INVESTIGADORES
VOJNOV Adrian Alberto
artículos
Título:
Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods.
Autor/es:
RIGANO L; MARANO MR; CASTAGNARO AP; ALEXANDRE MORAIS DO AMARAL; ADRIÁN A. VOJNOV
Revista:
BMC MICROBIOLOGY
Editorial:
BIOMED CENTRAL LTD
Referencias:
Año: 2010 vol. 10 p. 176 - 184
ISSN:
1471-2180
Resumen:
Abstract
Background: Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many
countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker,
named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative
agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type
B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of
those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation,
antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be
time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated
isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and
requires no specialized laboratory equipment.
Results: A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was
developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like
pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition,
amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick,
eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different
conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue.
The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected
plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of
detecting CBC-causing strains from several geographical origins and pathotypes.
Conclusions: The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus
Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide.