Validation of a microbead-based format for spoligotyping of Legionella pneumophila.
A 42-plex clustered regularly interspaced short palindromic repeat (CRISPR)-based typing technique (spoligotyping) was recently developed at the French National Reference Center for Legionella. It allows the subtyping of the Legionella pneumophila sequence type 1/Paris pulsotype. In this report, we present the transfer of the membrane-based spoligotyping technique to a microbead-based multiplexed format. This microbead-based high-throughput assay uses devices such as Luminex 200 or the recently launched Magpix system (Luminex Corp., Austin, TX). We designated this new technique LP-SPOL (for L. pneumophila spoligotyping). We used two sets of samples previously subtyped by the membrane-based spoligotyping method to set up and validate the transfer on the two microbead-based systems. The first set of isolates (n = 56) represented the whole diversity of the CRISPR patterns known to date. These isolates were used for transfer setup (determination of spacer cutoffs for both devices). The second set of isolates (n = 245) was used to validate the transfer to the two microbead-based systems. The results obtained by the Luminex 200 system were 100% concordant with those obtained by the Magpix system for the 2 sets of isolates. In total, 10 discrepant results were observed when comparing the membrane-based method to the microbead-based method. These discrepancies were further resolved by repeating either the membrane-based or the microbead-based assay. This new assay is expected to play an emerging role for surveillance of L. pneumophila, starting with one of the most frequent genotypes, the sequence type 1/Paris pulsotype. However, the generalization of this typing method to all L. pneumophila strains is not feasible, since not all L. pneumophila strains contain CRISPRs.
Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud, Paris, France Centre Muraz, Bobo-Dioulasso, Burkina Faso.
Michel Kiréopori
MK
Gomgnimbou
CIRI, International Center for Infectiology Research, Legionella Pathogenesis Team, Université de Lyon, Lyon, France INSERM, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France CNRS, UMR5308, Lyon, France Centre National de Référence des légionelles, Centre de Biologie Est, Hospices Civils de Lyon, Lyon, France.
Christophe
C
Ginevra
Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud, Paris, France.
Caroline
C
Peron-Cane
Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud, Paris, France.
Margaux
M
Versapuech
Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud, Paris, France.
Guislaine
G
Refrégier
CIRI, International Center for Infectiology Research, Legionella Pathogenesis Team, Université de Lyon, Lyon, France INSERM, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France CNRS, UMR5308, Lyon, France Centre National de Référence des légionelles, Centre de Biologie Est, Hospices Civils de Lyon, Lyon, France.
Nathalie
N
Jacotin
Institut de Génétique et Microbiologie, UMR8621, CNRS-Université Paris-Sud, Paris, France christophe.sola@u-psud.fr.
Christophe
C
Sola
CIRI, International Center for Infectiology Research, Legionella Pathogenesis Team, Université de Lyon, Lyon, France INSERM, U1111, Lyon, France Ecole Normale Supérieure de Lyon, Lyon, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon, France CNRS, UMR5308, Lyon, France Centre National de Référence des légionelles, Centre de Biologie Est, Hospices Civils de Lyon, Lyon, France.
Sophie
S
Jarraud
D057205:Automation, Laboratory; D005602:France; D057166:High-Throughput Screening Assays; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification* / Q000235:genetics*; D008863:Microspheres; D017720:Molecular Epidemiology / Q000379:methods; D058889:Molecular Typing / Q000379:methods*