The generation of specific DNA primers using random amplified polymorphic DNA and its application to Verticillium dahliae

被引：35

作者：

Li, KN ^{[1
]}

Rouse, DI ^{[1
]}

Eyestone, EJ ^{[1
]}

German, TL ^{[1
]}

机构：

[1] Univ Wisconsin, Dept Plant Pathol, Madison, WI 53706 USA

来源：

MYCOLOGICAL RESEARCH | 1999年 / 103卷

关键词：

D O I：

10.1017/S0953756299008357

中图分类号：

Q93 [微生物学];

学科分类号：

071005 ; 100705 ;

摘要：

A DNA fragment apparently unique to Verticillium dahliae was found by comparing RAPD profiles of V. dahliae to those of other closely related fungi. RAPD analyses were performed on eight V. dahliae, six V. albo-atrum and three V. tricorpus isolates to identify DNA sequences specific to V. dahliae. RAPD primer E20 (AACGGTGACC) yielded a 567 bp band shared only by V. dahliae isolates. This band from isolate V14 was cloned and sequenced. No significant sequence similarity was found between this amplicon and any other nucleic acid sequence in the databases. Based on the sequence information, a pair of PCR primers, VDS1 (5'-CACATTCAGTTCAGGAGACGGA-3') and VDS2 (5'-CCTTCTACTGGAGTATTTCGG-3') was designed. PCR tests showed that VDS1 and VDS2 amplified the expected fragment of DNA from 62 V. dahliae isolates from diverse hosts and geographical origins, but not from any other sources of DNA tested, including DNA from the most closely related species, V. albo-atrum. Southern blot analysis showed that the PCR product specifically hybridized to V, dahliae genomic DNA. An internal control was constructed for competitive PCR and used to develop a detection and quantification assay for V. dahliae, for which the detection limit was determined.

引用

页码：1361 / 1368

页数：8

共 24 条

[1]

Bej A K, 1992, PCR Methods Appl, V1, P151

[2]

Clementi M, 1993, PCR Methods Appl, V2, P191

[3]

COEN DM, 1988, CURRENT PROTOCOLS MO

[4] PCR JUMPING IN CLONES OF 30-MILLION-YEAR-OLD DNA FRAGMENTS FROM AMBER PRESERVED TERMITES (MASTOTERMES-ELECTRODOMINICUS) [J].

DESALLE, R ;

BARCIA, M ;

WRAY, C .

EXPERIENTIA, 1993, 49 (10) :906-909

[5] DETECTION OF POLYCHLORINATED BIPHENYL DEGRADATION GENES IN POLLUTED SEDIMENTS BY DIRECT DNA EXTRACTION AND POLYMERASE CHAIN-REACTION [J].

ERB, RW ;

WAGNERDOBLER, I .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1993, 59 (12) :4065-4073

[6] QUANTITATIVE RELATIONSHIP BETWEEN NAPHTHALENE CATABOLIC GENE-FREQUENCY AND EXPRESSION IN PREDICTING PAH DEGRADATION IN SOILS AT TOWN GAS MANUFACTURING SITES [J].

FLEMING, JT ;

SANSEVERINO, J ;

SAYLER, GS .

ENVIRONMENTAL SCIENCE & TECHNOLOGY, 1993, 27 (06) :1068-1074

[7] QUANTIFICATION OF VERTICILLIUM BIOMASS IN WILT DISEASE DEVELOPMENT [J].

HU, X ;

NAZAR, RN ;

ROBB, J .

PHYSIOLOGICAL AND MOLECULAR PLANT PATHOLOGY, 1993, 42 (01) :23-36

[8]

LESLIE JF, 1991, PHYTOPATHOLOGY, V81, P1058

[9]

Li K.-N., 1993, Phytopathology, V83, P1370

[10] PCR PRIMERS THAT ALLOW INTERGENERIC DIFFERENTIATION OF ASCOMYCETES AND THEIR APPLICATION TO VERTICILLIUM SPP [J].

LI, KN ;

ROUSE, DI ;

GERMAN, TL .

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 1994, 60 (12) :4324-4331

← 1 2 3 →