Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection

Abstract

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA–LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per μL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA–LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

Rapid detection of duck ingredient in adulterated foods by isothermal recombinase polymerase amplification assays

“…When adulteration is present, the level of sensitivity obtained is sufficient to cope with the actual demand in animal-derived ingredient detection. Some RPA-based methods for detection of animal-derived ingredient had been developed ( Chen et al, 2022 , Ivanov et al, 2021 , Kumar et al, 2021 ). All of them achieved good detection limits or adulteration sensitivity, but none of them studied the effect of background DNA on the detection ability at different adulteration ratio.…”

Meat Authenticity Made Easy: DNA Extraction-Free Rapid Onsite Detection of Duck and Pork Ingredients in Beef and Lamb Using Dual-Recombinase-Aided Amplification and Multiplex Lateral Flow Strips

“…Similarly, our dual-RAA-MLFS detection system has a detection limit of 10 1 copies of the target gene in animal-derived samples and can amplify adulterated meat samples with proportions as low as 1:999 within 16 min using instruments, such as a micro thermostatic electronic bath. This system is comparable in detection efficiency to Duplex PCR LFS (2 h) and digital PCR (1.5 h) but does not require complex instruments. , …”

“…The typical rapid nucleic acid lysis solution, consisting of NaOH, PBS, and other components, effectively lysed small samples, providing an alternative to traditional DNA extraction methods. The resistance of RPA/RAA to amplification inhibitors is advantageous for onsite applications, reducing the necessity for purification steps. ,− However, employing crude lysate as an RAA amplification template requires iterative system optimization to maintain detection sensitivity. , This study observed inhibition of RAA enzymatic activity due to rapid nucleic acid lysis and background noise. The addition of 2 μL of lysis supernatant per 50 μL of reaction effectively resolved this issue, allowing for the tolerance of impurities, such as hemoglobin, salt, sucrose, etc., with minimal impact on RAA enzyme activity while preserving detection sensitivity.…”

“…This system is comparable in detection efficiency to Duplex PCR LFS (2 h) and digital PCR (1.5 h) but does not require complex instruments. 16,22 3.3. Dual-RAA-MLFS Identification of Animal-Derived Components in Meat Products Using Rapid Nucleic Acid Lysis.…”

Raman spectroscopic techniques for meat analysis: A review

“…Their disadvantage resides in poor repeatability, dependence on taster's experience and difficulties in quantitative interpretation of results. Objective methods include various laboratory tests that evaluate physical and chemical properties of meat, including electrophoresis [7], enzyme-linked immunosorbent assay (ELISA) [8], mass-spectrometric methods [9], gas chromatography-mass spectrometry [10], high performance liquid chromatography (HPLC) [11,12] and methods based on the polymerase chain reaction (PCR) [13,14,15]. Although PCR and ELISA are the most specific and sensitive methods, they require expensive equipment and highly qualified specialists, which restricts their use.…”