Use of O-antigen gene cluster-specific PCRs for the identification and O-genotyping of Yersinia pseudotuberculosis and Yersinia pestis.
Yersinia pestis is a very recently evolved clone of Yersinia pseudotuberculosis serotype O:1b. This close relationship causes potential difficulties in DNA-based diagnostic methods. Analysis of the O-antigen gene clusters in these two organisms identified two regions that were used to specifically identify Y. pestis-Y. pseudotuberculosis as a group or Y. pestis alone. Both PCR assays were found to be 100% specific when tested on a large collection of Yersinia species and other Enterobacteriaceae. Furthermore, advantage was taken of the different setups of the O-antigen gene clusters of the 21 known Y. pseudotuberculosis serotypes to develop a multiplex PCR assay to replace the conventional serotyping method of Y. pseudotuberculosis by O-genotyping. The multiplex PCR assay contained nine sets of specific PCRs in a single tube and when used on Y. pseudotuberculosis reference strains allowed the distinction of 14 individual serotypes and two duplex serotypes (O:4a-O:8 and O:12-O:13). Serotype O:7, O:9, and O:10 strains required additional PCRs for O-genotyping. Once applied to Y. pseudotuberculosis strains of various origins, a very good correlation between classical serotypes and O-genotypes was observed, although some discrepancies were found. O-genotyping also proved useful to correct misidentification of some strains and to type Y. pseudotuberculosis isolates that had lost the expression of the O-antigen. The PCR-based O-genotyping can easily be applied in conventional laboratories, without the need for tedious preparation of a large set of specific antisera.
Bogdanovich
Tatiana
T
Department of Medical Biochemistry, University of Turku, Turku, Finland.
Carniel
Elisabeth
E
Fukushima
Hiroshi
H
Skurnik
Mikael
M
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004285:Dogs; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D005810:Multigene Family; D019081:O Antigens / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine; D015009:Yersinia Infections / Q000145:classification; D015010:Yersinia pestis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015011:Yersinia pseudotuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004269:DNA, Bacterial; D019081:O Antigens