Development and evaluation of multiplex pcr assay for the determination of cat, rabbit, rat and squirrel elements in food products / Mohammad Nasir Uddin Ahamad

Mohammad Nasir , Uddin Ahamad (2018) Development and evaluation of multiplex pcr assay for the determination of cat, rabbit, rat and squirrel elements in food products / Mohammad Nasir Uddin Ahamad. PhD thesis, University of Malaya.

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Abstract

Cat, rabbit, rat and squirrels are adulterated in meat and meat products for economic gain and exotic taste. However, most of these species are potential carriers of zoonotic threats and so pose huge threats to public health. Currently, several polymerase chain reaction (PCR) based methods have been proposed for authentication these species in separate assays which are costly and involved long-amplicon length biomarkers that breakdown during food processing. To overcome the need, for the first time, multiplex conventional PCR, PCR-RFLP and quantitative PCR assays with TaqMan Probes were developed here for the discriminatory identification of cat, rabbit, rat, and squirrel in food products. In conventional PCR and PCR-RFLP, rabbit (123 bp), rat (108 bp) and squirrel (243 bp) targets were amplified from ATP6 and cytb genes along with a eukaryotic internal control (141bp). The products were sequenced and cross-tested against 22 species. A total of 81 reference samples and 72 meatball specimens were screened to validate the assay. Analyte stability was evaluated through boiling, autoclaving and micro oven cooking. The lower limits of detection were 0.001ng DNA for pure meat and 0.1% for meatballs. Specificity was confirmed through sequencing and RFLP analysis. When PCR products were digested with BtsIMutI and BtsCI enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67 bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter formulation. Finally, a novel pentaplex real- time PCR assay with TaqMan probes was developed for identification and quantification of the squirrel, rat, rabbit and cat species in a single assay platform. For real-time quantitative PCR, species specific primers and probes were developed against ATP6, and cytochrome b genes to amplify 108, 123, 161and 176 bp DNA fragments from rat, rabbit, squirrel and cat meat products, respectivly under various states. A141 bp internal amplification control (IAC) of 18S rRNA was used to avoid any false negative results. Specificity of the assay was evaluated against 22 non- target species but no cross-reactivity was found. Each of the target species DNA was quantified, and PCR efficiency was determined based on standard curve that was generated using 10-fold serially diluted mixed DNA extract (1:1:1:1) from squirrel, rat, rabbit and cat species. The assay was valid both under pure, processed and admixed states with 10-0.1% (w/w) adulterant from each species. The limit of quantification was 0.003 ng DNA from each species. Analyses of 18 model burgers (9 chicken and 9 beef) and 18 frankfurters (9 chicken and 9 beef) revealed 91 - 122% target recovery at 0.1 - 10% adulteration. Finally, 72 commercial burgers (36 chicken and 36 beef) and 72 frankfurters (36 chicken and 36 beef) were screened but no target species was detected except IAC. Although, the study was validated using different food matrices, the shorter-aspects of amplicon length, exceptional stability under veracious treatment conditions convinced that the developed methods could be a useful tool in the identification and quantification of cat, rabbit, rat and squirrel species in any food matrices.

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