title: Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Luo
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Jian-Hua
authors.initials: JH
authors.lastname: Jiao
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Wen-Yue
authors.initials: WY
authors.lastname: Zhang
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Han-Ming
authors.initials: HM
authors.lastname: Pu
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Bao-Jin
authors.initials: BJ
authors.lastname: Qu
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Bing-Ya
authors.initials: BY
authors.lastname: Yang
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Min
authors.initials: M
authors.lastname: Hou
authors.affiliation: Xiao-Feng Luo, Wen-Yue Zhang, Bing-Ya Yang, Min Hou, Min-Jun Ji, Department of Pathogen Biology, Nanjing Medical University, Nanjing 211166, Jiangsu Province, China.
authors.forename: Min-Jun
authors.initials: MJ
authors.lastname: Ji
mesh_terms: D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017291:Clarithromycin / Q000494:pharmacology; D017931:DNA Primers; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D005753:Gastric Mucosa / Q000382:microbiology; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012338:RNA, Ribosomal, 23S / Q000235:genetics*; D012463:Saliva / Q000382:microbiology
chemical_list: D000900:Anti-Bacterial Agents; D017931:DNA Primers; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin
keywords: Clarithromycin resistance; Drug sensitivity testing; Helicobacter pylori; Nested-allele specific primer-polymerase chain reaction; Rapid urease test