3.3.8
PCR-ELISA FOR DETECTION OF DIDYMELLA BRYONIAE AND PHOMA SPP.

BM SOMAI 1, AP KEINATH 2 and RA DEAN 1

1Dept Plant Pathology, Clemson University, Clemson, SC 29634, USA; 2Coastal Research and Education Center, Clemson University, Charleston, SC 29414, USA

Background and objectives
Conventionally, agarose gel electrophoresis has been used for detection of PCR-amplified products. After electrophoresis, the gel has to be stained with ethidium bromide or silver and destained prior to viewing. Ethidium bromide is a carcinogen and silver staining results in high backgrounds. When a large number of samples has to be processed simultaneously, gel electrophoresis becomes labor intensive. PCR-ELISA is another colorimetric alternative that has been successfully used for detection of amplified DNA [1]. Therefore, the objective of this study was to develop an ELISA reaction for detecting PCR products specific to three fungal species Didymella bryoniae, Phoma medicaginis and P. exigua isolated from cucurbit plants with symptoms of gummy stem blight (GSB) [2].

Materials and methods
PCR primers specific to D. bryoniae, P. medicaginis and P. exigua were developed to enable rapid PCR detection and differentiation among these three groups of organisms. A fluorescein label and a biotin label were incorporated into the 5 end of the forward and reverse primers respectively. After amplification, PCR products were immobilized onto NeutrAvidin-coated microtiter plates. After incubation, horseradish peroxidase-conjugated anti-fluorescein antibody was added to the wells and incubated for a further 30 min. After washing off excess antibody, a color substrate was added to each well. The substrates were tetramethylbenzidine (TMB), O-phenylenediamine (OPD) and azino-bis-ethylbenzthiazoline sulfonic acid (ABTS) for detecting D. bryoniae, P. medicaginis and P. exigua respectively. After 15 min incubation the reactions were stopped either with 10% SDS (TMB and ABTS) or 3M sulfuric acid (OPD.

Results and conclusions
ELISA was used to successfully detect PCR products from all positive reactions. In the negative reactions primer-dimers formed during PCR contributed to high backgrounds. PCR conditions had to be adjusted to ensure minimal primer-dimer formation but this could not be totally eliminated. The best substrate was ABTS, because it had the lowest background. We used SDS as the termination reagent for the TMB and ABTS reactions because sulfuric acid precipitated the TMB substrate and falsely intensified the ABTS substrate over time. Overall, PCR-ELISA successfully and reproducibly detected PCR amplified products from 37 D. bryoniae, 15 P. medicaginis and 11 P. exigua isolates. Once conditions were optimized, no false positives or false negatives were obtained. All PCR-ELISA reactions had signal to noise ratios of at least 2:1 but were often about 6-7:1. Furthermore, PCR-ELISA was capable of detecting fungal DNA from crude plant lesion extracts. These results indicate that ELISA detection of PCR products is feasible. The method is easy, sensitive, makes use of well-established procedures and can be easily automated for multiple routine diagnostic assays.

Reference
1. Mutasa ES, Chwarszczynska DM, Asher, MJC, 1996. Phytopathology 86, 493-497.
2. Keinath AP, Farnham MW, Zitter TA, 1995. Phytopathology 85, 364-369.