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 Vinjé, J.
Right arrow Articles by Long, S. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Vinjé, J.
Right arrow Articles by Long, S. C.
Agricola
Right arrow Articles by Vinjé, J.
Right arrow Articles by Long, S. C.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2004, p. 5996-6004, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.5996-6004.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Molecular Detection and Genotyping of Male-Specific Coliphages by Reverse Transcription-PCR and Reverse Line Blot Hybridization

Jan Vinjé,1* Sjon J. G. Oudejans,1 Jill R. Stewart,2 Mark D. Sobsey,1 and Sharon C. Long3

Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina,1 National Ocean Service, Charleston, South Carolina,2 Civil and Environmental Engineering, University of Massachusetts, Amherst, Massachusetts3

Received 17 March 2004/ Accepted 12 June 2004

In recent years, there has been increased interest in the use of male-specific or F+ coliphages as indicators of microbial inputs to source waters. Sero- or genotyping of these coliphages can also be used for microbial source tracking (MST). Among the male-specific coliphages, the F+ RNA (FRNA) viruses are well studied, while little is known about the F+ DNA (FDNA) viruses. We have developed a reverse line blot hybridization (RLB) assay which allows for the simultaneous detection and genotyping of both FRNA as well as FDNA coliphages. These assays included a novel generic duplex reverse transcription-PCR (RT-PCR) assay for FRNA viruses as well as a generic PCR for FDNA viruses. The RT-PCR assays were validated by using 190 field and prototype strains. Subsequent DNA sequencing and phylogenetic analyses of RT-PCR products revealed the classification of six different FRNA clusters, including the well-established subgroups I through IV, and three different FDNA clusters, including one (CH) not previously described. Within the leviviruses, a potentially new subgroup (called JS) including strains having more than 40% nucleotide sequence diversity with the known levivirus subgroups (MS2 and GA) was identified. We designed subgroup-specific oligonucleotides that were able to genotype all nine (six FRNA, three FDNA) different clusters. Application of the method to a panel of 351 enriched phage samples from animal feces and wastewater, including known prototype strains (MS2, GA, Qß, M11, FI, and SP for FRNA and M13, f1, and fd for FDNA), resulted in successful genotyping of 348 (99%) of the samples. In summary, we developed a novel method for standardized genotyping of F+ coliphages as a useful tool for large-scale MST studies.

* Corresponding author. Mailing address: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 966-7317. Fax: (919) 966-4711. E-mail: janvinje{at}email.unc.edu.

Applied and Environmental Microbiology, October 2004, p. 5996-6004, Vol. 70, No. 10
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.10.5996-6004.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Love, D. C., Sobsey, M. D. (2007). Simple and Rapid F+ Coliphage Culture, Latex Agglutination, and Typing Assay To Detect and Source Track Fecal Contamination. Appl. Environ. Microbiol. 73: 4110-4118 [Abstract] [Full Text]  
  • Kirs, M., Smith, D. C. (2007). Multiplex Quantitative Real-Time Reverse Transcriptase PCR for F+-Specific RNA Coliphages: a Method for Use in Microbial Source Tracking. Appl. Environ. Microbiol. 73: 808-814 [Abstract] [Full Text]  
  • Nappier, S. P., Aitken, M. D., Sobsey, M. D. (2006). Male-Specific Coliphages as Indicators of Thermal Inactivation of Pathogens in Biosolids. Appl. Environ. Microbiol. 72: 2471-2475 [Abstract] [Full Text]  
  • Stewart, J. R., Vinje, J., Oudejans, S. J. G., Scott, G. I., Sobsey, M. D. (2006). Sequence Variation among Group III F-Specific RNA Coliphages from Water Samples and Swine Lagoons. Appl. Environ. Microbiol. 72: 1226-1230 [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»