Validation and improvement of a multiplex PCR method to detect murine Helicobacter species in feces samples of mice

Abstract

Background Murine Helicobacter species have gained increasing awareness in mouse facilities over the last years. Infections with Helicobacter species may have an impact effect on the health of mice and might pose a zoonotic risk to researchers. To minimize the interference with experiments and hence contribute to the 3Rs, a reliable method of monitoring Helicobacter infections in animal facilities needs to be available. The aim of this study was to improve and validate the detection of the most common murine Helicobacter species. Material and Methods A multiplex PCR assay was developed for identification of Helicobacter hepaticus, H. bilis, H. muridarum, H. rodentium, and H. typhlonius that could simultaneously detect these five strains in fecal samples. To ensure the quality of the results, the method was validated based on recommendations for in‐house developed tests. Results The method established was highly sensitive and specific. All five strains were detectable with a detection limit of 102 bacteria. Eight different mouse facilities were tested with the validated assay, and the following prevalence were found: H. rodentium 57%, H. hepaticus 46%, H. typhlonius 17%, H. bilis 12%, and H. muridarum 0%. Conclusion The multiplex PCR is a reliable, economic, and time‐saving diagnostic tool for routine health monitoring. Further prevalence studies are needed to confirm the high prevalence and hence importance of H. rodentium, as until now this agent is not yet listed in FELASA recommendations.

Validation of Multiplex PCR and Serology Detecting Helicobacter Species in Mice

“…The integration of a DNA quality control into the Helicobacter multiplex DNA finder gives information about the validity of samples and reduces the number of false-negative results. Analytical sensitivity of the Helicobacter DNA multiplex finder ranged from 10 to 1000 copies which is comparable to published qPCR assays and to other multiplex Helicobacter PCR assays, assuming that one bacterial cell contains at least one 16S rRNA copy [ 38 , 39 , 40 ]. We determined the sensitivity in detecting Helicobacter infections in comparison to published conventional singleplex PCR assays routinely applied for monitoring laboratory animals at DKFZ, and found the novel Helicobacter multiplex DNA finder to be highly comparable (kappa = 0.95 (95%CI 0.901–0.999)), with a more sensitive detection of H. hepaticus, H. bilis , and H. typhlonius , analyzing 241 samples with both assays.…”

“…So far, Helicobacter detection in routine health monitoring is mostly accomplished by PCR covering single or a small groups of Helicobacter spp. and subsequent gel electrophoresis or qPCR, both being time-, labor- and/or cost-intensive [ 2 , 17 , 18 , 19 , 38 , 39 , 40 ]. Moreover, these PCR assays most often target 16S rRNA, which is known to have homologies between Helicobacter spp.…”