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Journal of Clinical Microbiology, February 2009, p. 311-321, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01567-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Classification of Fowl Adenovirus Serotypes by Use of High-Resolution Melting-Curve Analysis of the Hexon Gene Region{triangledown}

Penelope A. Steer, Naomi C. Kirkpatrick, Denise O'Rourke, and Amir H. Noormohammadi*

Faculty of Veterinary Science, The University of Melbourne, Werribee, Victoria 3030, Australia

Received 12 August 2008/ Returned for modification 16 October 2008/ Accepted 18 November 2008

Identification of fowl adenovirus (FAdV) serotypes is of importance in epidemiological studies of disease outbreaks and the adoption of vaccination strategies. In this study, real-time PCR and subsequent high-resolution melting (HRM)-curve analysis of three regions of the hexon gene were developed and assessed for their potential in differentiating 12 FAdV reference serotypes. The results were compared to previously described PCR and restriction enzyme analyses of the hexon gene. Both HRM-curve analysis of a 191-bp region of the hexon gene and restriction enzyme analysis failed to distinguish a number of serotypes used in this study. In addition, PCR of the region spanning nucleotides (nt) 144 to 1040 failed to amplify FAdV-5 in sufficient quantities for further analysis. However, HRM-curve analysis of the region spanning nt 301 to 890 proved a sensitive and specific method of differentiating all 12 serotypes. All melt curves were highly reproducible, and replicates of each serotype were correctly genotyped with a mean confidence value of more than 99% using normalized HRM curves. Sequencing analysis revealed that each profile was related to a unique sequence, with some sequences sharing greater than 94% identity. Melting-curve profiles were found to be related mainly to GC composition and distribution throughout the amplicons, regardless of sequence identity. The results presented in this study show that the closed-tube method of PCR and HRM-curve analysis provides an accurate, rapid, and robust genotyping technique for the identification of FAdV serotypes and can be used as a model for developing genotyping techniques for other pathogens.

* Corresponding author. Mailing address: Faculty of Veterinary Science, The University of Melbourne, 250 Princes Highway, Werribee, Victoria 3030, Australia. Phone: 61 3 9731 2275. Fax: 61 3 9731 2366. E-mail: amirh{at}unimelb.edu.au

{triangledown} Published ahead of print on 26 November 2008.

Journal of Clinical Microbiology, February 2009, p. 311-321, Vol. 47, No. 2
0095-1137/09/$08.00+0     doi:10.1128/JCM.01567-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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