A HIGH THROUGHPUT MEMBRANCE BIO-PCR TECHNIQUE FROM ULTRA SENSITIVE DETECTION OF PSEUDOMONAS SYRINGAE PV. PHASEOLICOLA

Authors: Schaad, Norman; BERTHIER-SCHAAD, YVETTE - JOHNS HOPKINS UNIVERSITY; KNORR, DAVID - VELOCITY II

Submitted to: Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2007
Publication Date: 1/20/2007
Citation: Schaad, N.W., Berthier-Schaad, Y., Knorr, D. 2007. A high throughput membrance bio-pcr technique from ultra sensitive detection of pseudomonas syringae pv. phaseolicola. Plant Pathology. 56:1-8.

Interpretive Summary: Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of beans, is a regulated plant pathogen. We describe an ultra sensitive high throughput polymerase chair reaction (PCR)-based method for detecting the pathogen in seed and leaf washes even in the presence of numerous other organisms. One ml washings of seeds or leaves are filtered under vacuum in wells of a 96 well plate fitted with a membrane. The plate is incubated on a semi-selective agar medium to allow growth of the target organism (BIO-PCR) and well washes assessed using direct (no extraction of DNA) classical or real-time PCR. This 96 well plate membrane BIO-PCR technique requires less labor than the standard BIO-PCR and is at least 10 fold more sensitive.

Technical Abstract: Molecular-based methods, such as PCR, have greatly improved detection of bacteria in environmental samples. However, sensitivity of classical PCR is not high and inhibitors from plants are often a problem. BIO-PCR can increase the sensitivity of classical PCR by over 100% and reduce the effects of inhibitors. To further increase sensitivity and also reduce labor needed for BIO-PCR, we describe a high throughput 96 well membrane BIO-PCR technique for ultra-sensitive detection of Pseudomonas syringae pv. phaseolicola (PSP) in washings of seeds and leaves. Samples (1.2 ml) are filtered under vacuum in triplicate wells of each of two 96 well plates with special detergent-free membranes. After incubating on semi-selective soft agar medium for 48-52 h, each well is washed with 200ul of sterile water and used immediately for classical nested PCR or real-time PCR or stored at minus 20C. Results of assaying spiked seed washings showed that classical direct PCR was unable to detect PSP at concentrations of 40 colony forming units (cfu) per ml. BIO-PCR detected 5/6 samples at 40 cfu/ml but none at concentrations of 4.2 and 0.4 cfu/ml. In contrast, membrane BIO-PCR was able to detect 6/6 samples containing as few as 0.4 cfu/ml. Results with leaf washings and real-time PCR were similar; direct classical PCR and BIO-PCR failed to detect washings containing 80 cfu/ml whereas 3/3 and 1/3 samples containing 80 and 40 cfu/ml, respectively, were positive by membrane BIO-PCR.