Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of Clarithromycin‐Resistant Helicobacter pylori Infection in Children Using Stool Sample

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Abstract
To analyze clarithromycin-resistant Helicobacter pylori infection in children, we developed a method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using stool samples. Twenty-three children without significant upper abdominal symptoms were included (mean age 7.0 years). Of these, 18 and five were diagnosed as H. pylori-positive and -negative, respectively, by the H. pylori stool antigen test (HpSA). The DNA from the stool samples was purified using the QIAamp DNA Stool Minikit (QIAGEN). The PCR was performed on the purified DNA using oligonucleotide primers designed to amplify the 23S rRNA gene of H. pylori. The PCR products were reacted with restriction enzymes MboII, BceAI, and BsaI to detect mutations A2142G, A2142C, and A2143G, respectively. Sixteen of the 18 HpSA-positive samples were PCR-positive, and all five HpSA-negative samples were PCR-negative. Thus, the PCR had 89% sensitivity and 100% specificity, with 91% accuracy in reference to HpSA. Of the 16 PCR-positive samples, one and four were digested with MboII and BsaI, respectively, indicating 31% prevalence of CAM-resistance. We conclude that the PCR-RFLP using stool samples is a rapid and reliable method to noninvasively detect clarithromycin-resistant H. pylori infection in children. It may be useful before choosing regimens of H. pylori eradication.