Bio-Speedy® Brucella qPCR SNP-Genotyping Kit’s principle is based on the steps that consist of:
1) Reactions are setup by combining the “2X qPCR Mix” component, the “Target specific – Oligo Mix” component and the template DNA.
2) There are two different oligo sets targeting the same genomic position. Two of these oligonucleotide sets apply two different quantitative real-time PCR (qPCR) reactions using the same isolate DNA. If the number of threshold cycles (Cq) of oligo sets is low, then for that isolate, that genome position is the result of that SNP type. For example, as a result of the qPCR reactions performed with Oligo 1207539-T and Oligo 1207539-C, if the reaction with Oligo 1207539-T results in a lower number of Cq, the result is the T base at position 1207539 genome.
3) In reactions involving Brucella DNA isolate, at least one of two different sets of oligos applied for the same genomic position should be positive. Otherwise, the template DNA quality is not sufficient or the template DNA inhibits the reaction.
4) All reactions to which NC is added as template should be negative.
INTENDED USE
Bio-Speedy® Brucella qPCR SNP-Genotyping Kit is used for the genotyping of Brucella spp. isolates.