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Journal of Clinical Microbiology, April 2002, p. 1571-1572, Vol. 40, No. 4
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.4.1571-1572.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Improving Real-Time PCR Genotyping Assays by Asymmetric Amplification

LETTER
Rapid-cycle real-time PCR by the LightCycler (Roche Molecular
Biochemicals, Auckland, New Zealand) provides a very quick,
one-step means of genotyping herpes simplex virus (HSV) from
clinical samples and a number of assays involving this application
have been reported (
1,
2,
3).
However, weamong othershave found that a commonly used protocol (1) provides poor genotyping results when the published reagent concentrations are used. The PCR resulted in the "hook effect" phenomenon (Fig. 1) that is the sign of a very efficient PCR (Technical note no. LC 8/99, Roche Molecular Biochemicals, 1999). Simply, in later cycles of the PCR, the amplified strands reanneal before the probes can bind to generate fluorescence. For quantification, this is not an issue, since the crossing point (the cycle number where the reaction enters exponential phase) is of interest. However, subsequent genotyping by melting-curve analysis may be more difficult.
We overcame this phenomenon by the use of asymmetric primer
concentrations whereby a higher concentration of the reverse
primer (compared to that of the forward primer) was used in
the reaction. This resulted in more of the strand complementary
to the probes being amplified and allowed more signal to be
generated (Fig.
2). This in turn resulted in the easy genotyping
of the HSV samples via melting-curve analysis (Fig.
3).
We suggest that the use of asymmetric primer concentrations
be considered when new LightCycler genotyping assays that result
in the hook effect on the quantitation screen are being optimized.

ADDENDUM IN PROOF
Since the submission of this manuscript, Burggraf et al. have
described the benefits of asymmetric primer concentrations for
genotyping the M129V polymorphism associated with human prion
disease (S. Burggraf et al., Clin. Chem.
48:199-201, 2002).
However, equimolar primer concentrations in this assay result
in a less-pronounced hook effect than that seen with the HSV
assay used here.

REFERENCES
1
- Espy, M. J., J. R. Uhl, P. S. Mitchell, J. N. Thorvilson, K. A. Svien, A. D. Wold, and T. F. Smith. 2000. Diagnosis of herpes simplex virus infections in the clinical laboratory by LightCycler PCR. J. Clin. Microbiol. 38:795-799.[Abstract/Free Full Text]
2
- Kessler, H. H., G. Muhlbauer, B. Rinner, E. Stelzl, A. Berger, H.-W. Dorr, B. Santner, E. Marth, and H. Rabenau. 2000. Detection of herpes simplex virus DNA by real-time PCR. J. Clin. Microbiol. 38:2638-2642.[Abstract/Free Full Text]
3
- Schalasta, G., A. Arents, M. Schmid, R. W. Braun, and G. Enders. 2000. Fast and type-specific analysis of herpes simplex virus types 1 and 2 by rapid PCR and fluorescence melting-curve-analysis. Infection 28: 85-91.[CrossRef][Medline]
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Kevin Barratt*
Specialist Services Department, Health Waikato Laboratory, Waikato Hospital, Hamilton, New Zealand
John F. Mackay
Roche Diagnostics New Zealand, Ltd.
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* Phone: (64) 7 8398899, ext. 8537, Fax: (64) 7 8398759, E-mail: barrattk{at}hwl.co.nz |
Journal of Clinical Microbiology, April 2002, p. 1571-1572, Vol. 40, No. 4
0095-1137/02/$04.00+0 DOI: 10.1128/JCM.40.4.1571-1572.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
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