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Journal of Clinical Microbiology, August 2008, p. 2547-2554, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02428-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sensitive Detection of Multiple Rotavirus Genotypes with a Single Reverse Transcription-Real-Time Quantitative PCR Assay{triangledown}

Ion Gutiérrez-Aguirre,1* Andrej Steyer,2 Jana Boben,1 Kristina Gruden,1 Mateja Poljsak-Prijatelj,2 and Maja Ravnikar1

Department of Biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia,1 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia2

Received 18 December 2007/ Returned for modification 20 December 2007/ Accepted 28 May 2008

Rotaviruses are one of the major causes of diarrhea in infants and children under 5 years old, especially affecting developing countries. In natural disasters, fecal matter and potable waters can mix, allowing low, yet infective, concentrations of rotavirus to be present in water supplies, constituting a risk for the population. Any of the most commonly detected rotavirus genotypes could originate an outbreak. The development of a fast and sensitive method that could detect the broadest possible range of rotavirus genotypes would help with efficient diagnosis and prevention. We have designed a reverse transcription (RT)-real-time quantitative PCR approach targeted to the rotaviral VP2 gene, based on a multiple-sequence alignment of different human rotaviral strains. To overcome the high nucleotide sequence diversity, multiple forward and reverse primers were used, in addition to a degenerate probe. The performance of the assay was tested on isolates representing the most prevalent human genotypes: G1P[8], G2P[4], G3P[8], G4P[8], G9P[8], and G12P[8]. The developed method improved classical rotavirus detection by enzyme-linked immunosorbent assay and nested RT-PCR by 5 and at least 1 order of magnitude, respectively. A survey of 159 stool samples indicated that the method can efficiently detect a broad range of rotavirus strains, including different G-P genotype combinations of human, porcine, and bovine origin. No cross-reactivity was observed with other enteric viruses, such as astrovirus, sapovirus, and norovirus.

* Corresponding author. Mailing address: Department of Biotechnology and Systems Biology, National Institute of Biology, Vecna pot 111, 1000, Ljubljana, Slovenia. Phone: 386 1 4233388. Fax: 386 1 2573847. E-mail: ion.gutierrez{at}nib.si

{triangledown} Published ahead of print on 4 June 2008.

Journal of Clinical Microbiology, August 2008, p. 2547-2554, Vol. 46, No. 8
0095-1137/08/$08.00+0     doi:10.1128/JCM.02428-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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