Utilization of a real-time PCR approach for Haemophilus influenzae serotype determination as an alternative to the slide agglutination test

• Published in: Molecular and cellular probes Vol. 27; no. 2; pp. 86 - 89
• Main Authors: Wroblewski, Danielle, Halse, Tanya A., Hayes, Jill, Kohlerschmidt, Donna, Musser, Kimberlee A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.04.2013
• Subjects: Agglutination; Agglutination Tests; Bacterial Typing Techniques - methods; Haemophilus influenzae; Haemophilus influenzae - classification; Haemophilus influenzae - genetics; Molecular serotyping; Molecular Typing; Multiplex; Polymerase chain reaction; Probes; Real-time PCR; Real-Time Polymerase Chain Reaction - methods; Retrospective Studies; Sensitivity and Specificity; Serotypes; Serotyping; Serotyping - methods; Molecular serotyping; Real-time PCR; Multiplex; Haemophilus influenzae
• ISSN: 0890-8508; 1096-1194
• DOI: 10.1016/j.mcp.2012.11.003

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.