title: Utilization of a real-time PCR approach for Haemophilus influenzae serotype determination as an alternative to the slide agglutination test.
abstract: Our laboratory has developed a simple two-step multiplex real-time PCR for use on isolates of Haemophilus influenzae for molecular serotype identification and the detection of capsular gene targets. The assay consists of a 2-plex real-time PCR targeting the capsule transport gene (bexA), and serotype b specific gene (bcsB), and a 5-plex real-time PCR detecting serotypes a, c, d, e, and f targeting Region II serotype-specific genes. Both real-time PCR assays are highly sensitive (<8 CFU) for all serotypes and 100% specific when tested by a panel of more than 40 bacterial organisms. A retrospective study of 214 isolates received between 1998 and 2011 were tested and compared against the traditional slide agglutination test (SAT) resulting in 100% concordance. We demonstrate that this two-step real-time PCR approach is more sensitive than previously published PCR assays and provides a simple alternative to the SAT. Reliable, rapid and sensitive H. influenzae serotyping is critical for identifying new emerging strains for epidemiological surveillance.
authors.affiliation: Wadsworth Center, New York State Department of Health, PO Box 22002, Albany, NY 12201-2002, USA.
authors.forename: Danielle
authors.initials: D
authors.lastname: Wroblewski
authors.forename: Tanya A
authors.initials: TA
authors.lastname: Halse
authors.forename: Jill
authors.initials: J
authors.lastname: Hayes
authors.forename: Donna
authors.initials: D
authors.lastname: Kohlerschmidt
authors.forename: Kimberlee A
authors.initials: KA
authors.lastname: Musser
mesh_terms: D000372:Agglutination Tests; D015373:Bacterial Typing Techniques / Q000379:methods*; D006193:Haemophilus influenzae / Q000145:classification* / Q000235:genetics*; D058889:Molecular Typing; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D012703:Serotyping / Q000379:methods