Opracowanie szybkiego testu PCR-RFLP do identyfikacji pałeczek Yersinia enterocolitica bioserotypu 1B/O8 z epidemicznego szczepu tych drobnoustrojów izolowanych w Polsce od 2004 roku
[Development of molecular PCR-RFLP test for identification of the epidemic strain of Y. enterocolitica bioserotype 1B/O8 circulating in Poland since 2004]

Streszczenie

Od 2004 roku obserwowane jest w Polsce rozproszone ognisko zakażeń pałeczkami Y. enterocolitica należącymi do wysoce chorobotwórczego bioserotypu 1B/O8. Celem prowadzonych badań było opracowanie szybkiego, molekularnego testu do identyfikacji drobnoustrojów należących do tego epidemicznego szczepu. Zaproponowany schemat diagnostyczny obejmuje dwa etapy. Pierwszy z nich, test multipleks-PCR, pozwala na identyfikację pałeczek Y. enterocolitica bioserotypu 1B/O8 poprzez detekcję czterech markerów
genetycznych (ail, ystA, irp1, sekwencja 16S rDNA). Drugi etap, test dupleks-PCR-RFLP, pozwala wykryć wariant genu ysrR charakterystyczny dla epidemicznego szczepu. Zaproponowany test może stanowić przydatne narzędzie diagnostyczne w typowaniu izolatów epidemicznego szczepu Y. enterocolitica 1B/O8 w diagnostyce klinicznej, weterynaryjnej, badaniu żywności jak również w dochodzeniu epidemiologicznym.

Abstract

Introduction: Highly pathogenic Y. enterocolitica bioserotype 1B/O8 is considered to be an important etiological agent of yersiniosis in Poland. Infections caused by Y. enterocolitica 1B/O8 became an important public health problem in Poland, especially because of their high potential of virulence and the unknown source of the bacteria. Y. enterocolitica 1B/O8 isolates recovered in Poland are genetically highly related and constitute single epidemic sensu stricto strain. The aim of the present study was to develop a time- and money-effective molecular assay for rapid identification of pathogenic Y. enterocolitica 1B/O8 isolates belonging
to the epidemic strain. Methods: In the first stage we performed a multiplex-PCR for four genetic markers: ail, ystA, irp1 and 16S rDNA sequence. In the next stage we designed a duplex-PCR-RFLP assay with BtsI endonuclease to detect / identify specific variant of an ysrR gene that is characteristic for epidemic strain of Y. enterocolitica 1B/O8 strain. The assay was tested against a panel of a consisted of a variety Yersinia enterocolitica and Y. pseudotuberculosis strains.Results: All the tested Y. enterocolitica 1B/O8 strains were positive for all the genetic markers in multiplex-PCR assay what distinguished them from other tested Yersinia strains. In duplex-PCR-RFLP test all tested isolates of the epidemic strain were negative for ysrR digestion with BtsI endonuclease, while all tested reference strains of Y. enterocolitica 1B/O8 were positive. Conclusions: The assay developed in this study was two-stage / two-step molecular test efficiently distinguishing wild-type and the epidemic Y. enterocolitica 1B/O8 strain. Such test can be a useful screening tool for clinical, veterinary and food diagnostics, as well as for the purposes of epidemiological investigation.