We developed an alternative method to sequencing, focusing on the SSR8 locus (constituted by GGT triplets from three to six repeats). The approach is based on asymmetric quantitative PCR, followed by high-resolution melting analysis with unlabelled probes (UP-HRM). www.selleckchem.com/screening/anti-diabetic-compound-library.html Data showed perfect concordance between
direct sequencing and UP-HRM, which is faster, simpler and more cost effective. Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of paratuberculosis in ruminants and other species. This bacterium is characterized by a very slow growth rate and limited genomic diversity (Stevenson et al., 2009). Numerous methods have been proposed to sub-type Map strains, such as multiplex PCR for IS900 integration loci, IS900 restriction fragment length polymorphism, amplified fragment length polymorphism, pulsed field gel electrophoresis and genotyping microarrays (Motiwala et al., 2006; Pribylova et al., 2009; Stevenson et al., 2009). However, methods based on micro- and minisatellite analyses are the most widely used techniques in this regard (Motiwala www.selleckchem.com/products/VX-770.html et al., 2006; Thibault et al., 2007) because of their relative simplicity and efficacy. Short sequence repeat (SSR) loci, particularly SSR1, SSR2, SSR8 and SSR9, showed highest allelic diversity, making these loci very useful for the evaluation of discriminatory indices (Amonsin et al., 2004; Thibault et al.,
2008). However, at present, the only method available for the scanning of these loci is direct sequencing, which requires expensive systems and dedicated
facilities. To overcome this problem, we developed a new alternative approach to sequencing, for the identification of SSR loci repeat number. We focused on the SSR8 locus, which comprises GGT triplets. So far, four alleles (ranging from three to six repeats) have been described for this locus (Amonsin et al., 2004; Ghadiali et al., 2004; Motiwala et al., 2006; Thibault et al., 2008). The new method is based on an asymmetric quantitative PCR (qPCR), followed by high-resolution melting (HRM) analysis with unlabelled probes (hereafter UP-HRM) (Zhou et al., 2004). The efficiency and specificity of the asymmetric PCR reaction was Anacetrapib improved by designing primers according to the linear-after-the-exponential PCR (LATE-PCR) strategy (Pierce et al., 2005), whereas to avoid any elongation during the amplification, the unlabelled probe was blocked at its 3′ end. The shortness of the probe (32 bp) enhanced the ability of HRM analysis to differentiate between sequences with very similar melting temperature (Tm), allowing clear identification of typical Tm values for every single allele. Map strains were collected at the Italian National Reference Centre for Paratuberculosis. Briefly, DNA was extracted by suspending one colony of Map in 100 μL of PCR-grade water.