Identification of racehorse and sample contamination by novel 24-plex STR system

Abstract 

Proper identification of racehorses competing in an official race and maintenance of defensible chain of custody are important in doping control regulations. The purpose of this study was to develop a reliable multiplex PCR method for providing genetic evidence for matching donors to test samples by using short tandem repeat (STR) loci. Amplification of 21 STR loci from blood, urine or hair root was achieved in a single tube and STR length polymorphism was analyzed using fluorescent labeled capillary electrophoresis. This novel approach showed an allele confidence interval of 0.19–0.43bp and size estimation error of 0–0.48bp. In 90 thoroughbred (TB) and 171 standardbred (STB) horses, the method was highly discriminating and reproducible with probability of false identification of 1 in 10¹¹ (TB) and 1 in 10¹³ (STB). All loci were highly polymorphic with an average probability of identity of 0.18 (TB) and 0.13 (STB), heterozygosity of 0.65 (TB) and 0.68 (STB), and polymorphism information content (PIC) of 0.62 (TB) and 0.69 (STB). The highest allele frequency also reflected the degree of polymorphism due to high correlation with PIC. To obtain evidence of sample tampering with human material, three human specific STR markers were included in the panel. This method is the first in the horseracing industry, specifically designed for racehorse identification and detection of equine sample contamination by human DNA.

Abbreviations: STR, short tandem repeat, bp, base pair, TB, thoroughbred, SB, standardbred, CE, capillary electrophoresis, PIC, polymorphism information content, IACUC, Institutional Animal Care and Use Committee, PA, Pennsylvania, PAR, peak area ratio, PCR, polymerase chain reaction, PI, probability of identity

Keywords: Equine, Human, STR loci, Multiplex PCR, Probability of identity