| Summary: | 碩士 === 輔仁大學 === 生物學系 === 84 === The objective of this research was to develop a sensitive,
specific, and rapid DNA-based method for detection of
Pseudomonas solanacearum. Random amplified olymorphic DNA (RAPD)
technique was used as a first step to evaluate polymorphism
among different Psedomonas spp. A specific 0.7-kb DNA fragment
amplified from genomic DNA of P. solanacearum using primer R-424
was cloned. This 0.7-kb DNA fragment showed specificity to P.
solanacearum determined by Southern hybridization against total
DNA of different Pseudomoas spp. A 2.7-kb EcoRI fragment
containing the 0.7-kb fragment was also hybridized only to P.
solanacearum but not to other Pseudomonas spp., pathovars of
Xanthomonas campestris, Erwinia spp., and tested soil
saprophytic bacteria. Three sets of oligonucleotide primers were
designed based on the DNA sequences of 2.7-kb DNA fragment.
These primer sets only amplify specific DNA fragments from P.
solanacearum, and one set of primers can detect P. solanacearum
as low as 10 c.f.u/ml in polymerase chain reaction(PCR).The PCR
assay was used to detect P. solanacearum in soil and plant
tissue samples using these primer sets. No PCR product could be
found when soil extract with P. solanacearum was used directly
in the assay. DNA extraction from soil was needed for successful
PCR assay. One step method for DNA extraction from soil for PCR
assay was developed and can fastened the detection of P.
solanacearum in soil. The sensitivity of the PCR assay for soil
samples was between 7.5x104~106 c.f.u/ml so far.At least one
open reading frame was found in this 2.7-kb DNA fragment but the
function of this frame was not known. Restriction length
polymorphism (RFLP) was found in Southern hybridization using
2.7-kb fragment as a probe against strains of P. solanacearum
isolated from different host plants. There were more variations
among the strains isolatH3 from tomato.
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