Use of a Combination of Brushing Technique and the Loop-Mediated Isothermal Amplification Method as a Novel, Rapid, and Safe System for Detection of Helicobacter pylori

Abstract

Gastric mucosal biopsy is widely used in the detection of Helicobacter pylori but is associated with a number of problems, including false-negative results due to sampling error and massive bleeding after biopsy. Given the extended period required to culture H. pylori, detection would be further improved by the use of rapid detection methods such as PCR. Here, we developed a rapid, safe, and convenient method for collecting H. pylori which combines endoscopic brushing with the loop-mediated isothermal amplification (LAMP) method. The specificity and sensitivity of LAMP were examined using nine urease-generating non-H. pylori bacterial species, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Helicobacter hepaticus, and 51 H. pylori strains. Results showed that H. pylori-specific LAMP primers amplified H. pylori DNA only and that the lowest detection limit of the LAMP reaction was 10 2 CFU. Brushing and biopsy samples taken from 200 patients with peptic ulcer at Nagoya University Hospital and a regional health care center were subjected to both LAMP and culturing. No adverse effects such as severe bleeding or penetration occurred during the procedure. By LAMP assay, 123 patients were confirmed as H. pylori positive when brushing technique samples were assayed, whereas only 100 were positive when biopsy samples were assayed. Culture assay detected H. pylori in 117 patients when it was combined with the brushing technique and in 96 when it was combined with biopsy. Combination of the endoscopic brushing technique with LAMP is considered a useful and safe system for identifying H. pylori infection.Helicobacter pylori infection is recognized as a major cause of peptic ulcer disease and gastric cancer (8,29). H. pylori occurs beneath the mucus layer, predominantly in the antrum (34). Infection is generally confirmed by histology (21), culturing (13), and the rapid urease test (RUT) (23) performed on endoscopic gastric mucosal biopsy samples or by the urea breath test (14). This latter test is a noninvasive method that detects 13 CO 2 generated by H. pylori urease (5); because it is indirect, however, it does not allow the measurement of antibiotic susceptibility, a disadvantage given that the recent gradual increase in the number of antibiotic-resistant strains has mandated susceptibility testing before eradication therapy (9, 25). The collection and identification of strains thus remain a valuable confirmation of infection and is necessary for the investigation of virulence (12, 25).Nevertheless, biopsy carries the risk of causing bleeding and cannot be used for patients with a bleeding tendency, resulting, for example, from liver cirrhosis or idiopathic thrombocytopenic purpura, notwithstanding the possible association of these diseases with H. pylori infection (11). A second problem is the existence of urease-generating bacteria other than H. pylori; a number of bacteria produce urease and may colonize the stomach in an acid suppression environment (24, 25), potentially confounding the differentiati...

Helicobacter pylori and Helicobacter heilmannii in untreated Bulgarian children over a period of 10 years

“…The number of positive results for DGS, culture and RUT in gastric mucus specimens decreased by 22.2 , 19.8 and 9.9 %, respectively, compared to those in antral biopsy specimens (Table 2). However, Minami et al (2006) have reported that combination of the endoscopic brushing technique with the loop-mediated isothermal amplification method is a useful and safe system for identifying H. pylori infection.…”

Sensitive and Rapid Detection of Cholera Toxin-Producing Vibrio cholerae Using Loop-Mediated Isothermal Amplification

“…Also, the increase in the turbidity of the reaction mixture according to the production of precipitate correlates with the amount of DNA synthesized [10,11]. Various LAMP assays for the identification of pathogenic organisms have been developed [10][11][12][13]15,16], however, no assay for the detection of CT-producing V. cholerae has been described.…”

Molecular Detection of New Delhi Metallo-Beta-Lactamase-1 (NDM-1) Positive Bacteria from Environmental and Drinking Water Samples by Loop Mediated Isothermal Amplification of bla NDM-1

“…LAMP products could be detected & M. Parani parani.m@ktr.srmuniv.ac.in 1 visually by turbidity or by using fluorescent dyes such as ethidium bromide, calcein, and SYBR Green [13]. LAMP was successfully established as a tool for detecting pathogenic bacteria and viruses from clinical and environmental samples [14][15][16][17][18][19]. LAMP was used to detect the NDM-1 gene in sputum, urine and fecal samples [20].…”