1). Therefore, a 166-bp fragment was chosen for the design of the primer pair. A panel of closely related micro-organisms as well as the Listeria species (53 Listeria species and 45 non-Listeria species) listed in Tables 1 and 2 were analyzed through Q-PCR to further evaluate the specificity of the primer set (Fig. 2a). The results showed that the assay specifically displayed only positive amplification curves
when Listeria species were present, and there were no cross-reactions with any of the other Everolimus species tested. We also used the blast program for evaluating the specificity of the primer set by comparing other bacterial DNA. Based upon the earlier results, the primer set was chosen and performed well for Q-PCR amplification and specific detection of the ssrA gene fragment in Listeria species. As shown in Fig. 1, there were ≥ 1 different bases in the amplified products between the forward and reverse primer in each Listeria species, inducing differences in GC content and melting temperature
(Tm) values. There was more variability in the L. welshimeri sequence than in the others, and therefore, it was supposed that this species should be more easily distinguished from the other members. HRM analysis was then employed for identification of the differences in Protein Tyrosine Kinase inhibitor the ssrA gene. The specificity of the Q-PCR and HRM analysis was evaluated by testing the Listeria isolates and reference strains and other organisms listed in Tables 1 and 2. Positive Q-PCR amplification and HRM curves were obtained for the following: 25 isolates and eight standard strains of L. monocytogenes (n = 33) including serotypes 1/2a, 1/2b, 1/2c, 3a, 3c, 4a, 4b and 4e; nine isolates and two standard strains of L. innocua (n = 11) including serotypes 6a and 6b; L. welshimeri strains (n = 3) including serotypes 1/2a and 6a; L. seeligeri
strains (n = 2) including serotype 1/2b; L. ivanovii strains (n = 2); and L. grayi strains (n = 2). However, when all the non-Listeria (n = 45) and blank control were identified, no amplification curve appeared and no melting curve in a certain range was produced. All the 53 Listeria species in Tables 1 and 2 had been sequenced directly, and there were no nucleotide differences from the sequences obtained next from GenBank. The earlier results demonstrated that the sequence variations observed in the ssrA gene were species specific. HRM curves were analyzed, and the species-specific dissociation profiles are displayed in Fig. 2b. The results indicated that each species had a unique melting profile, and that L. innocua possessed a lower melting temperature than that of other Listeria species. Furthermore, L. welshimeri had a distinctive HRM curve attributed to a greater number of different sequences than the others. We tested all target Listeria species and calculated the Tm values from each experiment, and the value corresponding to each Listeria species was stable. The Tm values for the six Listeria spp. of interest were L.
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