Genotyping horse epithelial cells from fecal matter by isolation of polymerase chain reaction products

Abstract

Aim To show that application of the polymerase chain reaction (PCR) method modified for amplification of a lowcopy number DNA samples, ie, the isolation of PCR products (IPCRp), would represent improvement in obtaining genotypes from a fecal DNA compared with previously used genotyping methods. Methods The DNA from the horse fecal matter was extracted by modified Qiagen DNA Stool Mini Kit protocol. Following the extraction, the DNA genotypes from fecal samples were obtained by the most powerful PCR amplification method, the IPCRp. The IPCRp-based multiplex kit amplified biotin-labeled strands were captured on streptavidin- coated plates, where everything but the dye-labeled target sequence was washed, eliminating all the background noise, released, and run on a genotyping instrument in a single-strand configuration. Results The IPCRp-based multiplex kit (6 loci) revealed equine DNA full genotype profiles, ie, appearance of all six loci, when sampled from fresh feces in 87% of the samples and partial genotype profile (appearance of one to five loci) in 13% of the samples, for a total of 100% genotyping success rate. Conclusion These results indicate that the IPCRp amplification method, coupled with the Qiagen DNA Stool Mini Kit extraction can maximize the likelihood of obtaining horse DNA genotypes from fecal samples.