chen, Xiaoyun Lu, Yuwen Xu, Junfeng Peng, Cheng Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title | Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title_full | Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title_fullStr | Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title_full_unstemmed | Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title_short | Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods |
title_sort | qualitative and quantitative detection of crispr-associated cas gene in gene-edited foods |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10572612/ https://www.ncbi.nlm.nih.gov/pubmed/37835336 http://dx.doi.org/10.3390/foods12193681 |
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