Barbora Šeligová1, Ľudovít Lukáč2, Michaela Bábelová1, Silvia Vávrová3, Pavol Sulo1. 1. Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia. 2. First Department of Internal Medicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia. 3. Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
Abstract
BACKGROUND: The aim of this work was to find a reliable nested PCR for the detection of Helicobacter pylori in biopsy, stool, and saliva specimens. MATERIALS AND METHODS: 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), 13 C-urea breath test (UBT). RESULTS: 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 × 103 cells/g or ml of specimen. The sensitivity for nested PCR from stomach biopsies was on the same scale as 13 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. CONCLUSIONS: 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 13 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.
BACKGROUND: The aim of this work was to find a reliable nested PCR for the detection of Helicobacter pylori in biopsy, stool, and saliva specimens. MATERIALS AND METHODS: 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), 13 C-urea breath test (UBT). RESULTS: 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 × 103 cells/g or ml of specimen. The sensitivity for nested PCR from stomach biopsies was on the same scale as 13 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. CONCLUSIONS: 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 13 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.
Authors: Hyun Soo Seo; Hyung Sun Chin; Yeon-Hee Kim; Hye Su Moon; Kyungnam Kim; Le Phuong Nguyen; Dongeun Yong Journal: Ann Lab Med Date: 2021-07-01 Impact factor: 3.464