Differential detection of five mouse-infecting helicobacter species by multiplex PCR.
Several species of helicobacter have been isolated from laboratory mice, including H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, which appear to be the most common. The most widely used published method for molecular detection of these agents is PCR amplification of a conserved region of 16S rRNA, but differential speciation requires restriction enzyme digestion of the amplicons. This study was undertaken to determine PCR conditions that would simultaneously and specifically identify each of the five common species without restriction enzyme analyses. First, we designed novel and specific PCR primers for H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius, using sequences from the heterologous regions of 16S rRNA. Because of comigration of amplified products, we next identified P17, an H. bilis-specific protein; P25, an H. hepaticus-specific protein; and P30, an H. muridarum-specific protein by screening genomic DNA expression libraries of each species. Primers were designed from these three genes, plus newly designed, species-specific 16S rRNA primers for H. rodentium and H. typhlonius that could be utilized for a five-plex PCR. The sizes of the amplicons from H. bilis, H. hepaticus, H. muridarum, H. rodentium, and H. typhlonius were 435, 705, 807, 191, and 122 bp, respectively, allowing simultaneous detection and effective discrimination among species.
Feng
Sunlian
S
Center for Comparative Medicine, School of Medicine, University of California, Davis, Davis, California 95616, USA.
Ku
Karin
K
Hodzic
Emir
E
Lorenzana
Edward
E
Freet
Kim
K
Barthold
Stephen W
SW
D000818:Animals; D001431:Bacteriological Techniques; D017931:DNA Primers / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D003937:Diagnosis, Differential; D016998:Helicobacter / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016481:Helicobacter Infections / Q000175:diagnosis; D051379:Mice; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000032:analysis; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012313:RNA; D004262:DNA Restriction Enzymes