Detection of rodent Helicobacter spp. by use of fluorogenic nuclease polymerase chain reaction assays.
Polymerase chain reaction (PCR) analysis is the standard method for detection of Helicobacter spp. infections in laboratory rodents, with H. hepaticus, H. bilis, and H. typhlonius considered primary pathogens. Fluorogenic nuclease PCR assays that detect all known rodent Helicobacter spp., or that specifically detect H. hepaticus, H. bilis, or H. typhlonius were developed to eliminate post-PCR processing, enhance specificity, and provide quantitative data on starting template concentration. Each fluorogenic PCR assay detected a minimum of 10 copies of target template, had comparable or greater sensitivity when compared directly with corollary gel detection PCR assays, and detected only targeted species when numerous Helicobacter spp. and other enteric bacteria were analyzed. Fluorogenic nuclease PCR analysis of fecal DNA samples obtained from numerous laboratory mice sources detected all samples with positive results by use of Helicobacter spp., H. hepaticus, H. bilis, and/or H. typhlonius gel detection PCR analysis, except for one sample that had positive results by H. typhlonius gel detection PCR but negative results by H. typhlonius fluorogenic nuclease PCR analysis. Among fecal DNA samples that were Helicobacter spp. negative by use of all gel detection PCR assays, the fluorogenic nuclease PCR assays detected target template in only one sample that was positive by use of the Helicobacter spp. and the H. bilis fluorogenic nuclease PCR assays. In conclusion, fluorogenic nuclease PCR assays provide sensitive, specific, and high-throughput diagnostic assays for detection of Helicobacter spp., H. hepaticus, H. bilis, and H. typhlonius in laboratory rodents, and the quantitative data generated by these assays make them potentially useful for bacterial load determination.
Drazenovich
Nicole L
NL
Department of University Animal Care, University of Arizona, Tucson 85721-0101, USA.
Franklin
Craig L
CL
Livingston
Robert S
RS
Besselsen
David G
DG
D000818:Animals; D004269:DNA, Bacterial / Q000032:analysis; D005243:Feces / Q000382:microbiology; D005456:Fluorescent Dyes; D016998:Helicobacter / Q000235:genetics / Q000302:isolation & purification*; D016481:Helicobacter Infections / Q000175:diagnosis / Q000662:veterinary*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012376:Rodent Diseases / Q000175:diagnosis* / Q000235:genetics; D012680:Sensitivity and Specificity
D004269:DNA, Bacterial; D005456:Fluorescent Dyes