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

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.

Rapid Detection of Porcine DNA in Meatball Using Recombinase Polymerase Amplification Couple with Lateral Flow Immunoassay for Halal Authentication.

Point-of-care diagnostic methods for animal species determination are critical for rapid, simple, and accurate enforcement of food labelling. PCR is the most common method for species identification. However, the requirement of using a thermal cycler created drawbacks for the PCR application, particularly in low-resource settings. Hence, in this study, a method for porcine DNA detection using recombinase polymerase amplification (RPA), coupled with nucleic acid lateral flow immunoassay (NALFIA), was developed. Porcine-specific primers targeting pig (Sus scrofa) cytochrome b gene fragments specifically amplify a 197 bp fragment of the mitochondrial gene as being visualized by 2% agarose gel and PCRD NALFIA. The reaction temperature and time were 39 °C and 20 min, respectively. Herein, the specificity of the primers to porcine was confirmed after being assayed against six animal species, namely cow, goat, chicken, duck, dog, and rabbit. The porcine-specific RPA assay shows a high limit of detection of 0.01 ng/µL pork DNA. Based on the preliminary performance data obtained from this study, the potential of this method as a rapid and sensitive tool for porcine DNA detection in meat-based products is foreseen.

Future perspectives on aptamer for application in food authentication.

Food fraudulence and food contamination are major concerns, particularly among consumers with specific dietary, cultural, lifestyle, and religious requirements. Current food authentication methods have several drawbacks and limitations, necessitating the development of a simpler, more sensitive, and rapid detection approach for food screening analysis, such as an aptamer-based biosensor system. Although the use of aptamer is growing in various fields, aptamer applications for food authentication are still lacking. In this review, we discuss the limitations of existing food authentication technologies and describe the applications of aptamer in food analyses. We also project several potential targets or marker molecules to be targeted in the SELEX process. Finally, this review highlights the drawbacks of current aptamer technologies and outlines the potential route of aptamer selection and applications for successful food authentication. This review provides an overview of the use of aptamer in food research and its potential application as a molecular reporter for rapid detection in food authentication process. Developing databases to store all biochemical profiles of food and applying machine learning algorithms against the biochemical profiles are urged to accelerate the identification of more reliable biomarker molecules as aptamer targets for food authentication.

Next Generation Sequencing-based DNA metabarcoding for animal species profiling in fish feed.

The expansion of worldwide aquaculture has been accompanied by extensive growth in the fish feed industry. However, improper labelling of many commercially available fish feeds has raised security and safety concerns over the species' origin of the ingredients. The inclusion of ruminants-derived ingredients in fish feed is prohibited according to EU legislation while porcine inclusion in fish feed has been a great concern among Muslim farmers. In contrast to the limited species that could be simultaneously determined using multiplex PCR, this study utilised Next Generation Sequencing-based DNA metabarcoding assay to determine the compositional profiles of animal species in fish feed samples in a more holistic manner. In relation to the religious issue associated with porcine-derived ingredients in fish feed, this study firstly aimed to determine the sensitivity of the methods in profiling fish feed adulterated with porcine blood and muscle tissues. Next, 10 commercially available fish feed samples were analysed. As a result, a detection limit of as low as 3% (w/w) porcine muscle and blood in the laboratory-prepared fish feed was obtained. The analysis of 10 commercial fish feeds shows surprising findings: 50% of the feeds contain Sus scrofa and 80% contain Bos taurus, a ruminant. Only one commercial fish feed was found to be solely composed of marine species. This study shows that commercial fish feeds sold in Malaysia contain undesirable animal species, and emphasises the need for accurate and legally enforced labelling of mammalian species in fish feed products.

The Discovery of New Antilisterial Proteins From Paenibacillus polymyxa Kp10 via Genome Mining and Mass Spectrometry.

The inhibitory properties of novel antimicrobial proteins against food-borne pathogens such as Listeria monocytogenes offer extensive benefits to the food and medical industries. In this study, we have identified antimicrobial proteins from a milk curd-derived bacterial isolate that exhibits antilisterial activity using genome mining and mass spectrometry analysis. The analysis of the draft genome sequence identified the isolate as Paenibacillus polymyxa Kp10, and predicted the presence of antimicrobial paenibacillin, paenilan, paeninodin, sactipeptides, thiazole-oxazole modified microcin, and histone-like DNA binding protein HU encoded in its genome. Interestingly, nanoLC-MS/MS analysis identified two histone-like DNA binding proteins HU as predicted in silico earlier, exhibiting antilisterial activity. Additionally, translation initiation factor IF-1 and 50S ribosomal protein L29 were also discovered by the mass spectrometry in the active fractions. The antilisterial activity of the four proteins was verified through heterologous protein expression and antimicrobial activity assay in vitro. This study has identified structural regulatory proteins from Paenibacillus possessing antilisterial activity with potential future application in the food and medical industries.

Current analytical methods for porcine identification in meat and meat products.

Authentication of meat products is critical in the food industry. Meat adulteration may lead to religious apprehensions, financial gain and food-toxicities such as meat allergies. Thus, empirical validation of the quality and constituents of meat is paramount. Various analytical methods often based on protein or DNA measurements are utilized to identify meat species. Protein-based methods, including electrophoretic and immunological techniques, are at times unsuitable for discriminating closely related species. Most of these methods have been replaced by more accurate and sensitive detection methods, such as DNA-based techniques. Emerging technologies like DNA barcoding and mass spectrometry are still in their infancy when it comes to their utilization in meat detection. Gold nanobiosensors have shown some promise in this regard. However, its applicability in small scale industries is distant. This article comprehensively reviews the recent developments in the field of analytical methods used for porcine identification.