Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by the polymerase chain reaction.
AUTOR(ES)
Seal, S E
RESUMO
A subtraction hybridization technique was employed to make a library enriched for Pseudomonas solanacearum-specific sequences. One cloned fragment, PS2096, hybridized under stringent conditions to DNA of 82 P. solanacearum strains representing all subgroups of the species. Other plant-associated bacteria, including closely related species such as Pseudomonas capacia, Pseudomonas picketti, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10(5) and 4 x 10(6) P. solanacearum cells could routinely be detected with PS2096 labelled either with [32P]dCTP or with digoxigenin-11-dUTP. To improve the sensitivity of detection, PS2096 was sequenced to allow the construction of specific oligonucleotide primers to be used for polymerase chain reaction (PCR) amplification. After 50 cycles of amplification, 5 to 116 cells, depending on the strain, could reproducibly be detected by visualization of a 148-bp PCR product on an agarose gel. A preliminary field trial in Burundi with the probe and PCR primers has confirmed that they are sensitive tools for specifically detecting low-level infections of P. solanacearum in potato tubers.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=183169Documentos Relacionados
- Isolation and characterization of a species-specific DNA fragment for detection of Candida albicans by polymerase chain reaction.
- Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis.
- Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.
- Construction of a DNA probe and detection of Actinobacillus pleuropneumoniae by using polymerase chain reaction.
- A species-specific DNA probe for the detection of Mycoplasma gallisepticum.