Wykrywanie pałeczek Bordetella w ramach międzynarodowego sprawdzianu Eupert-labnet Bordetella PCR EQA
[Detection of Bordetella within the framework of the Eupert-labnet Bordetella PCR EQA]

Streszczenie

Celem badania była molekularna identyfikacja DNA Bordetella sp. w ramach pierwszego międzynarodowego sprawdzianu External Quality Assessment (Eupert-labnet Bordetella PCR EQA) oraz porównanie wyników uzyskanych w Laboratorium Zakładu Bakteriologii NIZP-PZH z wynikami uzyskanymi w specjalistycznych laboratoriach w innych krajach EU. Materiał do badań stanowił panel 10 zakodowanych próbek genomowego DNA, w tym DNA Bordetella pertussis w różnym stężeniu. Molekularną identyfikację przeprowadzono
przy użyciu metody: standardowy PCR, multiplex-PCR i real-time PCR. Wśród 10 próbek DNA nadesłanych do identyfikacji znajdowały się 4 zawierające DNA B. pertussis i po jednej próbce zawierającej DNA odpowiednio: B. parapertussis, B. holmesii i B. bronchiseptica. Wyniki sprawdzianu potwierdziły kompetencje naszego laboratorium w zakresie molekularnego wykrywania materiału genetycznego Bordetella sp.

Abstract

The aim of this study was evaluation of molecular identification results of samples including genomic DNA of Bordetella by using PCR method, obtained by laboratory of Department of Bacteriology NIZP-PZH and their comparison with the results obtained by other reference laboratories in EU. The study was conducted within the framework of the first external quality assessment (Eupert-labnet Bordetella PCR EQA).Methods: The panel of ten coded samples of purified genomic DNA was investigated. The panel was designed to include dilution of genomic DNA from B. pertussis at the three concentrations 2 pg/μl (high), 0,2 pg/μl (medium) and 0,02 pg/μl (low).The panel included as well DNA of other Bordetella species (B. parapertussis, B. holmesii, B. bronchiseptica) and H. influenzae at concentrations 2 pg/μl. There was also two ,,blank” samples containing only Tris Buffet (10mM, pH 8,0). Presence or absence of B. pertussis DNA in the tested samples was determined by using four PCR assays: conventional in-house PCR (detection of IS481 B. pertussis and IS1001 B. parapertussis), commercial multiplex PCR (detection of DNA B. pertussis), conventional in-hause real-time PCR (detection of IS481 B. pertussis) and commercial real-time PCR (detection of IS1001 B. parapertussis). Results: All but one samples were correctly identified in our laboratory. Laboratory of Department of Bacteriology NIZP-PZH correctly detected DNA of B. pertussis at both the ,,high” and ,,medium” dilution. In addition, the distinction between B. pertussis and other Bordetella species was correctly obtained by our laboratory. The negative samples, the two blank samples and one containing H. influenzae were correctly detected.Conclusions: Results of the first international external quality assessment have confirmed competences of laboratory of Department of Bacteriology NIZP-PZH in molecular identification of Bordetella pertussis.