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Applied and Environmental Microbiology, May 2000, p. 2071-2078, Vol. 66, No. 5
Instituto Valenciano de Investigaciones
Agrarias, 46113 Moncada (Valencia),1 and
Instituto de Tecnologia Agroalimentaria, Laboratorio de
Producción Vegetal, Universitat de Girona, 17071 Girona,2 Spain
Received 16 August 1999/Accepted 7 February 2000
A novel method, which involves a nested PCR in a single closed
tube, was developed for the sensitive detection of Erwinia amylovora in plant material. The external and internal primer pairs used had different annealing temperatures and directed the amplification of a specific DNA fragment from plasmid pEA29. The procedure involved two consecutive PCRs, the first of which was performed at a higher annealing temperature that allowed amplification only by the external primer pair. Using pure cultures of E. amylovora, the sensitivity of the nested PCR in one tube was
similar to that of a standard nested PCR in two tubes. The specificity
and sensitivity were greater than those of standard PCR procedures that
used a single primer pair. The presence of inhibitors in plant
material, very common in E. amylovora hosts, is overcome
with this system in combination with a simple DNA extraction protocol
because it eliminates many of the inhibitory compounds. In addition, it
needs a very small sample volume (1 µl of DNA extracted). With 83 samples of naturally infected material, this method achieved better
results than any other PCR technique: standard PCR detected 55% of
positive samples, two-tube nested PCR detected 71% of positive
samples, and nested PCR in a single closed tube detected 78% of
positive samples. When analyzing asymptomatic plant material, the
number of positive samples detected by the developed nested PCR was
also the highest, compared with the PCR protocols indicated previously (17, 20, and 25% of 251 samples analyzed, respectively). This method
is proposed for the detection of endophytic and epiphytic populations
of E. amylovora in epidemiological studies and for routine
use in quarantine surveys, due to its high sensitivity, specificity,
speed, and simplicity.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Development of a Highly Sensitive Nested-PCR Procedure Using a
Single Closed Tube for Detection of Erwinia amylovora
in Asymptomatic Plant Material
*
Corresponding author. Mailing address: Departamento de
Protección Vegetal y Biotecnología, Instituto Valenciano
de Investigaciones Agrarias, Apartado Oficial, Carretera
Moncada-Náquera km 4,5, 46113 Moncada, Valencia, Spain. Phone:
34-96-1391000. Fax: 34-96-1390240. E-mail: mlopez{at}ivia.es.
Applied and Environmental Microbiology, May 2000, p. 2071-2078, Vol. 66, No. 5
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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