A digital PCR method for identifying and quantifying adulteration of meat species in raw and processed food

Abstract

Meat adulteration is a worldwide concern. In this paper, a new droplet digital PCR (ddPCR) method was developed for the quantitative determination of the presence of chicken in sheep and goat meat products. Meanwhile, a constant (multiplication factor) was introduced to transform the ratio of copy numbers to the proportion of meats. The presented ddPCR method was also proved to be more accurate (showing bias of less than 9% in the range from 5% to 80%) than real-time PCR, which has been widely used in this determination. The method exhibited good repeatability and stability in different thermal treatments and at ultra-high pressure. The relative standard deviation (RSD) values of 5% chicken content was less than 5.4% for ultra-high pressure or heat treatment. Moreover, we confirmed that different parts of meat had no effect on quantification accuracy of the ddPCR method. In contrast to real-time PCR, we examined the performance of ddPCR as a more precise, sensitive and stable analytical strategy to overcome potential problems of discrepancies in amplification efficiency discrepancy and to obtain the copy numbers directly without standard curves. The method and strategy developed in this study can be applied to quantify the presence and to confirm the absence of adulterants not only to sheep but also to other kinds of meat and meat products.

Accurate determination of meat mass fractions using DNA measurements for quantifying meat adulteration

“…For quantitative measurements, therefore, the use of β-actin gene is more suitable than multiple copy number genes such as mitochondrial DNA and 18s rRNA genes, as these genes can vary among tissue types and animal species (Barakat et al 2014). In order to transform the ratio of DNA copy numbers to the mass proportion, a constant number factor has been applied (Ren et al 2017). However, when this factor was applied to the mixture of the unknown meat, the method has to be monitored for accuracy since the constant factor depends on the type of meats (Ren et al 2017).…”

“…This technique is highly sensitive, sequence-specific, amendable to a type of samples and has a vast dynamic range of detection (Nixon et al 2015). However, the need for reference standards to generate a calibration curve is one of the downsides of qPCR (Ren et al 2017). Recently, digital PCR (dPCR) has been adopted for quantifying the amount of nucleic acid targets by partitioning them into a number of reaction chambers (Whale et al 2016).…”

“…As a result, each reaction contains one or no copy of nucleotide sequences of interest that can be assayed individually and counted without requiring a standard curve and correcting by Poisson statistics (Baker 2012). Compared to qPCR, dPCR is more advantageous in terms of its sensitivity, specificity and precision (Baker 2012;Köppel et al 2019;Manoj 2016;Ren et al 2017;Cao et al 2020, Wang et al 2018.…”

“…Moreover, the DNA measurements may not reflect the actual quantity (mass fractions) if the selected target are multiple genes such as mitochondrial gene (Nixon et al 2015). Ren and colleagues tried to use the k constant to transform the copy number ratio to the mass fraction by ddPCR method (Ren et al 2017). However, the known types of meat were also required in order to use the correctly k constant and assays to determine meat species in the fact that meat products in markets may contain with an unknown and variety of meat type, resulting in missed quantification.…”

Integration of Artificial Neural Network Modeling and Hyperspectral Data Preprocessing for Discrimination of Colla Corii Asini Adulteration

“…As a kind of traditional food, authenticity of CCA is usually identified by its external properties; however, it needs rich personal experience and professional knowledge. Nowadays, some physical and chemical inspection methods were used to discriminate adulteration in food like meat [5,6], oil [7,8], honey [9,10], milk [11,12], and so on. As for CCA, polymerase chain reaction method has been reported [4].…”