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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...

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Published in:Journal of food science and technology 2021-04, Vol.58 (4), p.1286-1294
Main Authors: Kumar, Dhananjay, Kumar, Rajiv Ranjan, Rana, Preeti, Mendiratta, S. K., Agarwal, R. K., Singh, Praveen, Kumari, Sarita, Jawla, Jyoti
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description 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%.
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subjects Amplification
Animals
Assaying
Beef cattle
Buffalo
Cattle
Chemistry
Chemistry and Materials Science
Chemistry/Food Science
Cytochrome b
Cytochromes
Deoxyribonucleic acid
Diagnostic systems
DNA
Electrophoresis
Food
Food Science
Gel electrophoresis
Gene sequencing
Homology
Meat
Mitochondria
Nutrition
Original
Original Article
Recombinase
Species
Swine
Target recognition
title On point identification of species origin of food animals by recombinase polymerase amplification-lateral flow (RPA-LF) assay targeting mitochondrial gene sequences
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