Diagnostic reliability of nested PCR depends on the primer design and threshold abundance of Helicobacter pylori in biopsy, stool, and saliva samples.

Abstract

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), 13 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 1-5×103 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. 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.