Development of species-specific multiplex PCR assays of mitochondrial 12S rRNA and 16S rRNA for the identification of animal species

Abstract

Species-specific PCR assay was developed for detection of cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey using mitochondrial 12S rRNA and 16S rRNA as target genes. Also, an internal positive control was used to detect possible false negatives by using 18S rRNA gene. We designed species-specific primers with amplicon length of 190, 219, 350, 467, 241, 119, 171, 229, 111 and 268 bp for cattle, sheep, goat, horse, dog, pig, chicken, duck, goose, and turkey respectively. The specificity of the primers was tested against the other 10 non-target animal species and a cross-reaction was not observed. We developed two multiplex PCR assays for the simultaneous identification of Korea's major livestock species (cattle, pig, chicken and duck) and poultry species (chicken, duck, goose and turkey) from analogous samples, retaining the same specificity. The limit of detection of the multiplex PCR assay (cattle, pig, chicken and duck) ranged between 1 pg and 0.1 pg of template DNA extracts from raw meat. Applying multiplex PCR assays to DNA extracts from experimental pork/beef and pork/chicken tested raw and heat-treated (120 o C for 30 min) mixtures respectively, detection limit was 0.1% level beef in pork, pork in beef and chicken in pork and 1.0% level pork in chicken. In conclusion, this assay using gel-based capillary electrophoresis would be very useful in highly sensitive and rapid identification of animal species or ingredients in minced meat and other meat products.

Species-specific loop-mediated isothermal amplification (LAMP) assay for identification of tissue of cattle origin by targeting mitochondrial gene sequences

“…DNA-based methods have raised interest as a useful tool in the identification of species in animal products and other food materials, due to the unique features of DNA, such as its structure remains conserved within all tissues of an individual (Girish et al 2004;Saini et al 2007). A number of researchers working for species discrimination in meat have targeted the mitochondrial DNA sequences because of its unique properties such as maternal inheritance (Rojas et al 2011), multiple numbers of copies (approximately 1000 copies) than nuclear DNA (Koh et al 2011), relatively high tolerance for environmental stresses such as heat, salt and pressure, 10-times higher mutation rate than in nuclear genes, rare possibility of recombination and high efficiency of amplification than nuclear markers (Rastogi et al 2007). They act as naturally amplified source of genetic variation (Girish et al 2004;Fajardo et al 2006) and due to their stability attributes, simplicity, abundance in multiple copies per cells, additional protection given by the specialized mitochondrial membranes and short sequence (Ghovvati et al 2009), increase the probability of positive result even in highly fragmented DNA (Mane et al 2011).…”

Loop‐Mediated Isothermal Amplification (LAMP): A Rapid and Sensitive Tool for Quality Assessment of Meat Products

“…The mtDNA is more stable than nDNA during heat‐, pressure‐, and salt‐processing; thus, it is suitable for PCR and LAMP reactions (Yang and others ). Moreover, the higher copies (approximately 1000 copies) of mtDNA are present in a cell (Koh and others ), which can be extracted in intact form from heat‐treated processed meat products (Dooley and others ).…”

Meat Species Identification using Loop-mediated Isothermal Amplification Assay Targeting Species-specific Mitochondrial DNA

“…In the raw and cooked meat admixtures, target species content was tested in triplicate from 10 to 0.0.00001%. Previously reported primers and conditions were used for the PCR-based comparators ( Koh et al ., 2011 ) with minor modification. Briefly, the reaction mixture contained 1× Emerald Master Mix (Takara Biotechnology, Japan), 0.5 μM each primer, and 1 μL template.…”

“…Nucleotide amplification methods have been developed as an alternative to protein-based methods ( Dooley et al ., 2004 ; Koh et al ., 2011 ; Wolf et al ., 1999 ). Mitochondrial DNA (mtDNA) has been widely used for species identification, as it is relatively undamaged by processing and can thus be extracted intact from cooked and processed meats and meat products ( Dooley et al ., 2004 ).…”

“…mtDNA sequences are frequently used instead of nuclear DNA for species- specific identification for several reasons. mtDNA exists in multiple copies (approximately 1,000 copies) per cell ( Koh et al ., 2011 ) and is relatively tolerant of environmental stresses such as heat, salt, and pressure. In addition, its rate of evolution facilitates discrimination between closely related species ( Avise et al ., 1987 ).…”