Nanoplate-based digital PCR for highly sensitive pork DNA detection targeting multi-copy nuclear and mitochondrial genes

放大器 低拷贝数 数字聚合酶链反应 生物 核DNA 线粒体DNA 基因 DNA 分子生物学 核基因 限制性酶 计算生物学 聚合酶链反应 生物化学 基因组
作者
Nur Fadhilah Khairil Mokhtar,Ying Quah Shun,Raja Kamil,Nurhidayatul Asma Mohamad,Nur Maisarah Shahidan,Irwan Hanish Warsanah,Amalia Mohd Hashim
出处
期刊:Food Additives & Contaminants: Part A [Taylor & Francis]
卷期号:41 (2): 120-133 被引量:1
标识
DOI:10.1080/19440049.2023.2298476
摘要

The inclusion of ingredients derived from pigs in highly processed consumer products poses a significant challenge for DNA-targeted analytical enforcement, which could be overcome by using digital PCR. However, most species detection methods use digital PCR to target single-copy nuclear genes, which limits their sensitivity. In this work, we examined the performance of a nanoplate-based digital PCR method that targets multi-copy nuclear (MPRE42) and mitochondrial (Cytb) genes. Poor separation of positive and negative partitions, as well as a 'rain effect' were obtained in the porcine-specific MPRE42 assay. Among the optimization strategies examined, the inclusion of restriction enzymes slightly improved the separation of positive and negative partitions, but a more extensive 'rain effect' was observed. The high copy number of the MPRE42 amplicon is hypothesized to contribute to the saturation of the positive signal. In contrast, the porcine-specific Cytb assay achieved perfect separation of positive and negative partitions with no 'rain effect'. This assay can detect as little as 0.4 pg of pork DNA, with a sensitivity of 0.05% (w/w) in a pork-chicken mixture, proving its applicability for detecting pork in meat and meat-based products. For the MPRE42 assay, potential applications in highly degraded products such as gelatin and lard are anticipated.
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