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.

Single-primer PCR correction: a strategy for false-positive exclusion

“…PCR technology plays an important role in molecular biology research (Vallette et al, 1989;Ono et al, 2007;Wen et al, 2008;Löfström et al, 2008;Fabi et al, 2009). We found that even the use of strict PCR conditions can result in nonspecific amplification, false-positive results, and mixed single-/double-primer amplification fragments caused by single-primer PCR amplification.…”

Species identification and authentification of human and rodent cell cultures using polymerase chain reaction analysis of vomeronasal receptor genes

“…RNase free water was used as negative control. Arrows indicate the DNA molecular weight marker of 500 bp (Hyperladder IV; Bioline) sensitivity or ability to detect contamination (Liu et al 2003;Ono et al 2007;Ramya et al 2009;Higgins et al 2010;Parodi et al 2002). Inexpensive assays for the identification and authentication of cell lines are therefore clearly necessary and should be relatively easily developed using the vast DNA databases as is demonstrated here.…”

A suspected case of hunting accident. Case report

“…The fragment coding for 28S presents regions variables, some of which are significantly different in size between diverse animal species [6,7,8,9,10,11].…”