This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by DiStefano, D. J.
Right arrow Articles by Shaw, A. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by DiStefano, D. J.
Right arrow Articles by Shaw, A. R.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2005, p. 5876-5880, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.5876-5880.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Novel Rotavirus VP7 Typing Assay Using a One-Step Reverse Transcriptase PCR Protocol and Product Sequencing and Utility of the Assay for Epidemiological Studies and Strain Characterization, Including Serotype Subgroup Analysis

Daniel J. DiStefano,1* Nikolai Kraiouchkine,1 Laura Mallette,2 Marianne Maliga,2 Gregory Kulnis,2 Paul M. Keller,1 H. Fred Clark,3 and Alan R. Shaw1

Vaccine & Biologics Research, Merck Research Laboratories, Merck & Co. Inc., West Point, Pennsylvania,1 Vaccine & Biologics Research, Merck Research Laboratories, Merck & Co., Inc., Wayne, Pennsylvania,2 University of Pennsylvania, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania3

Received 27 April 2005/ Returned for modification 21 June 2005/ Accepted 9 September 2005

Rotavirus is the most common cause of severe dehydrating gastroenteritis in infants. To date, 10 different serotypes of rotavirus have been identified in human stools. While four or five serotypes dominate, serotype circulation varies with season and geography. Since our laboratory has been involved in the development of a multivalent rotavirus vaccine, it is important to identify the serotypes of rotavirus encountered during our clinical trials. We have developed methodologies for the molecular identification of rotavirus strains based on VP7 gene segment sequence. A 365-bp reverse transcriptase PCR product was generated from the VP7 gene segment using a pair of novel degenerate primers. All serotypes tested (both animal and human) yielded an identically sized product after amplification. Sequencing of these products is performed using truncated versions of the original primers. The sequence generated is compared against a database of rotavirus VP7 sequences, with the G type determined, based on the sequence homology. Using this assay, we have correctly identified human VP7 strains from a panel of available serotypes, as well as numerous animal strains. The assay was qualified using rotavirus positive stool samples, negative stool samples, and rotavirus-spiked stool samples. In addition, samples from cases of acute gastroenteritis collected at Children's Hospital of Philadelphia have been evaluated and indicate that the assay is able to discriminate subtle differences within serotypes. The assay has been utilized in the testing of >3,000 antigen-positive (enzyme immunoassay) samples collected during clinical trials of a rotavirus vaccine (RotaTeq) and identified a serotype in ~92% of samples (3, 17, 19).

* Corresponding author. Mailing address: Vaccine & Biologics Research, Merck Research Laboratories, Merck & Co., Inc., West Point, PA 19486. Phone: (215) 652-1747. Fax: (215) 652-2142. E-mail: daniel_distefano{at}merck.com.

Journal of Clinical Microbiology, December 2005, p. 5876-5880, Vol. 43, No. 12
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.12.5876-5880.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • van Doorn, L.-J., Kleter, B., Hoefnagel, E., Stainier, I., Poliszczak, A., Colau, B., Quint, W. (2009). Detection and Genotyping of Human Rotavirus VP4 and VP7 Genes by Reverse Transcriptase PCR and Reverse Hybridization. J. Clin. Microbiol. 47: 2704-2712 [Abstract] [Full Text]  
  • Martinez-Laso, J., Roman, A., Rodriguez, M., Cervera, I., Head, J., Rodriguez-Avial, I., Picazo, J. J. (2009). Diversity of the G3 genes of human rotaviruses in isolates from Spain from 2004 to 2006: cross-species transmission and inter-genotype recombination generates alleles. J. Gen. Virol. 90: 935-943 [Abstract] [Full Text]  
  • Clark, H F., Lawley, D., Mallette, L. A., DiNubile, M. J., Hodinka, R. L. (2009). Decline in Cases of Rotavirus Gastroenteritis Presenting to The Children's Hospital of Philadelphia after Introduction of a Pentavalent Rotavirus Vaccine. CVI 16: 382-386 [Abstract] [Full Text]  
  • Banyai, K., Bogdan, A., Szucs, G., Arista, S., De Grazia, S., Kang, G., Banerjee, I., Iturriza-Gomara, M., Buonavoglia, C., Martella, V. (2009). Assignment of the group A rotavirus NSP4 gene into genotypes using a hemi-nested multiplex PCR assay: a rapid and reproducible assay for strain surveillance studies. J Med Microbiol 58: 303-311 [Abstract] [Full Text]  
  • Ajjampur, S. S. R., Rajendran, P., Ramani, S., Banerjee, I., Monica, B., Sankaran, P., Rosario, V., Arumugam, R., Sarkar, R., Ward, H., Kang, G. (2008). Closing the diarrhoea diagnostic gap in Indian children by the application of molecular techniques. J Med Microbiol 57: 1364-1368 [Abstract] [Full Text]  
  • Block, S. L., Vesikari, T., Goveia, M. G., Rivers, S. B., Adeyi, B. A., Dallas, M. J., Bauder, J., Boslego, J. W., Heaton, P. M., for the Pentavalent Rotavirus Vaccine Dose Confirm, (2007). Efficacy, Immunogenicity, and Safety of a Pentavalent Human-Bovine (WC3) Reassortant Rotavirus Vaccine at the End of Shelf Life. Pediatrics 119: 11-18 [Abstract] [Full Text]  
  • Logan, C., O'Leary, J. J., O'Sullivan, N. (2006). Real-Time Reverse Transcription-PCR for Detection of Rotavirus and Adenovirus as Causative Agents of Acute Viral Gastroenteritis in Children.. J. Clin. Microbiol. 44: 3189-3195 [Abstract] [Full Text]  
  • Vesikari, T., Matson, D. O., Dennehy, P., Van Damme, P., Santosham, M., Rodriguez, Z., Dallas, M. J., Heyse, J. F., Goveia, M. G., Black, S. B., Shinefield, H. R., Christie, C. D.C., Ylitalo, S., Itzler, R. F., Coia, M. L., Onorato, M. T., Adeyi, B. A., Marshall, G. S., Gothefors, L., Campens, D., Karvonen, A., Watt, J. P., O'Brien, K. L., DiNubile, M. J., Clark, H F., Boslego, J. W., Offit, P. A., Heaton, P. M., the Rotavirus Efficacy and Safety Trial (REST) Stu, (2006). Safety and Efficacy of a Pentavalent Human-Bovine (WC3) Reassortant Rotavirus Vaccine. NEJM 354: 23-33 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»