A COMBINED BIOLOGICAL AND ENZYMATIC AMPLIFICATION (BIO-PCR) TECHNIQUETO DETECT PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA IN BEAN SEED EXTRACTS

A polymerase chain reaction (PCR) method, hereafter referred to as BIO -PCR, that combines biological and enzymatic amplification of PCR targ ets and simplified procedures for sample processing is described for t he detection of Pseudomonas syringae pv. phaseolicola in bean seed ext racts. Seeds are soaked overnight following standard protocols and ali quots of the extracts are plated onto a general agar medium. After 45- 48 hr of incubation, the plates are washed with water to remove bacter ial cells and aliquots of a pooled washing are subjected to two consec utive rounds of PCR, without prior DNA extraction, using ''nested'' pa irs of primers designed to amplify a segment of the organism's tox (ph aseolotoxin) gene region. Positive detection was reproducibly obtained at near-limit concentrations of the pathogen in the seed wash. Advant ages of BIO-PCR over existing PCR techniques include the elimination o f false positives resulting from the presence of dead cells that may b e present in the seed, elimination of false negatives due to potential PCR inhibitors in seed extracts, increased sensitivity of detection, and no need for DNA extraction prior to amplification. Accordingly, BI O-PCR should prove useful for routine detection of other bacterial pat hogens of quarantine importance. The primers detected some nontoxigeni c strains of the pathogen, which evidently contain part of or the enti re tox gene cluster.