On point identification of species origin of food animals by recombinase polymerase amplification-lateral flow (RPA-LF) assay targeting mitochondrial gene sequences.

The present study was aimed to develop and standardize Recombinase polymerase amplification-lateral flow (RPA-LF) assays for on point identification of species origin of food animals viz: cattle, buffalo and pig. Species specific RPA primers sets for cattle, buffalo and pig were designed by homology comparisons of the sequences of mitochondrial cytochrome b gene and d-loop region from common food species viz: cattle, buffalo, sheep, goat, pig and chicken. The RPA assays for designed primers sets were optimized using the reaction components from Twist Amp basic kit and instructions in its manual. Endpoint detection of species specific amplified RPA products were made by gel electrophoresis and designed species specific RPA-LFA strips. The developed assays were evaluated for their specificity, diagnostic sensitivity, and validated on coded samples and binary meat admixtures with relative percentage of 20, 10, 5 & 1% target species. The developed RPA assays resulted in amplification of DNA template exclusively of cattle, buffalo and pig origin to product sizes of 294, 405 and 283 bp respectively. The diagnostic sensitivities of developed assays were up to 10 pg of genomic DNA and highly correlated with species specific PCR assays taken as gold standard. Developed species specific RPA assays also identified the target species in coded samples and binary meat admixture up to 1%.

Identification of species origin of meat and meat products on the DNA basis: a review.

The adulteration/substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness, and unhealthy competition in meat market. Consumer should be protected from these malicious practices of meat adulterations by quick, precise, and specific identification of meat animal species. Several analytical methodologies have been employed for meat speciation based on anatomical, histological, microscopic, organoleptic, chemical, electrophoretic, chromatographic, or immunological principles. However, by virtue of their inherent limitations, most of these techniques have been replaced by the recent DNA-based molecular techniques. In the last decades, several methods based on polymerase chain reaction have been proposed as useful means for identifying the species origin in meat and meat products, due to their high specificity and sensitivity, as well as rapid processing time and low cost. This review intends to provide an updated and extensive overview on the DNA-based methods for species identification in meat and meat products.

Development of ion torrent-based targeted next-generation sequencing panel for identification of animal species in pet foods.

Manufacturers may intentionally or unintentionally incorporate ingredients not specified on the label of canned pet foods. Including any unacknowledged ingredients in a food product is considered food fraud or misbranding. Contamination of pet foods may occur in the processing of the foods, including potential cross-contamination in packaging facilities. Of the methods available to identify meat species in food products, Sanger sequencing and several next-generation sequencing methods are available, but there are limitations including the number of targets analyzed at a time and the method specificity. In this study, we developed a targeted next-generation sequencing panel to detect meat species in canned pet foods using Ion Torrent technology. The panel contains multiple primers targeting mitochondrial genes from as many as 27 animal species, of which 7 major animal species were validated. The meat species targets could be identified from samples spiked with as low as 0.01% w/w of the contaminating meat species in a vegetarian food matrix material. Targeted NGS in the current study enriches species-specific multiple target areas in the mitochondrial genome of the target material, which gives high accuracy in the sequencing results.

Quantification of Pork, Chicken, Beef, and Sheep Contents in Meat Products Using Duplex Real-Time PCR.

Accurate methods for meat speciation and quantification are essential for ensuring the supply of safe and wholesome meat and composite products with animal origins to negate the potential associated hazards, aid classification of consignments at the import control system, and thwart food fraud committed for financial gain. To better enhance meat safety control and combat food fraud, this study developed two duplex real-time polymerase chain reaction (real-time PCR) systems specifically designed for chicken, pork, sheep, and beef, using single-copy, chromosomally encoded, species-specific gene sequences to accurately measure the content of each meat type in meat products. DNA extracted from the raw and boiled reference materials prepared in varying proportions (ranging from 1% to 75%) were used in the development of the duplex assay to derive calibration factors to determine the meat content in different meat products. The method was further validated using proficiency test samples and market monitoring samples. Our findings showed that this method exhibits high specificity and sensitivity, with a significant accuracy range of 0.14% to 24.07% in quantifying the four meat types in both raw and processed meat products. Validation results further confirmed the effectiveness of our method in accurately quantifying meat content. Thus, we have demonstrated the duplex qPCR assays as promising approaches for implementation in routine analysis to strengthen meat safety control systems and combat meat fraud, thereby safeguarding consumer health and trust in the meat industry.

