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Journal of Clinical Microbiology, September 2009, p. 2737-2743, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00823-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Identification of 20 Common Human Enterovirus Serotypes by Use of a Reverse Transcription-PCR-Based Reverse Line Blot Hybridization Assay {triangledown}

Fei Zhou,1,2 Fanrong Kong,1 Kenneth McPhie,1 Mala Ratnamohan,1 Linda Donovan,1 Frank Zeng,1 Gwendolyn L. Gilbert,1,2 and Dominic E. Dwyer1,2*

Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales, Australia,1 The University of Sydney, Sydney, New South Wales, Australia2

Received 24 April 2009/ Returned for modification 29 May 2009/ Accepted 25 June 2009

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs. Twenty HEV serotypes accounted for 87.1% of all HEVs isolated at an Australian reference virology laboratory from 1979 to 2007. VP1 sequences of all known HEV prototype strains were aligned to design one sense primer and three antisense primers for RT-PCR. After sequencing of the complete VP1 genes of 37 previously serotyped examples of the commonest 20 serotypes and alignment of these VP1 sequences with GenBank sequences, four serotype-specific probes for each serotype were designed for RLB. The RT-PCR-RLB assay was then applied to 132 HEV isolates, made up of the previously sequenced 37 isolates and another 95 serotyped clinical isolates. The RT-PCR-RLB genotypes corresponded with the serotypes for 131/132 isolates; the one exception was confirmed by VP1 sequencing, and the genotype was confirmed by repeat conventional serotyping. Genotyping by RT-PCR-RLB complements traditional serotyping methods and VP1 sequencing and has the advantages of convenience, speed, and accuracy. RT-PCR-RLB allows detection of specific enteroviral serotypes or genotypes associated with HEV outbreaks and significant disease.

* Corresponding author. Mailing address: Centre for Infectious Diseases and Microbiology (CIDM), Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Darcy Road, Westmead, New South Wales, 2145 Australia. Phone: (612) 9845 6255. Fax: (612) 9893 8659. E-mail: dominic_dwyer{at}wmi.usyd.edu.au

{triangledown} Published ahead of print on 1 July 2009.

Journal of Clinical Microbiology, September 2009, p. 2737-2743, Vol. 47, No. 9
0095-1137/09/$08.00+0     doi:10.1128/JCM.00823-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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