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Development and Application of a Novel Real‐Time PCR Assay for Citrobacter rodentium Quantification
Author(s) -
Sagaidak* Sofia,
Wen* Bijun,
Taibi Amel,
Villa Christopher,
Comelli Elena
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.757.5
Subject(s) - citrobacter rodentium , bifidobacterium bifidum , microbiology and biotechnology , feces , citrobacter , biology , real time polymerase chain reaction , pathogen , bacteria , escherichia coli , enterobacteriaceae , bifidobacterium , lactobacillus , gene , biochemistry , genetics
Citrobacter rodentium is a murine pathogen causing transmissible colonic hyperplasia and used to model intestinal colitis including foodborne Escherichia coli O157:H7 pathogenesis. Several probiotics have been found to alleviate infection and Bifidobacterium bifidum MIMBb75 may be effective because it strongly adheres to colonocytes and modifies the gut microbiota. C. rodentium is typically quantified by classical culturing; the aim of this study was to establish a novel molecular assay based on quantitative real‐time PCR. Primers were designed to target the C. rodentium‐ specific espB gene. Specificity was validated in silico and experimentally with a range of intestinal bacteria. A standard curve was constructed using serially diluted C. rodentium DNA depicting a linearity range (R 2 > 0.99) of 10 2 to 10 7 cells per PCR reaction (efficiency 96%) and sensitivity of 10 4 cells/g feces. To assess effects of B. bifidum MIMBb75 on C. rodenti um fecal load, C57BL/6J mice were gavaged with 10 9 cells of B. bifidum MIMBb75 in PBS or PBS alone daily from 7 days before to 10 days post infection (PI) with 10 9 cells of C. rodentium. Fecal counts of C. rodentium at day 10 PI were quantified with the novel qPCR assay and did not differ significantly between the treatment and control group (6.0 ± 0.4 and 6.4 ± 0.1 log cells/g feces, respectively). This was confirmed by classical culturing. This is the first real‐time PCR assay specific for C. rodentium and it can be used to determine its load and distribution along the intestinal tract in response to preventive and therapeutic interventions. Other probiotics were unable to reduce fecal load yet mitigated inflammation thus, further research is warranted. *co‐first authors Funding JP Bickell Foundation, NSERC, NSERC USRA