Utility of normalized genome quantification of Helicobacter pylori in gastric mucosa using an in-house real-time polymerase chain reaction

Abstract

Traditional diagnostic assays for Helicobacter pylori detection have their limitations. Molecular methods can improve both diagnosis and understanding of gastric diseases. Here we describe an in-house quantitative real-time polymerase chain reaction (q-rt-PCR) for the detection of H. pylori in gastric biopsies which has been developed and has a detection limit of 10 copies, the specificity of which was tested against other gastric colonizer bacteria. In this study, 199 gastric biopsies from adults with different clinical gastric symptoms were examined. Biopsies were obtained during endoscopy and the following tests performed: rapid urease testing (RUT), culture and q-rt-PCR. H. pylori bacterial load expressed as bacterial load per 105 cells was calculated using a standard curve. H. pylori was isolated in 41% of patients, RUT was positive in 32% and bacterial genome was detected in 45% (p = 0.010). Concordance between traditional invasive microbiological methods used together and q-rt-PCR was almost 100%. Bacterial load in patients with positive RUT was significantly higher than those where it was negative (p<0.0001). There were also significant differences between bacterial load in patients with more than one positive assay versus those where only one method was positive (p = 0.006). The in-house q-PCR developed here is quick and inexpensive, and allows accurate diagnosis of H. pylori infection. It also permits normalized bacterial load quantification, which is important to differentiate between asymptomatic colonisation and infection.

Evaluating the spectrum of histomorphological patterns on endoscopic biopsy in patients with upper gastrointestinal tract disorders

“…Electron microscopic examination showed that the bacilli are adherent to the surface epithelium [20,21]. It has been confirmed that the development of gastric cancer arises from background of H. pylori infection [22,23].…”

Validation of loop‐mediated isothermal amplification to detect Helicobacter pylori and 23S rRNA mutations: A prospective, observational clinical cohort study

“…First, the sensitivity of the LAMP assay for H pylori and 23S rRNA point mutants was assessed with commercial heat‐killed H pylori . When 10 6 copies of stock DNA from H pylori were serially diluted, the LAMP assay could detect as few as 10 copies of H pylori , which was a similar sensitivity to that of qPCR 20 . The LAMP primers targeting the 2143G and 2182C mutations showed a lower degree of sensitivity than the primers for H pylori and could detect as few as 100 copies of mutant H pylori (Figure 1A).…”

Detection of Epstein-Barr Virus DNA in Gastric Biopsies of Pediatric Patients with Dyspepsia

“…Evidence of Hp genomic sequences was found in 46 patients (24.7%). Some studies have looked into Hp load by PCR, finding 25–46% and 17–26% positivity in adult and pediatric dyspeptic patients, respectively [ 19 , 20 , 21 , 22 , 23 , 24 , 25 ]. PCR detection of Hp infection has not been previously reported for this cohort of patients.…”