high throughput membrane BIO-PCR technique for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola

Molecular-based methods such as PCR have greatly improved detection of bacteria in environmental samples. However, the sensitivity of PCR is not high when compared to agar plating assays, and inhibitors from plants are often a problem. Pre-enriching bacteria on agar media (BIO-PCR) can increase the sensitivity of PCR by more than 100% and reduce the effects of inhibitors. To further increase the sensitivity and also reduce the labour needed for BIO-PCR, a high throughput 96-well membrane BIO-PCR technique is described for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola (PSP) (syn. P. phaseolicola) in washings of seeds and leaves of Phaseolus vulgaris, using available classical PCR primers and newly designed real-time primers and probe. The primers and probe, designed from a tox-argK chromosomal cluster of the PSP-specific phaseolotoxin gene, were confirmed to be specific to PSP. Samples (1·2 mL) were filtered under vacuum in 96-well membrane plates. After incubating on soft agar medium for 48-52 h, each well is washed with 200 μL of sterile water and used immediately for nested (two-step) PCR or real-time PCR or stored at -20°C. Results of assaying spiked seed washings showed that classical PCR was unable to detect PSP at mean concentrations of 40 colony forming units (cfu) mL⁻¹. BIO-PCR detected PSP in five out of six samples at 40 mean cfu mL⁻¹ but none at mean concentrations of 4·2 and 0·4 mean cfu mL⁻¹. In contrast, membrane BIO-PCR detected the bacterium in all six samples tested containing as few as 0·4 mean cfu mL⁻¹. The sensitivity of detection from leaf washings was lower but the results were similar, classical and BIO-PCR were negative from all three levels of inoculum while membrane BIO-PCR detected three out of three samples at 80 mean cfu mL⁻¹ and one out of three at 40 mean cfu mL⁻¹.