ORIGINAL ARTICLE Validation of a real-time PCR for Haemophilus parasuis C. Turni, M. Pyke and P.J. Blackall Animal Research Institute, Queensland Primary Industries and Fisheries, Yeerongpilly, Qld, Australia Introduction In 1906, a disease of young pigs associated with poly- serositis and arthritis was described by Karl Gla ¨sser (Sutherland and Simmons 1947). This disease is now known as Gla ¨sser’s disease and is caused by the bacterium Haemophilus parasuis (Oliveira and Pijoan 2004). Haemo- philus parasuis is a fastidious and delicate organism, char- acteristics which cause problems in the diagnosis of the disease (Ferri et al. 2000). Recommendations on the best sampling sites and on transport media for swabs and temperatures are available in the literature and from labo- ratories involved in the processing of samples (Oliveira 2004; Turni and Blackall 2007a). However, the conditions in the field are not always optimal, which makes culturing samples difficult and creates a dependence on a sensitive and specific PCR to use on DNA templates extracted from tissues, fluids and swabs directly. The PCR devel- oped by Oliveira et al. (2001) has problems in specificity giving a weak positive with Actinobacillus indolicus, lower- ing the value of the test when it is applied to samples from upper respiratory sites where both H. parasuis and A. indolicus can be present. The PCR developed by Angen et al. (2007) does not report problems with specificity, but seems not as sensitive as the one of Oliveira et al. (2001). The PCR of Oliveira et al. (2001) can detect a minimum concentration of 1 · 10 2 CFU ml )1 of H. para- suis. When the conventional PCR of Oliveira et al. (2001) was compared to the culture method for swabs taken from pigs challenged with H. parasuis, the culture method was more sensitive than the conventional PCR (Turni and Blackall 2007a). According to Espy et al. (2006), the sensitivity of the real-time PCR is in some cases greater than the conventional methods used for bacteriological diagnostics (culture and conventional PCR). A real-time PCR has the potential to be highly sensitive and specific (Mackay 2004; Valasek and Repa 2005; Espy et al. 2006). The primers developed for the two conventional PCRs for H. parasuis target the 16S ribosomal RNA gene, which is conventionally used as a taxonomic and phylogenetic marker (Wilson et al. 1990). However, a preliminary study in our laboratory could not differentiate Pasteurella mairii from H. parasuis for the short amplification prod- uct of the real-time PCR technology selected, when using the 16S rRNA as the target. A possible alternate gene to the 16S rRNA gene is the inf B gene. Hedegaard et al. (2000) concluded that the inf B gene might be useful as a genetic marker for phylogenetic studies. The inf B gene codes for the two forms of the translation initiation factor IF2 – IF2 alpha and IF2 beta (Hedegaard et al. 2000). Keywords Gla ¨ sser’s disease, Haemophilus parasuis, inf B, PCR, real-time PCR. Correspondence Conny Turni, Animal Research Institute, Queensland Primary Industries and Fisheries, Locked Mail Bag No 4, Moorooka, QLD 4105, Australia. E-mail: Conny.Turni@deedi.qld.gov.au 2009 ⁄ 0832: received 12 May 2009, revised 16 July 2009 and accepted 11 August 2009 doi:10.1111/j.1365-2672.2009.04526.x Abstract Aims: To validate a real-time PCR test for the diagnosis of Gla ¨sser’s disease, a major pig disease caused by Haemophilus parasuis. Methods and Results: The specificity of a real-time PCR amplifying the inf B gene was validated with 68 H. parasuis isolates and 36 strains of closely related species. As well, 239 samples of DNA from tissues and fluids of 16 experimen- tally challenged animals were tested with the real-time PCR, and the results were compared with culture and a conventional PCR. The real-time PCR produced significantly more positive results than the conventional PCR (165 vs 86). Conclusions: The sensitivity of the real-time PCR combined with high specific- ity makes it a very valuable tool for the diagnosis of Gla ¨sser’s disease. Significance and Impact of Study: This new method will improve the ability of laboratories to diagnose Gla ¨sser’s disease, especially in laboratories where the culture method for H. parasuis is not optimal. Journal of Applied Microbiology ISSN 1364-5072 ª 2009 The State of Queensland (through the Department of Primary Industries and Fisheries) Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 108 (2010) 1323–1331 1323