采用基于肽核酸探针的熔点分析检测克拉霉素耐药相关突变的幽门螺杆菌。

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

机构信息

Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea.

Brain Korea 21 PLUS Projects for Medical Science, Yonsei University College of Medicine, Seoul, South Korea.

出版信息

Helicobacter. 2019 Oct;24(5):e12634. doi: 10.1111/hel.12634. Epub 2019 Jul 14.

DOI:10.1111/hel.12634

PMID:31304664

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.

摘要

背景

检测胃活检中的幽门螺杆菌对于适当的治疗和预防胃癌和淋巴瘤非常重要。本研究开发了一种基于新型肽核酸探针（PNA）的实时聚合酶链反应（PCR）方法，用于检测 H pylori 以及与克拉霉素耐药相关的 23S rRNA 基因 A2142G/A2143G 突变。

方法

为了评估 PNA 探针 PCR 方法的性能，共分析了 409 例胃活检样本，采用 PNA 探针 PCR 与其他 H pylori 检测方法（包括苏木精和伊红（HE）和沃辛氏（WS）染色、免疫组织化学（IHC））进行比较。采用双重引物寡核苷酸（DPO）-基于 PCR 和 Sanger 测序检测 23S rRNA 基因的 A2142G/A2143G 突变，以评估 PNA 探针 PCR。

结果

在 271 例被认为是标准方法的 IHC 阳性的病例中，HE/WS 和 PNA 探针 qPCR 分别检测到 264 例（97.4%）和 259 例（95.6%）为阳性。在 100 例经 IHC 检测为 H pylori 阳性的患者中，PNA 探针 PCR 检测到 93 例（93.0%），DPO 基于 PCR 检测到 86 例（86.0%），常规 PCR 检测到 93 例（93.0%）。PNA 探针 qPCR 的敏感性、特异性、准确性、阳性预测值和阴性预测值分别为 93.0%、94.9%、93.9%、94.9%和 93.0%，均高于 DPO 基于 PCR。当 Sanger 测序被确定为检测 A2142G/A2143G 突变的标准方法时，PNA-和 DPO-基于方法的敏感性分别为 100%和 94.4%，特异性均为 100%。