Developmental validation of SpeID: A pyrosequencing-based assay for species identification. (November 2021)

• Record Type: Journal Article
• Authors: Ghemrawi, Mirna; Fischinger, Frank; Duncan, George; Dukes, Mary Jones; Guilliano, Mark; McCord, Bruce
• Is Part Of: Forensic science international. Volume 55(2021)
• Publication Date: 2021-11
• Subjects: Species identification -- Pyrosequencing -- 12S rRNA -- Real-time PCR; Forensic genetics -- Periodicals; Génétique légale -- Périodiques; Forensic genetics; Electronic journals; Periodicals; 614.1
• DOI: 10.1016/j.fsigen.2021.102560
• Languages: English
• ISSNs: 1872-4973

Abstract

Abstract: In crime scenes, biological exhibits are often human in origin, yet biological stains from other fauna may also be present at a crime scene, creating confusion during an investigation. Furthermore, identifying the source of a biological sample can be critical during an investigation. To identify the presence of biological material from non-human sources, it is common to use genetic markers within mitochondrial DNA such as cytochrome b, 16S rRNA, and 12S rRNA genes. This process usually requires DNA sequencing, a process that is neither quick nor easy. In general, a faster, more standardized method for species identification from tissue and body fluids is desirable.For this reason, we have developed a vertebrate specific real-time quantitation method that is followed by an automated pyrosequencing-based procedure that sequences a short fragment within the 12S rRNA gene. Using no more than 35 bases, the assay can distinguish between 32 different species commonly found in and around a household with a turnaround time of 6 h from extraction to sequencing. -Using this procedure, up to 48 samples can be run at a time without the need for expensive reagents or bioinformatic skills. Highlights: Rapid species identification based on 12S rRNA mtDNA and pyrosequencing. Vertebrate-specific primers coupled using SYBR Green as a real-time quantification method to optimize DNA input. Thirty two species (n = 103) from five classes: Mammalia, Aves, Pisces, Amphibia and Reptilia have been examined. The assay is suitable for forensically relevant samples that are degraded, exposed to inhibitors and can detect mixtures.