Paper-based loop-mediated isothermal amplification and lateral flow (LAMP-LF) assay for identification of tissues of cattle origin.

The present study was made with the objectives of development and standardization of cattle specific paper-based loop-mediated isothermal amplification cum lateral flow assay (LAMP-LFA), as a Point-of-care test (POCT) for identification of tissue of cattle origin. The components of standardized LAMP reaction utilizing cattle specific primer sets were lyophilized over paper buttons, identified best as the carrier of LAMP reagents. Based on probable LAMP amplicon, a pair of probes was designed, tagged and its hybridization with the amplified product of paper LAMP reaction was optimized. The components of lateral flow assay for detection of probe hybridized LAMP products were standardized. Analysis of successful amplification was made by using HNB dye, LAMP-LFA strip, and also by the typical ladder-like pattern on gel electrophoresis. The assay was found highly specific for cattle with an analytical sensitivity of 0.1 pg of absolute DNA. Laboratory validation carried out on samples from different individuals of cattle, coded samples, binary meat admixture, and heat-processed cattle tissues substantiated the accuracy of the assay. Comparison with pre-standardized species-specific PCR assay taken as gold standards revealed 100% conformity. The field utility of the developed assay was further established by its compatibility with the commercial kit eliminating the lengthy DNA extraction step and storage stability of LAMP reagent carrier buttons for 4 months under refrigeration. Thus, the developed assay capable of the result within 3 h in resource-limited settings can be used as POCT for identification of tissue of cattle origin.

Simultaneous determination of heat stable peptides for eight animal and plant species in meat products using UPLC-MS/MS method.

Food adulteration and fraud is driven by economic interests; it is thus necessary to establish a high-through method that allows quantitative identification of familiar animal and plant proteins for global use. In this study, a sensitive mass spectrometric approach for the detection of eight species, including pork, beef, lamb, chicken, duck, soy, peanut, and pea, is presented and the heat stability and specificity of screened peptides are verified. To improve screening efficiency of specific peptides, several key data searching parameters, including peptides, sequence lengths, sequence coverage, and unique peptides, are investigated. Using this approach, it is possible to detect a 0.5% contamination of any of the eight species. The method is proven to have high sensitivity, specificity, repeatability, and a low quantitative detection limit with respect to adulteration of diverse types of meat products.

OFFGEL electrophoresis and tandem mass spectrometry approach compared with DNA-based PCR method for authentication of meat species from raw and cooked ground meat mixtures containing cattle meat, water buffalo meat and sheep meat.

The present study compared the accuracy of an OFFGEL electrophoresis and tandem mass spectrometry-based proteomic approach with a DNA-based method for meat species identification from raw and cooked ground meat mixes containing cattle, water buffalo and sheep meat. The proteomic approach involved the separation of myofibrillar proteins using OFFGEL electrophoresis, SDS-PAGE and protein identification by MALDI-TOF MS. Species-specific peptides derived from myosin light chain-1 and 2 were identified for authenticating buffalo meat spiked at a minimum 0.5% level in sheep meat with high confidence. Relative quantification of buffalo meat mixed with sheep meat was done by quantitative label-free mass spectrometry using UPLC-QTOF and PLGS search engine to substantiate the confidence level of the data. In the DNA-based method, PCR amplification of mitochondrial D loop gene using species specific primers found 226bp and 126bp product amplicons for buffalo and cattle meat, respectively. The method was efficient in detecting a minimum of 0.5% and 1.0% when buffalo meat was spiked with cattle meat in raw and cooked meat mixes.