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158 Olmos et al.
8. The sensitivity of nested RT-PCR in a single closed tube can be compared with the
sensitivity obtained by conventional RT-PCR using internal or external primers.
9. The background of the nested RT-PCR must be reduced. Excessive cycling often results
in the generation of multimeric PCR product that usually appears as a smear in the lane
from the slot to the expected size of the amplified product in the agarose gel. Background
also appears if the annealing temperature is not sufficient and/or the primer concentration
is too high.
Acknowledgments
The authors wish to thank Dr. Mats Ohlin, University of Lund, Lund, Sweden, and
Dr. Nuria Duran-Vila from IVIA, Valencia, Spain, for critical reading of the manuscript.
The development of nested RT-PCR in a single closed tube has been funded by IVIA,
INIA (SC98-060), CICYT (OLI96-2179), and EU-BIO96-0773 grants. O. Esteban
and E. Bertolini were the recipients of PhD fellowships from Instituto Valenciano
de Investigaciones Agarias and Agencia Española de Cooperación Internacional,
respectively.
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