Species Identification Study Through Pyrosequencing On 12S RRNA

Objective: The purpose of this study was to screen species-specific loci of mitochondrial DNA for 37 species, and to establish a simple and accurate system for species identification including human, chicken, duck, cattle, sheep, dog, SD rat, mouse, goose, pig, horse, cat, rabbit, donkey, turtle, pigeon and deer by pyrosequencing technology.Methods: The different regions among above 37 species were screened by software Dna SP v5.10.1 and three target regions were named 12S-1, 12S-2 and 16S-1. Then primers were designed by Primer Primer 5 software. Selected 12S-2 as final target after optimizing and sequencing primer was designed by software PyroMarker Assay Design 2.0. Then amplified and sequenced the 100-bp fragment from 17 different species by Pyromarker Q96 instrument, and established a system for species identification. Partial of the 17 species were selected to perform forensic validation study such as reproducibility, sensitivity and accuracy.Results:(1) A method of short PCR products of 12 S rRNA gene tested by pyrosequencing was successfully developed for species identification. Specimen origin could be accurately determined by only 12 bases according to results of pyrosequencing.(2) The sensitivity of the method was 250-pg template DNA. Tested all samples of unknown species, cooked meats and materials of several years ago were identified successfully by this method.Conclusions: A species identification system tested 12 S rRNA gene by pyrosequencing was constructed, which can successfully identify human, chicken, duck, cattle, sheep, dog, SD rat, mouse, goose, pig, horse, cat, rabbit, donkey, turtle, pigeon and deer. This system can successfully identify special samples and unknown sources with characteristic of reproducibility, accuracy and high sensitivity of 250 pg DNA. It satisfies the requirements of species identification practice in forensic science with its easily-operated, quick and low cost.