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Titolo:
Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers
Autore:
Cullen, DW; Lees, AK; Toth, IK; Duncan, JM;
Indirizzi:
Scottish Crop Res Inst, Unit Mycol Bacteriol & Nematol, Dundee DD2 5DA, Scotland Scottish Crop Res Inst Dundee Scotland DD2 5DA Dundee DD2 5DA, Scotland
Titolo Testata:
EUROPEAN JOURNAL OF PLANT PATHOLOGY
fascicolo: 4, volume: 107, anno: 2001,
pagine: 387 - 398
SICI:
0929-1873(2001)107:4<387:CPARQP>2.0.ZU;2-3
Fonte:
ISI
Lingua:
ENG
Soggetto:
BETA-TUBULIN GENE; MICROBIAL DNA; PLANT-TISSUE; QUANTIFICATION; AMPLIFICATION; EXTRACTION; RESISTANCE; IDENTIFY; PRIMERS; SEED;
Keywords:
diagnostics; internal transcribed spacer regions; potato; quantitative (TaqMan (TM)) PCR; silver scurf; soil;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Agriculture,Biology & Environmental Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Duncan, JM Scottish Crop Res Inst, Unit Mycol Bacteriol & Nematol, Dundee DD2 5DA, Scotland Scottish Crop Res Inst Dundee Scotland DD2 5DA 5DA, Scotland
Citazione:
D.W. Cullen et al., "Conventional PCR and real-time quantitative PCR detection of Helminthosporium solani in soil and on potato tubers", EUR J PL P, 107(4), 2001, pp. 387-398

Abstract

Silver scurf is an economically important blemish disease of potato causedby the fungus Helminthosporium solani. Two sets of PCR primers, Hs1F1/Hs2R1 (outer) and Hs1NF1/Hs2NR1 (nested) were designed to unique sequences of the nuclear ribosomal internal transcribed spacer (ITS1 and ITS2) regions ofH. solani. Nested PCR was used to increase the specificity and sensitivityof single round PCR. Each primer set amplified a single product of 447 bp and 371 bp respectively, with DNA from 71 European and North American isolates of H. solani, and the specificity of primers was confirmed by the absence of amplified product with DNA from other fungal and bacterial plant pathogens. A simple and rapid procedure for direct extraction of DNA from soilsand potato tubers was modified and developed to yield DNA of a purity and quality suitable for PCR within 3 h. The sensitivity of PCR for the specific detection of H. solani in seeded soils was determined to be 1.5 spores g(-1) of soil. H. solani was also detected by PCR in naturally infested soil and from peel and peel extract from infected and apparently healthy tubers. Specific primers and a TaqMan(TM) fluorogenic probe were designed using the original primer sequences to perform real-time quantitative (TaqMan(TM)) PCR. The same levels of sensitivity for specific detection of H. solani in soil and tubers were obtained during first round mboxTaqMan-based PCR as with conventional nested PCR and gel electrophoresis. This rapid and quantitative PCR assay allows an accurate estimation of tuber and soil contamination by H. solani, thus providing a tool to study the ecology of the organism and to serve as a crucial component for disease risk assessments.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 25/09/20 alle ore 06:15:16