Meat adulteration has recently become a global problem (X. Chen, Lu, Xiong, Xiong, & Liu, 2020; Y. Kumar & Narsaiah, 2021; O'Mahony, 2013), seriously affecting the relationship between consumers and the food industry and undermining public confidence in the entire food industry. Meat adulteration often involves adding inferior meat to expensive one to make enormous profits. These animals used for adulterated meats are usually fed without food hygiene standards and may be given prohibited antibiotics or psychotropic drugs to reduce costs further. For example, the horse meat disguised as beef in the European horsemeat scandal was sourced from sick or old horses. It was detected to contain phenylbutazone, which is harmful to humans even in trace amounts (Abbas, Zadravec, Baeten, Mikus, Lesic, Vulic, et al., 2018). Even adulterants could negatively affect health-related aspects (Bansal, Singh, Mangal, Mangal, & Kumar, 2017) and religious diets (Hossain, Uddin, Sultana, Wahab, Sagadevan, Johan, et al., 2022). Therefore, adulteration identification plays an important role in the economic area and consumer’s physical and mental health.
Duck is one of the common species used as adulteration for lamb and beef because of their similar texture and color (Fu, Zhang, Zhou, & Liu, 2020; Qin, Qiao, Xu, Song, Yao, Lu, et al., 2019; Zheng, Li, Wei, & Peng, 2019), especially the beef/lamb skewers and slices that appear in barbecue stalls and hot pot restaurants in China. Duck-derived products are difficult for consumers to distinguish from beef and lamb due to additives such as using butter to mask the smell and supported with beef paste and mutton essence to smell like them (X. Chen, Yu, Ji, Wei, Peng, Wang, et al., 2022). Therefore, it is desired to establish a simple, rapid, sensitive, and affordable method to detect duck ingredient in meat-related products.
Several methods have been developed for meat adulteration identification, including chromatography (Di Stefano, Avellone, Bongiorno, Cunsolo, Muccilli, Sforza, et al., 2012) and spectroscopy (Weng, Guo, Tang, Yin, Pan, Zhao, et al., 2020). However, their application in the food industry for routine analysis is rarely discussed. Currently, the techniques for meat identification and adulteration are protein-based methods (Mandli, El Fatimi, Seddaoui, & Amine, 2018; Yamasaki, Hirakawa, Momma, Yamaguchi, Kotoura, Miyake, et al., 2021) and DNA-based methods (A. Chen, Wei, Chen, Zhao, & Yang, 2015; W. Liu, Tao, Xue, Ji, Zhang, Zhang, et al., 2019) culminating in Polymerase Chain Reaction (PCR)-based methods and enzyme-linked immunosorbent assay (ELISA) are the two main methods used to detect meat adulteration (X. Chen, et al., 2022; Yin, Sun, Wang, Feng, Zhang, & Xiao, 2020), and the latter being extensively studied in the literature. ELISA is less expensive and time-saving, but it cannot discriminate between closely-related species (Li, Liu, Meng, Liu, Zhang, Wang, et al., 2020). The target protein denaturation caused by harsh processing conditions (Zhao, Hu, Liu, Wu, Xiao, Zhang, et al., 2020) and the poor reproducibility for frozen products (Zia, Alawami, Mokhtar, Nhari, & Hanish, 2020) also hinder the ELISA application in processed meat products. PCR-based methods, especially real-time PCR, are more optimal for species identification in processed meat products because of the DNA thermostability. However, each procedure of the PCR method requires precise temperature control, so sophisticated equipment is indispensable. This reason makes it inconvenient to use outside the laboratory.
Therefore, nucleic acid isothermal amplification techniques independent of thermal cycling instruments become increasingly important. Isothermal amplification techniques have a high potential for on-site detection because they require only a single constant temperature to complete the amplification process. Several promising isothermal amplification methods have emerged, while some of them need relatively high temperatures and/or a phase for complex primers design, such as loop-mediated isothermal amplification (LAMP) requires three primer pairs targeting different regions of the amplified fragment and working at 65 ℃ (Cai, Kong, & Xu, 2020) and cross-priming amplification (CPA) requires 63 ℃ and relies on five primers to detect the target sequence (Feng, Li, Wang, & Pan, 2018). In contrast, the RPA method works successfully over a wide temperature ranging from 37∼42 ℃ with a specific primer pair (Yogesh Kumar, 2021). Thus, the amplification could be achieved even at room or human body temperature (Cherkaoui, Huang, Miller, Turbe, & McKendry, 2021).
RPA reaction primarily relies on three pivotal enzymes: recombinase, DNA polymerase, and single-stranded DNA-binding protein (SSB). An oligonucleotide-protein complex consisting of primers and recombinase browses the template for homologous sequences. Then, the complex separates, a polymerase with strand-displacing activity binds the primers to the template, and the extension begins while SSB stabilizes another single strand. The amplification is initiated simultaneously from the forward and reverse primers to form a new double strand. Once this reaction is initiated, the template amplifies exponentially within 30 min. Amplification at constant temperature allows RPA can be used in conjunction with portable detection equipment, making detection simpler and faster. Therefore, RPA technology is promising for on-site detection and has the potential to be applied in a resource-limited setting.
In this study, we aim to develop a simple, rapid, sensitive, and affordable technique for duck ingredient detection in meat-related products. Cytb will be used as a target gene, and real-time RPA and LFS RPA techniques will be applied.