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 Gardner, S. N.
Right arrow Articles by Slezak, T. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gardner, S. N.
Right arrow Articles by Slezak, T. R.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, June 2003, p. 2417-2427, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2417-2427.2003

Limitations of TaqMan PCR for Detecting Divergent Viral Pathogens Illustrated by Hepatitis A, B, C, and E Viruses and Human Immunodeficiency Virus

Shea N. Gardner,* Thomas A. Kuczmarski, Elizabeth A. Vitalis, and Tom R. Slezak

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551

Received 7 February 2002/ Returned for modification 14 April 2002/ Accepted 8 February 2003

Recent events illustrate the imperative to rapidly and accurately detect and identify pathogens during disease outbreaks, whether they are natural or engineered. Particularly for our primary goal of detecting bioterrorist releases, detection techniques must be both species-wide (capable of detecting all known strains of a given species) and species specific. Due to classification restrictions on the publication of data for species that may pose a bioterror threat, we illustrate the challenges of finding such assays using five nonthreat organisms that are nevertheless of public health concern: human immunodeficiency virus (HIV) and four species of hepatitis viruses. Fluorogenic probe-based PCR assays (TaqMan; Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.) may be sensitive, fast methods for the identification of species in which the genome is conserved among strains, such as hepatitis A virus. For species such as HIV, however, the strains are highly divergent. We use computational methods to show that nine TaqMan primer and probe sequences, or signatures, are needed to ensure that all strains will be detected, but this is an unfeasible number, considering the cost of TaqMan probes. Strains of hepatitis B, C, and E viruses show intermediate divergence, so that two to three TaqMan signatures are required to detect all strains of each virus. We conclude that for species such as hepatitis A virus with high levels of sequence conservation among strains, signatures can be found computationally for detection by the TaqMan assay, which is a sensitive, rapid, and cost-effective method. However, for species such as HIV with substantial genetic divergence among strains, the TaqMan assay becomes unfeasible and alternative detection methods may be required. We compare the TaqMan assay with some of the alternative nucleic acid-based detection techniques of microarray, chip, and bead technologies in terms of sensitivity, speed, and cost.

* Corresponding author. Mailing address: Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, P.O. Box 808, L-448, 7000 East Ave., Livermore, CA 94551. Phone: (925) 422-4317. Fax: (925) 422-2133. E-mail: gardner26{at}llnl.gov.

Journal of Clinical Microbiology, June 2003, p. 2417-2427, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2417-2427.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Duitama, J., Kumar, D. M., Hemphill, E., Khan, M., Mandoiu, I. I., Nelson, C. E. (2009). PrimerHunter: a primer design tool for PCR-based virus subtype identification. Nucleic Acids Res 37: 2483-2492 [Abstract] [Full Text]  
  • Malanoski, A. P., Lin, B., Wang, Z., Schnur, J. M., Stenger, D. A. (2006). Automated identification of multiple micro-organisms from resequencing DNA microarrays. Nucleic Acids Res 34: 5300-5311 [Abstract] [Full Text]  
  • Welzel, T. M., Miley, W. J., Parks, T. L., Goedert, J. J., Whitby, D., Ortiz-Conde, B. A. (2006). Real-time PCR assay for detection and quantification of hepatitis B virus genotypes a to g.. J. Clin. Microbiol. 44: 3325-3333 [Abstract] [Full Text]  
  • Gardner, S. N., Lam, M. W., Smith, J. R., Torres, C. L., Slezak, T. R. (2005). Draft versus finished sequence data for DNA and protein diagnostic signature development. Nucleic Acids Res 33: 5838-5850 [Abstract] [Full Text]  
  • Garcia, E. P., Dowding, L. A., Stanton, L. W., Slepnev, V. I. (2005). Scalable Transcriptional Analysis Routine--Multiplexed Quantitative Real-Time Polymerase Chain Reaction Platform for Gene Expression Analysis and Molecular Diagnostics. J. Mol. Diagn. 7: 444-454 [Abstract] [Full Text]  
  • Swanson, P., de Mendoza, C., Joshi, Y., Golden, A., Hodinka, R. L., Soriano, V., Devare, S. G., Hackett, J. Jr. (2005). Impact of Human Immunodeficiency Virus Type 1 (HIV-1) Genetic Diversity on Performance of Four Commercial Viral Load Assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT. J. Clin. Microbiol. 43: 3860-3868 [Abstract] [Full Text]  
  • Gardner, S. N., Kuczmarski, T. A., Zhou, C. E., Lam, M. W., Slezak, T. R. (2005). System To Assess Genome Sequencing Needs for Viral Protein Diagnostics and Therapeutics. J. Clin. Microbiol. 43: 1807-1817 [Abstract] [Full Text]  
  • Gardner, S. N., Lam, M. W., Mulakken, N. J., Torres, C. L., Smith, J. R., Slezak, T. R. (2004). Sequencing Needs for Viral Diagnostics. J. Clin. Microbiol. 42: 5472-5476 [Abstract] [Full Text]  
  • Patolsky, F., Zheng, G., Hayden, O., Lakadamyali, M., Zhuang, X., Lieber, C. M. (2004). Electrical detection of single viruses. Proc. Natl. Acad. Sci. USA 101: 14017-14022 [Abstract] [Full Text]  
  • Shu, P.-Y., Huang, J.-H. (2004). Current Advances in Dengue Diagnosis. CVI 11: 642-650 [Full Text]  
  • Papin, J. F., Vahrson, W., Dittmer, D. P. (2004). SYBR Green-Based Real-Time Quantitative PCR Assay for Detection of West Nile Virus Circumvents False-Negative Results Due to Strain Variability. J. Clin. Microbiol. 42: 1511-1518 [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»