A new set of 16S rRNA universal primers for identification of animal species

Abstract

In this study, bioinformatics were used to specifically design universal primers within 16S rRNA gene according to the following criteria: the priming sites needed to be sufficiently conserved to permit a reliable amplification (pooled samples) and the genetic marker needed to (a) be sufficiently variable to discriminate among most species and sufficiently conserved within than between species, (b) be short enough to allow also accurate amplification from processed samples (food) and non-invasive approaches (fur, feathers, faeces, etc.) (c) convey sufficient information to assign samples to species and (d) be amplified under variable lab conditions and protocols. Furthermore, short sequences allow the accurate massive inter- and intra-species identification of point mutations by the SSCP technique. The size of the amplified segment ranged from 222 to 252 bp. Amplification and identification success were 100% with all kinds of tissue tested in both raw and processed samples in a wind range of species, mammals (n = 27), fishes (n = 32) birds (n = 19), coleoptera (n = 23), reptiles (n = 5), crustaceans (n = 5) and cephalopods (n = 2), including almost all European mammal and avian game species. In addition, no intra-specific polymorphism was detected. Finally, gene fragments, homologous to those amplified by the primers used herein and retrieved from the GenBank for three animal sets [mammals (n = 248), birds (n = 231) and fishes (n = 644)] showed a particular precise percentage of correct identifications. Therefore, this short segment of the 16S rRNA mitochondrial gene could be a good candidate for a rapid, accurate, low-cost and easy-to-apply and interpret method to identify mammal and avian game species by PCR amplification and sequencing that can be easily incorporated in integrated conservation and forensic programmes. (c) 2014 Elsevier Ltd. All rights reserved.