Development of event‐specific <scp>PCR</scp> detection methods for genetically modified tomato Huafan No. 1

Yang, Chao; Zhang, Dabing; Liu, Yang

doi:10.1002/jsfa.5908

Cited by 10 publications

(5 citation statements)

References 15 publications

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“…In GM component detection, the LOD is a crucial parameter that must be determined. According to literature reports, the LOD for conventional PCR is generally around 20 copies, while for qPCR, it is around 10 copies [25,26]. The LOD of the detection system established in this study is consistent with the reported results.…”

Section: Discussionsupporting

confidence: 91%

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods

Zhu,

Lin,

Yang

et al. 2023

Genes

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Endogenous reference genes play a crucial role in the qualitative and quantitative PCR detection of genetically modified crops. Currently, there are no systematic studies on the banana endogenous reference gene. In this study, the MaSPS1 gene was identified as a candidate gene through bioinformatics analysis. The conservation of this gene in different genotypes of banana was tested using PCR, and its specificity in various crops and fruits was also examined. Southern blot analysis showed that there is only one copy of MaSPS1 in banana. The limit of detection (LOD) test showed that the LOD of the conventional PCR method is approximately 20 copies. The real-time fluorescence quantitative PCR (qPCR) method also exhibited high specificity, with a LOD of approximately 10 copies. The standard curve of the qPCR method met the quantitative requirements, with a limit of quantification (LOQ) of 1.14 × 10−2 ng—about 20 copies. Also, the qPCR method demonstrated good repeatability and stability. Hence, the above results indicate that the detection method established in this study has strong specificity, a low detection limit, and good stability. It provides a reliable qualitative and quantitative detection system for banana.

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Section: Discussionsupporting

confidence: 91%

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods

Zhu,

Lin,

Yang

et al. 2023

Genes

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“…The qPCR method has the advantages of real-time analysis, high sensitivity, less time consumption, and detection of low levels of gene-edited components. Thus, the combination of the two methods in a complementary assay could yield accurate and rapid detection [28], It will also provide some technical support to establish a detection and identification system of gene-edited foods in the future.…”

Section: Discussionmentioning

confidence: 99%

“…The amplification efficiency was calculated according to the slope of the standard curve: E = 10 −1/slope −1. The quantitative detection of gene editing exogenous components requires the slope of the standard curve to be in the range of −3.6 ≤ slope ≤ −3.1, which implies that the amplification efficiency is 90-110%, and the correlation coefficient (R 2 ) is ≥0.98 [28]. The results showed that the slope of Cas12-real-1 was −3.340, the amplification efficiency E was 99.3%, and the correlation coefficient R 2 was 1.…”

Section: Primer Design and Screening For Qpcrmentioning

confidence: 99%

Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods

Ding,

Xu,

Wang

et al. 2023

Foods

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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.

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“…Cry1Ac transgene insertion sites in tomato genome were determined using thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) in four selected transgenic lines (T 1 -18, T 1 -20, T 1 -22, T 1 -25). Five arbitrary primers (ad1, ad2, ad3, ad20 and w4) (Yang et al 2013) were tested with three Cry1Ac gene-specific sequential nested primers for amplifying the 5′ upstream (Cry1Ac51, Cry1Ac52, Cry1Ac53) and 3′ downstream regions (Cry1Ac31, Cry1Ac32, Cry1Ac33). A three step sequential PCR method was used for TAIL-PCR.…”

Section: Tail-pcr Determination Of Cry1ac Transgene Insertion Sitementioning

confidence: 99%

Cry1Ac-mediated resistance to tomato leaf miner (Tuta absoluta) in tomato

Şelale

Dağlı

Mutlu

et al. 2017

Plant Cell Tiss Organ Cult

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the transgenic plants. Moreover, it was found that a single copy of the gene in the hemizygous condition is sufficient to confer tolerance to leaf miner. This is the first reported development of tomato plants resistant to T. absoluta. These transgenic plants are promising for development of commercial tomato cultivars resistant to leaf miner, which will limit the use of environmentally harmful chemicals for control of this pest.

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Development of event‐specific PCR detection methods for genetically modified tomato Huafan No. 1

Abstract: Our results suggest that the developed event-specific PCR methods can be routinely used for identification and quantification of GM Huafan No. 1 tomato.

Cited by 10 publications

References 15 publications

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods

The Identification of the Banana Endogenous Reference Gene MaSPS1 and the Construction of Qualitative and Quantitative PCR Detection Methods

Qualitative and Quantitative Detection of CRISPR-Associated Cas Gene in Gene-Edited Foods

Cry1Ac-mediated resistance to tomato leaf miner (Tuta absoluta) in tomato

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