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Journal of Clinical Microbiology, October 2005, p. 5408-5409, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5408-5409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Errors in Published Sequences of Human Cytomegalovirus Primers and Probes: Do We Need More Quality Control?

Top Letter References

 
Over the past decade, many authors have focused on PCR as a powerful technique for the evaluation of human cytomegalovirus (CMV). The key to PCR lies in the design of oligonucleotides, as the specific sequences largely affect PCR's efficacy and sensitivity. This study was designed to examine the quality of published sequences of CMV primers and probes.

PubMed was searched in the National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov) for English peer-reviewed articles using CMV and PCR as keywords. Articles reporting on virus genotyping or species-level identification, as well as letters to editors and reviews were excluded. The full texts of 91 papers published between 1993 and 2004 were studied. Of these, 34 papers did not describe the detailed nucleotide sequence, including 17 papers using commercially available kits. The remaining 57 papers with a total of 199 CMV-specific oligonucleotides were examined. Oligonucleotides with identical sequences or with one additional nucleotide at either the 3' or 5' end of the sequence were identified as synonymous.

Using The Sequence Manipulation Suite web-based programs (written by Paul Stothard, University of Alberta, Canada), the binding sites of all 199 oligonucleotides were identified using GenBank strain AD169 genome sequence (GenBank accession no. X17403 and NC_001347). Mismatches to all published sequences of CMV were analyzed by the Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/BLAST/). Papers containing oligonucleotide errors were studied for technical reasons for employment of mutated oligonucleotides or subsequently published errata. Moreover, information on identified errors was conveyed to all corresponding authors and coauthors.

Ten oligonucleotides did not match any CMV strain, and reasons were specified neither in the respective articles nor in authors' replies (Table 1). Primers R2, F6, and F9 were incorrectly transferred from previous publications (2, 5, 7) as indicated by the authors (V. H. Aquino and X. L. Pang, personal communication). The oligonucleotides F1, R3, and P10 were apparently also incorrectly transferred from prior publications (14). Moreover, primers F4 and R5 possessed mismatches at their 3'-end triplets, which may reduce PCR efficiency (21). Furthermore, the two degenerated probes (P7 and P8) contained Y (C/T) instead of R (A/G) in P7 and vice versa in P8. Obviously, these mismatches may drastically affect the efficacies of these probes (17).

View this table: [in this window] [in a new window]

TABLE 1. Published CMV oligonucleotides that showed errors with all published CMV strains

In summary, we show that 5% of the CMV oligonucleotides included mismatches to all published sequences half of which were incorrectly reported mostly in secondary publications. Moreover, since we considered all sequences correct when they matched at least one CMV strain, even when the authors used a different strain for design or admitted an error, the number of errors is underestimated by our approach. These observations point to the possibility that the report of erroneous primers and probes is a widespread problem irritating other researchers. It might therefore be advisable for authors and reviewers alike to pay special attention to the verification of such sequences and for researchers citing or following published work.

 
We thank Xiaoli Pang, Victor Hugo Aquino, Julio Mendez, Kristin Lund, Jean-Pierre Allain, Ursula Meyer-König, Yi-Wei Tang, Stephen Garner, Lars Schaade, Otávia Caballero, Junko Ohyashiki, Peter Schäfer, Andrew Simpson, and Svein Nordbø for their cooperative correspondence. We are indebted to Klaus Roemer for critical review of the manuscript.

Top Letter References

 

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						Wafa Habbal Fawza Monem Faculty of Pharmacy Damascus University Damascus, Syria Barbara Christine Gärtner Department of Virology University of Saarland Medical School Haus 47 D-66421 Homburg/Saar, Germany *Phone: 49-6841-1623950, Fax: 49-6841-1623980, E-mail: vibgae@uniklinikum-saarland.de

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Journal of Clinical Microbiology, October 2005, p. 5408-5409, Vol. 43, No. 10
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.10.5408-5409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Habbal, W., Monem, F., Gartner, B. C. (2009). Comparative evaluation of published cytomegalovirus primers for rapid real-time PCR: which are the most sensitive?. J Med Microbiol 58: 878-883 [Abstract] [Full Text]  

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