A Combined Biological and Enzymatic Amplification (BIO-PCR) Technique to DetectPseudomonas syringaepv.phaseolicolain Bean Seed Extracts

Abstract

A polymerase chain reaction (PCR) method, hereafter referred to as BIO-PCR, that combines biological and enzymatic amplification of PCR targets and simplified procedures for sample processing is described for the detection of Pseudomonas syringae pv. phaseolicola in bean seed extracts. Seeds are soaked overnight following standard protocols and aliquots of the extracts are plated onto a general agar medium. After 45-48 hr of incubation, the plates are washed with water to remove bacterial cells and aliquots of a pooled washing are subjected to two consecutive rounds of PCR, without prior DNA extraction, using «nested» pairs of primers designed to amplify a segment of the organism's tox (phaseolotoxin) gene region. Positive detection was reproducibly obtained at near-limit concentrations of the pathogen in the seed wash. Advantages of BIO-PCR over existing PCR techniques include the elimination of false positives resulting from the presence of dead cells that may be 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, BIO-PCR should prove useful for routine detection of other bacterial pathogens of quarantine importance. The primers detected some nontoxigenic strains of the pathogen, which evidently contain part of or the entire tox gene cluster