PCR using 3'-mismatched primers to detect A2142C mutation in 23S rRNA conferring resistance to clarithromycin in Helicobacter pylori clinical isolates

Abstract

Twenty-five clarithromycin-resistant Helicobacter pylori strains (selected by agar dilution) were studied to detect A2142G and A2143G mutations in the 23S rRNA gene by a PCR-restriction fragment length polymorphism method and an A2142C mutation by PCR using a 3'-mismatched specific primer. A 700-bp amplified fragment was obtained by the mismatched PCR only in strains without an A2142G or A2143G mutation, indicating that those strains had the A2142C mutation.

FIG. 1

PCR-RFLP patterns obtained after digestion with BsaI or MboII. BsaI digested the 1.4-kbp fragment, producing 1,000- and 400-bp fragments in either susceptible or resistant strains. However, if the A2143G mutation was present, the 1,000-bp fragment was converted to 700- and 300-bp fragments. MboII digested the fragment, producing two fragments of 700 bp only when the A2142G mutation was present. Lanes: 1 and 2, strain 1 digested with BsaI and MboII, respectively; 3 and 4, strain 2 digested with BsaI and MboII, respectively; 5 and 6, strain 3 digested with BsaI and MboII, respectively; 7 and 8, strain 4 digested with BsaI and MboII, respectively; 9, DNA markers. BsaI digested the fragment in strains 1 and 2. BsaI or MboII did not digest strains 3 and 4.

FIG. 2

A 3′-mismatched PCR pattern after amplification of different strains. Lanes 1 to 5, 3′-mismatched positive PCRs; lanes 6 to 8, 3′-mismatched negative PCRs; lane 9, DNA markers.