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 Knepp, J. H.
Right arrow Articles by Valsamakis, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Knepp, J. H.
Right arrow Articles by Valsamakis, A.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, August 2003, p. 3532-3536, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3532-3536.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Comparison of Automated and Manual Nucleic Acid Extraction Methods for Detection of Enterovirus RNA

Julia H. Knepp, Melissa A. Geahr, Michael S. Forman, and Alexandra Valsamakis*

Division of Microbiology, Department of Pathology, The Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-7093

Received 27 January 2003/ Returned for modification 7 April 2003/ Accepted 6 May 2003

Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type 2 Sabin in cerebrospinal fluid. The sensitivity of PCR was equivalent after RNA extraction with QIAamp, BioRobot M48, and MagNA Pure. All 18 replicates of 100 PFU/ml were detected after extraction by the four methods. Fewer replicates of each successive dilution were detected after extraction by each method. At 10-1 PFU/ml, 17 of 18 replicates were positive by QIAamp, 15 of 18 replicates were positive by BioRobot M48, and 12 of 18 replicates were positive by MagNA Pure; at 10-2 PFU/ml, 4 of 17 replicates were positive by QIAamp, 2 of 18 replicates were positive by BioRobot M48, and 0 of 18 replicates were positive by MagNA Pure. At 10-3 PFU/ml, no replicates were detected. Evaluation of TRIzol was discontinued after nine replicates due to a trend of lower sensitivity (at 10-3 PFU/ml eight of nine replicates were positive at 100 PFU/ml, four of nine replicates were positive at 10-1 PFU/ml, and zero of nine replicates were positive at 10-2 PFU/ml). Concordant results were obtained in 24 of 28 clinical specimens after extraction by all methods. No evidence of contamination was observed after extraction by automated instruments. The data indicate that the sensitivity of enterovirus PCR is largely similar after extraction by QIAamp, BioRobot M48, and MagNA Pure; a trend of decreased sensitivity was observed after TRIzol extraction. However, the results of enterovirus PCR were largely concordant in patient samples, indicating that the four extraction methods are suitable for detection of enteroviruses in clinical specimens.

* Corresponding author. Mailing address: Division of Microbiology, The Johns Hopkins Medical Institutions, Meyer B1-193, 600 North Wolfe St., Baltimore, MD 21287-7093. Phone: (410) 955-5077. Fax: (410) 614-8087. E-mail: avalsam1{at}jhmi.edu.

Journal of Clinical Microbiology, August 2003, p. 3532-3536, Vol. 41, No. 8
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.8.3532-3536.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Anwar, A., Wan, G., Chua, K.-B., August, J. T., Too, H.-P. (2009). Evaluation of Pre-Analytical Variables in the Quantification of Dengue Virus by Real-Time Polymerase Chain Reaction. J. Mol. Diagn. 11: 537-542 [Abstract] [Full Text]  
  • Dundas, N., Leos, N. K., Mitui, M., Revell, P., Rogers, B. B. (2008). Comparison of Automated Nucleic Acid Extraction Methods with Manual Extraction. J. Mol. Diagn. 10: 311-316 [Abstract] [Full Text]  
  • Mapes, S., Leutenegger, C. M., Pusterla, N. (2008). Nucleic acid extraction methods for detection of EHV-1 from blood and nasopharyngeal secretions. Vet Rec. 162: 857-859 [Full Text]  
  • Ginocchio, C. C., Manji, R., Lotlikar, M., Zhang, F. (2008). Clinical Evaluation of NucliSENS Magnetic Extraction and NucliSENS Analyte-Specific Reagents for Real-Time Detection of Human Metapneumovirus in Pediatric Respiratory Specimens. J. Clin. Microbiol. 46: 1274-1280 [Abstract] [Full Text]  
  • Fotedar, R., Stark, D., Beebe, N., Marriott, D., Ellis, J., Harkness, J. (2007). Laboratory Diagnostic Techniques for Entamoeba Species. Clin. Microbiol. Rev. 20: 511-532 [Abstract] [Full Text]  
  • Loens, K., Bergs, K., Ursi, D., Goossens, H., Ieven, M. (2007). Evaluation of NucliSens easyMAG for Automated Nucleic Acid Extraction from Various Clinical Specimens. J. Clin. Microbiol. 45: 421-425 [Abstract] [Full Text]  
  • Espy, M. J., Uhl, J. R., Sloan, L. M., Buckwalter, S. P., Jones, M. F., Vetter, E. A., Yao, J. D. C., Wengenack, N. L., Rosenblatt, J. E., Cockerill, F. R. III, Smith, T. F. (2006). Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing. Clin. Microbiol. Rev. 19: 165-256 [Abstract] [Full Text]  
  • Beuselinck, K., van Ranst, M., van Eldere, J. (2005). Automated Extraction of Viral-Pathogen RNA and DNA for High-Throughput Quantitative Real-Time PCR. J. Clin. Microbiol. 43: 5541-5546 [Abstract] [Full Text]  
  • Schuurman, T., van Breda, A., de Boer, R., Kooistra-Smid, M., Beld, M., Savelkoul, P., Boom, R. (2005). Reduced PCR Sensitivity Due to Impaired DNA Recovery with the MagNA Pure LC Total Nucleic Acid Isolation Kit. J. Clin. Microbiol. 43: 4616-4622 [Abstract] [Full Text]  
  • Wilson, D., Yen-Lieberman, B., Reischl, U., Warshawsky, I., Procop, G. W. (2004). Comparison of Five Methods for Extraction of Legionella pneumophila from Respiratory Specimens. J. Clin. Microbiol. 42: 5913-5916 [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»