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

Abstract

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 cepacia, Pseudomonas pickettii, or Pseudomonas syzygii, did not hybridize to PS2096. A minimum number of between 4 x 10i and 4 x 10' 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 Burndi 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.

PCR-based detection of Xanthomonas campestris pv. phaseoli var. fuscans in plant material and its differentiation from X. c. pv. phaseoli

“…The detection limits for both sets of primers are in good agreement with those determined for other PCR-based detection systems for plant pathogenic bacteria (Seal et al 1992;De Boer and Ward 1995;Leite et al 1995;Smid et al 1995;Hartung et al 1996;Sulzinski et al 1996;Pan et al 1997;Zhang and Goodwin 1997), although some workers have reported even greater sensitivity (Seal et al 1993;Maes et al 1996). Detection limits may be further improved by the method of Hyman et al (1997) using prior enrichment of the organism on selective medium.…”

Analysis of a VMP-like sequence (vls) locus in Borrelia garinii and Vls homologues among four Borrelia burgdorferi sensu lato species

“…A 10-kb HindIII fragment containing DNA homologous to the vls locus of B. burgdorferi B31 was identi¢ed in B. garinii A87SA by genome subtractive hybridization [16]. Brie£y, DNA from strain A87SA (tester) was partially digested with Sau3A and hybridized to DNA from strain A87SB (driver) which had been sheared by ultrasonication [17]. The reassociated DNA mixture was ligated into a vector and resulting clones were tested for their reactivity with strains A87SA and A87SB.…”

Detection, Identification and Phylogenetic Analysis of Indian Ralstonia solanacearum Isolates by Comparison of Partial hrpB Gene Sequences

“…Gene-based methods have proved very efficient to rapidly identify and detect the pathogens with high sensitivity and specificity. Designing and development of specific primers for identification and detection of R. solanacearum have already been reported by many workers (Seal et al 1992;Opina et al 1997;Boudazin et al 1999;Weller et al 2000;Wolf 2000;Schonfeld et al 2003). Most of the existing primers show positive signals to closely related species instead of specific detection of R. solanacearum.…”