title: Species identification of animal cells by nested PCR targeted to mitochondrial DNA.
abstract: We developed a highly sensitive and convenient method of nested polymerase chain reaction (PCR) targeted to mitochondrial deoxyribonucleic acid (DNA) to identify animal species quickly in cultured cells. Fourteen vertebrate species, including human, cynomolgus monkey, African green monkey, mouse, rat, Syrian hamster, Chinese hamster, guinea pig, rabbit, dog, cat, cow, pig, and chicken, could be distinguished from each other by nested PCR. The first PCR amplifies mitochondrial DNA fragments with a universal primer pair complementary to the conserved regions of 14 species, and the second PCR amplifies the DNA fragments with species-specific primer pairs from the first products. The species-specific primer pairs were designed to easily distinguish 14 species from each other under standard agarose gel electrophoresis. We further developed the multiplex PCR using a mixture of seven species-specific primer pairs for two groups of animals. One was comprised of human, mouse, rat, cat, pig, cow, and rabbit, and the other was comprised of African green monkey, cynomolgus monkey, Syrian hamster, Chinese hamster, guinea pig, dog, and chicken. The sensitivity of the PCR assay was at least 100 pg DNA/reaction, which was sufficient for the detection of each species of DNA. Furthermore, the nested PCR method was able to identify the species in the interspecies mixture of DNA. Thus, the method developed in this study will provide a useful tool for the authentication of animal species.
authors.affiliation: Health Science Research Resources Bank (HSRRB), Japan Health Sciences Foundation, 2-11 Rinku-minamihama, Sennan-shi, Osaka 590-0535, Japan.
authors.forename: Kazumi
authors.initials: K
authors.lastname: Ono
authors.forename: Motonobu
authors.initials: M
authors.lastname: Satoh
authors.forename: Touho
authors.initials: T
authors.lastname: Yoshida
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ozawa
authors.forename: Arihiro
authors.initials: A
authors.lastname: Kohara
authors.forename: Masao
authors.initials: M
authors.lastname: Takeuchi
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mizusawa
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Sawada
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000235:genetics*; D004587:Electrophoresis, Agar Gel; D016678:Genome; D006801:Humans; D006824:Hybridization, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial