Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Ding, Lin, Xu, Xiaoli, Wang, Xiaofu, Chen, Xiaoyun, Lu, Yuwen, Xu, Junfeng, Peng, Cheng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
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
_version_ 1785120273926717440
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
work_keys_str_mv AT dinglin qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT xuxiaoli qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT wangxiaofu qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT chenxiaoyun qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT luyuwen qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT xujunfeng qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods
AT pengcheng qualitativeandquantitativedetectionofcrisprassociatedcasgeneingeneeditedfoods