Detection of Helicobacter pylori with clarithromycin resistance‐associated mutations using peptide nucleic acid probe‐based melting point analysis

Abstract Background Detection of Helicobacter pylori in gastric biopsy is important for appropriate treatment and prevention of gastric carcinoma and lymphoma. A novel peptide nucleic acid probe (PNA)‐based real‐time polymerase chain reaction (PCR) method was developed for detection of H pylori and A2142G/A2143G mutation of the 23S rRNA gene, which is associated with clarithromycin resistance. Methods To evaluate the performance of the PNA probe‐based PCR method, a total of 409 gastric biopsy samples were analyzed by PNA probe‐based PCR and compared with other H pylori detection methods, including hematoxylin and eosin (HE) and Warthin‐Starry (WS) staining, immunohistochemistry (IHC). A2142G/A2143G mutation of the 23S rRNA gene was tested by dual priming oligonucleotide (DPO)‐based PCR and Sanger sequencing to evaluate PNA probe‐based PCR. Results Among 271 cases that were positive for H pylori on IHC which was considered as a standard method, 264 cases (97.4%) and 259 cases (95.6%) were positively detected by HE/WS and PNA probe‐based qPCR, respectively. Of 100 H pylori‐ positive patients tested by IHC, H pylori was detected in 93 cases (93.0%) by PNA probe‐based PCR, 86 cases (86.0%) by DPO‐based PCR, and 93 cases (93.0%) by conventional PCR. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PNA probe‐based qPCR were 93.0%, 94.9%, 93.9%, 94.9%, and 93.0%, respectively, which were all higher than those of DPO‐based PCR. When Sanger sequencing was determined as a standard method to detect A2142G/A2143G mutations, the sensitivity of the PNA‐ and DPO‐based methods was 100% and 94.4%, respectively, and the specificity was 100% for both methods. Conclusion PNA probe‐based qPCR is an appropriate method for detecting H pylori and the clarithromycin resistance‐associated mutation type.