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

• Main Authors: Mokhtar, Nur Fadhilah Khairil, Shun, Ying Quah, Raja Nhari, Raja Mohd Hafidz, Mohamad, Nurhidayatul Asma, Shahidan, Nur Maisarah, Warsanah, Irwan Hanish, Mohd Hashim, Amalia
• Format: Article
• Published: Taylor and Francis 2024
• Online Access: http://psasir.upm.edu.my/id/eprint/105707/; https://www.scopus.com/inward/record.uri?eid=2-s2.0-85181680757&doi=10.1080%2f19440049.2023.2298476&partnerID=40&md5=f0b824031bfa10cdeba005581aacd323

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.