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 was 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 intraspecific 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-toapply 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.

Benchmarking DNA Metabarcoding for Biodiversity-Based Monitoring and Assessment

“…The obtained barcodes are then high-throughput sequenced and compared to a previously generated DNA sequence reference database from well-characterized species for taxonomic assignment (Taberlet et al, 2012a). In the case of animals, different barcodes such as portions of the small and large subunits of the nuclear ribosomal RNA (18S and 28S rRNA) genes (Machida and Knowlton, 2012) and of the mitochondrial cytochrome oxidase I (COI; Meusnier et al, 2008) and 16S rRNA genes (Sarri et al, 2014) have been proposed for metabarcoding. The COI gene is by far the most commonly used marker for metazoan metabarcoding (Ratnasingham and Hebert, 2013), for which thousands of reference sequences are available in public databases [the Barcode of Life Database (BOLD) contains >1,000,000 COI sequences belonging to animal species] and several amplification primers have been designed [more than 400 COI primers are published in the Consortium for the Barcode of Life (CBOL) primer database].…”

Host use patterns of Culicoides spp. biting midges at a big game preserve in Florida, U.S.A., and implications for the transmission of orbiviruses

“…Bloodmeal analysis of field‐collected engorged females through PCR amplification of host DNA present in a bloodmeal is a valuable method to determine host use of many blood‐feeding arthropods, including biting midges (Slama et al, ), ticks (Allan et al, ), sandflies (Chaskopoulou et al, ), tsetse flies (Muturi et al, ), and mosquitoes (Burkett‐Cadena et al, ) amongst others. Bloodmeal analysis targets regions of certain genes that are well conserved throughout the animal kingdom but show sufficient interspecific variation for identification at the species level, including 16 s rRNA (Sarri et al, ), cytochrome b oxidase (Garros et al, ; Slama et al, ), and cytochrome c oxidase I (Ferri et al, ). As contact (biting) between vectors and susceptible hosts is a critical variable in determining vectorial capacity of a putative vector species, clearly delineating patterns of host use is important for inferring vector status.…”

Species Authentication of Common Meat Based on PCR Analysis of the Mitochondrial COI Gene

“…It is reported that DNA as the most appropriate biomarker to identify the source of animal-derived materials and DNA-based techniques are reliable, robust, and rapid, especially species-specific PCR which has the potential to reach higher identification simplicity, sensitivity, and specificity. The target genes and DNA fragments used as markers for identifying animal species are mainly coming from the mitochondrial genome, such as cytochrome oxidase subunit I (COI) [8], the mitochondrial D-loop region [9], and cytochrome b [10], 16 s rRNA [11], and 12 s rRNA [12]. Many techniques based on the DNA fractions, such as species-specific PCR [12], direct PCR [13], real-time PCR [14], polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [15], and sequencing [16].…”