Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
First Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia.
Helicobacter. 2020 Apr;25(2):e12680. doi: 10.1111/hel.12680. Epub 2020 Feb 14.
The aim of this work was to find a reliable nested PCR for the detection of Helicobacter pylori in biopsy, stool, and saliva specimens.
Novel nested PCR was elaborated and validated on 81 clinical biopsy, stool, and saliva samples from the same individual and compared to available H pylori assays: histology, rapid urease test (RUT), stool antigen test (SAT), C-urea breath test (UBT).
The efficiency and selectivity of 17 published nested polymerase chain reactions (PCR) available for Helicobacter pylori detection were re-evaluated. Most of them had serious limitations and mistakes in primer design. Hence, we elaborated a nested PCR for the unambiguous identification of H pylori in biopsy, stool, and saliva, using primers targeted to variable regions of the 16S ribosomal RNA (rRNA) gene. Moreover, we determined the detection limit by adding a known number of cells. This number was as low as 0.5 cells in a PCR vial, but due to the DNA isolation procedures, it required 1-5 × 10 cells/g or ml of specimen. The sensitivity for nested PCR from stomach biopsies was on the same scale as C-UBT (93.8%), but it was much lower in amplifications from stool (31.3%). Sequencing of all obtained PCR products exclusively confirmed H pylori-specific DNA sequences.
Elaborated nested PCR assay can serve as an auxiliary method for controversial samples (patients with bleeding or taking proton-pump inhibitor) in laboratories with basic equipment. The sensitivity and specificity for the amplification from gastric biopsies was almost like C-UBT. Despite the good sensitivity, the threshold occurrence and the ability to survive in the oral cavity aside from and independent of the stomach is the reason why H pylori DNA cannot be reliably detected in saliva, stool, and some biopsy samples.
本研究旨在寻找一种可靠的巢式 PCR 方法，用于检测活检、粪便和唾液标本中的幽门螺杆菌。
我们设计并验证了一种新型的巢式 PCR 方法，并与现有的幽门螺杆菌检测方法（组织学、快速尿素酶试验、粪便抗原检测、C-尿素呼气试验）进行了比较。该方法使用针对 16S 核糖体 RNA 基因可变区的引物，对 81 份来自同一个体的活检、粪便和唾液样本进行了检测。
我们重新评估了 17 种已发表的用于幽门螺杆菌检测的巢式聚合酶链反应（PCR）方法的效率和选择性。大多数方法在引物设计上存在严重的局限性和错误。因此，我们设计了一种巢式 PCR 方法，用于明确鉴定活检、粪便和唾液中的幽门螺杆菌，该方法使用针对 16S 核糖体 RNA 基因可变区的引物。此外，我们通过添加已知数量的细胞来确定检测限。这个数量在 PCR 管中低至 0.5 个细胞，但由于 DNA 提取程序的原因，需要 1-5×10 个细胞/g 或 ml 标本。从胃活检标本中进行巢式 PCR 的敏感性与 C-UBT 相同（93.8%），但从粪便标本中进行扩增的敏感性要低得多（31.3%）。对所有获得的 PCR 产物进行测序，仅证实了幽门螺杆菌特异性 DNA 序列。
设计的巢式 PCR 方法可作为基本设备实验室中存在争议的样本（出血或正在服用质子泵抑制剂的患者）的辅助方法。从胃活检标本中进行扩增的敏感性和特异性与 C-UBT 几乎相同。尽管敏感性良好，但由于幽门螺杆菌在口腔中的存在和生存能力是独立于胃之外的，因此无法在唾液、粪便和一些活检样本中可靠地检测到 H pylori DNA。
