Reliable diagnostics are a major challenge for the detection and treatment of () infection. Currently at the forefront are non-invasive urea breath test (UBT) and stool antigen test (SAT). Polymerase chain reaction (PCR) is not endorsed due to nonspecific primers and the threat of false-positives. The specificity of DNA amplification can be achieved by nested PCR (NPCR), which involves two rounds of PCR. If the primers are properly designed for the variable regions of the 16S rRNA gene, it is not difficult to develop an NPCR assay for the unambiguous identification of . Elaborate NPCR for a 454 bp amplicon was validated on 81 clinical biopsy, stool, and saliva samples, each from the same individuals, and compared with available assays, namely histology, rapid urease test, SAT, and C-UBT. The assay was much more sensitive than simple PCR, and it was equally sensitive in biopsy samples as the C-UBT test, which is considered the gold standard. In addition, it is sufficiently specific because sequencing of the PCR products exclusively confirmed the presence of -specific DNA. However, due to the threshold and lower abundance, the sensitivity was much lower in amplifications from stool or saliva. Reliable detection in saliva also complicates the ability of to survive in the oral cavity aside from and independent of the stomach. The reason for the lower sensitivity in stool is DNA degradation; therefore, a new NPCR assay was developed to obtain a shorter 148 bp 16S rRNA amplicon. The assay was validated on stool samples from 208 gastroenterological patients and compared to SAT results. Surprisingly, this NPCR revealed the presence of in twice the number of samples as SAT, indicating that many patients are misdiagnosed, not treated by antibiotics, and their problems are interpreted as chronic. Thus, it is unclear how to properly diagnose in practice. In the first approach, SAT or UBT is sufficient. If samples are negative, the 148 bp amplicon NPCR assay should be performed. If problems persist, patients should not be considered negative, but due to threshold abundance, they should be periodically tested. The advantage of NPCR over UBT is that it can be used universally, including questionable samples taken from patients with achlorhydria, receiving proton pump inhibitors, antibiotics, bismuth compound, intestinal metaplasia, or gastric ulcer bleeding.