The Folmer COI mtDNA universal primers that are considered standard for DNA
barcoding of life contain so many mismatches against the target sequences of vertebrate origin
that they often end in failure to amplify many of vertebrate DNA extractions. This discrepancy
favors for the selection and designing of new metabarcode primers that can be used to identify all
individuals of vertebrates or at least all individuals represented in a class of Vertebrata such as
Class Amphibia. The current study embarks on such an endeavor. In this study development of
new mtDNA metabarcode (16SrRNA) that can be used as universal primers to amplify almost all
species of Class Amphibia for different forensic and molecular biodiversity analyses.
Tissue samples were collected from order Urodela of Class Amphibia (Toads , Bull frog
and skittering frogs sample were collected from Punjab, Pakistan). DNA was extracted from the
collected specimens through standard organic method, qualified and quantified and then PCRamplified
using novel universal primers selected from aligned mtDNA sequences originating
from order Urodela mitochondrial DNA genomes submitted to different online sequence
databases such as NCBI nucleotide database. The sensitivity of PCR also be assessed using a
range of DNA concentrations. The amplified products were sequenced on ABI Genetic Analyzer
following Sanger’s dideoxy method of sequencing. The correctness of obtained mtDNA
sequences were examined visually in Chromas Lite 2.1 software and then alignment of these
sequences were performed against highly similar DNA sequences in NCBI nucleotide databases
using BLAST in order to identify origin of unknown mtDNA sequences. With the help of
sequencing and phylogenetic studies specificity of the universal primer set confirmed and
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presented as a novel metabarcode (16SrRNA) for species level identification of large number of
Amphibian species.
In summary, we present universal method for species classification of Amphibia using a targeted
parallel sequencing approach. Both sequencing and phylogenetic studies experiments confirm
specificity of universal primer set. Although promising results were obtained with current
settings, rapid improvement of bench top instruments will further develop method with less
hands-on, fewer sequencing errors and lower detection limit. So, in future, this barcode can be
used for species identification in various fields of study such as meat adulteration, illegal trade,
food mislabeling and molecular estimation of biodiversity.