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

Abstract

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.

Keywords:

• Authenticity
• species identification
• molecular biology
• PCR
• porcine DNA

Acknowledgement

We would like to appreciate technical advices and constructive manuscript’s review from Qiagen Biotechnology Malaysia Sdn Bhd.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was funded by Geran Putra - Inisiatif Putra Muda (GP-IPM/2023/8749500) (Vote No: 9749500) awarded to Nur Fadhilah Khairil Mokhtar by Universiti Putra Malaysia.