Browsing by Author "Bailey, A. M."
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- Asymmetric PCR ELISA: increased sensitivity and reduced costs for the detection of plant virusesPublication . Nolasco, Gustavo; Sequeira, Z.; Soares, C.; Mansinho, A.; Bailey, A. M.; Niblett, C. l.PCR ELISA is the immunodetection of the products of a polymerase chain reaction (PCR). It is effective for detecting and differentiating plant viral nucleic acids, but as currently performed, it is laborious and expensive. The procedure has been modified and simplified by using asymmetric PCR. This eliminated the need to denature and neutralize samples prior to hybridization. It also increased the relative concentration of the target DNA species, making PCR ELISA more sensitive than TaqMan(TM), a fluorescence-based detection method. Reducing the reaction volumes to half and the concentration of the dNTPs and the digoxigenin label by tenfold significantly reduced the costs of PCR ELISA without reducing its sensitivity. The usefulness of these modifications was demonstrated for the detection of Citrus tristeza virus and Rupestris stem pitting-associated virus. We expect that with only minor modifications asymmetric PCR ELISA could be used effectively for the detection of most nucleic acid molecules of interest.
- Differentiation of Citrus tristeza virus (CTV) isolates by cleavase fragment length polymorphism (CFLP) analysis of the major coat protein genePublication . Marques, N T.; Bailey, A. M.; Niblett, C. L.; Nolasco, GustavoA panel of Citrus tristeza virus (CTV, genus Closterovirus, family Closteroviridae) isolates of different origins and with different biological properties were compared for polymorphisms in the major coat protein (CP) gene by cleavase fragment length polymorphism (CFLP) and single stranded conformation polymorphism (SSCP) analysis. The similarity between the CFLP patterns, which consisted of 15 to 20 bands, was estimated by the Pearson coefficient. The clustering patterns from the CFLP data were very similar to those from sequence data in an experiment with 16 cloned standards of the CP gene. By SSCP analysis on the other hand, most of the clones were not clustered in the same way. To assess the ability of CFLP to analyse biological samples, which may consist of a mixture of genomic variants, the CP gene of 12 CTV isolates was obtained directly from infected plants by immunocapture/RT-PCR and analysed. With few exceptions, the isolates were correctly clustered according to the sequences of the variants composing the isolates. In artificial mixed infections of mild and severe isolates the patterns obtained were more closely related to the severe isolate. Thus the CFLP method was an accurate method for the identification, typing and clustering of CTV isolates. The usefulness of this technique as an alternative to SSCP analysis is suggested and discussed.
- Identification to the species level of the plant pathogens Phytophthora and Pythium by using unique sequences of the ITS1 region of ribosomal DNA as capture probes for PCR ELISAPublication . Bailey, A. M.; Mitchell, D. J.; Manjunath, K. L.; Nolasco, Gustavo; Niblett, C. L.The ribosomal internal transcribed spacer 1 region was sequenced for 10 species of Phythium and eight species of Phytophthora. Alignment of the sequences revealed considerable sequence microheterogeneity, which was utilized to prepare a capture probe of unique sequence for each species. The capture probes were tested by PCR ELISA, combining the sensitivity and specificity of the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). The probes were entirely species specific, enabling the detection and identification of the amplified DNA of species from individual Cultures or front mixed samples of the DNAs of two different species. This approach to species identification, which provides a molecular technology to process large numbers of samples and still identify the fungi with a high level of confidence, may greatly reduce the resources and the time of highly trained specialists currently needed to identify these important species of plant pathogenic fungi. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.