Development and validation of a novel 13-plex PCR system for commonly used short tandem repeats in horses (Equus caballus).

BACKGROUND: Due to the thriving development of the modern horse industry and the occurrence of horse related crimes, the demand for methods of individual horse identification, parentage tests and other genetic analyses is increasing. Previous methods had disadvantages that decreased the accuracy of the results, lacked the inclusion of all commonly used short tandem repeats (STR) or increased the experimental cost and time.

OBJECTIVES: We aimed to develop a novel 13-plex STR typing system to resolve the above issues.

STUDY DESIGN: Experimental study.

METHODS: Twelve autosomal and most commonly used di-nucleotide STRs (AHT4, AHT5, ASB2, ASB17, ASB23, HMS2, HMS3, HMS6, HMS7, HTG4, HTG10 and VHL20), and a Y-chromosomal STR (YJ10) were included. We redesigned the primers of eight STRs to establish a novel multiplex PCR system and tested this system for species specificity, sensitivity and repeatability.

RESULTS: Full profiles were easily generated in one fast PCR reaction using a low-cost polymerase, as little as 1 ng of horse DNA template and 13 pairs of primers labelled with fluorescent dyes. No full profile was generated from DNA templates of humans or other commonly encountered animals. We also established an allelic ladder that contained 110 alleles based on 200 horses from 12 breeds and calculated standard population genetic parameters based on 150 Thoroughbreds. Stutter analysis showed that the averages of the stutter ratios were distinctly lower than those of lower allele ratios and the combined probability of paternity exclusion for this system were 0.994659935 (CPEduo ) and 0.999854032 (CPEtrio ).

MAIN LIMITATIONS: A nonspecific and relatively low peak at 316 bp was frequently observed in locus HMS2, which is a nonexistent allele in all horses and should be ignored.

CONCLUSIONS: Our results indicate that this 13-plex STR genotyping system is sensitive, species-specific, cost-effective and robust for applications in the horse industry and forensic investigation.