Based on the sequence variation in the wzx genes of all E. albertii and other Escherichia/Shigella species, we designed three primer sets, containing 12, 14 and 14 primer pairs (from the first to third sets), to specifically detect each of the 40 E. albertii O-genotypes (EAOgs) (Table S5). Previously designed primer pairs targeting an E. albertii-specific region [9 (link)] were also included in each primer set as a positive control and a genetic marker of E. albertii (E_al_1_OF in the first set; E_al_1_NF in the second and third sets). Template DNA for PCR was prepared by the alkaline-boiling method, as described previously [33 (link)]. KOD -Multi and Epi- DNA polymerase (Toyobo) was used for PCR. Each reaction mixture (15 μl) contained 1 μl template DNA, 4.5 μM each primer and 0.3 U polymerase. PCR was conducted with initial denaturation for 2 min at 94 °C, followed by 25 cycles of 10 s at 94 °C, 30 s at 60 °C and 30 s at 68 °C. PCR products were analysed by agarose gel electrophoresis using 2 % agarose S (Nippon Gene).
Free full text: Click here