Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

Abstract

Aim: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23SrRNA gene in Helicobacter pylori (H. pylori) by nested-allele specific primer-polymerase chain reaction (nested-ASP-PCR).

Methods: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test (RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nested-ASP-PCR, bacterial culture and disk diffusion.

Results: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23SrRNA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates than ASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87 (87.88%) and 67 (67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin.

Conclusion: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

Keywords: Clarithromycin resistance; Drug sensitivity testing; Helicobacter pylori; Nested-allele specific primer-polymerase chain reaction; Rapid urease test.

Figure 1

PCR products of wild-type Helicobacter pylori strain in gastric mucosa. A: PCR product amplified with the external primers (M: 1000 bp DNA marker, N: Helicobacter pylori negative sample, 1: Wild-type strains); B: PCR products amplified with the different inner primers; C: The products amplified by nested-ASP-PCR [M: 1000 bp DNA marker (1000, 750, 500, 400, 300, 200, 100), W2: 2142 wild-type primers (2142A), M2: 2142 mutation primers (2142G), W3: 2143 wild-type primers (2143A), M3: 2143 mutation primers (2143G), W4: 2144 wild-type primers (2144A), M4: 2144 mutation primers (2144G)]. ASP-PCR: Allele-specific primer-polymerase chain reaction.

Figure 2

PCR products of Helicobacter pylori clinical strains in gastric mucosa with 2142, 2143 and 2144 positions mutation assayed by nested-ASP-PCR. A: Helicobacter pylori-negative control; B: A2142G mutation and A2143G mutation strains; C: A2144G mutation; C: wild-type and A2143G mutation mixture strains [M: 1000 bp DNA marker (1000, 750, 500, 400, 300, 200, 100), E: Outer PCR primers, W2: 2142 wild-type primers (2142A), M2: 2142 mutation primers (2142G), W3: 2143 wild-type primers (2143A), M3: 2143 mutation primers (2143G), W4: 2144 wild-type primers (2144A), M4: 2144 mutation primers (2144G)]. ASP-PCR: Allele-specific primer-polymerase chain reaction.

Figure 3

Mutation subtypes of each site in gastric mucosa and saliva. (1) “2142G, 2143G and 2144G” indicates the three-sites mutation sub-type; (2) “2142G and 2143G, 2142G and 2144G, 2143G and 2144G” indicate the two-sites mutation subtypes; (3) “Single 2142G, single 2143G and single 2144G” indicate the single-site mutation subtypes; (4) “Total 2142G, total 2143G and total 2144G” indicate the sum of corresponding site mutations in the single-site, two-sites and three-sites mutations.