Comparison of PCR, ELISA and DNA hybridization for the detection of Clavibacter michiganensis subsp. sepedonicus
Comparison studies were carried out in USA on the use of PCR, ELISA and DNA hybridization to detect Clavibacter michiganensis subsp. sepedonicus (EPPO A2 quarantine pest) in crude extracts of field-grown potatoes. The PCR method applied uses a synthetic oligomer which amplifies a fragment of the bacterial plasmid pCS1. In pure bacterial cultures, this PCR method can detect all strains of C. m. subsp. sepedonicus and has a sensitivity of ; 10 CFU. The field material tested corresponded to sections of potato stems that were grown in New York and North Dakota from seed pieces of potato cultivars BelRus (tolerant) and Russet Burbank (susceptible) inoculated with 0, 102 or 109 CFU of the bacterium and destructively sampled at 35 and 90 days after planting. As overall results, 36.2, 35.8 and 29.1 % of ;inoculated samples tested positive by PCR, ELISA and DNA hybridization, respectively. The experimental design in the field allow for the evaluation of these tests over a wide range of conditions which can be faced in practice, including a 'best case scenario' (high inoculum, susceptible cultivar, late sampling date) and a 'worse case scenario' (low inoculum, tolerant cultivar, early sampling date). In this study, each test was significantly affected by these three factors (inoculum level, cultivar susceptibility and sampling date). When these factors were taken into account, the general trend for greater frequency of detection with PCR than with the other tests remained. In the best case scenario, C. m. subsp. sepedonicus was detected in samples to a level approaching 100 %. However, in the worst case scenario, detection was low and, in many cases, the bacterium was not detected at all. The authors pointed out that these low rates of detection are of concern but that it is unclear whether the negative results represented an inability to detect low levels of bacteria or they resulted from a lack of infection following inoculation. None of the control plants (inoculated with buffer only) gave positive result with either PCR or DNA hybridization, whereas ELISA results were highly dependant upon the positive-negative threshold used.
Slack, S.A.; Drennan, J.L.; Westra, A.A.G.; Gudmestad, N.C.; Oleson, A.E. (1996) Comparison of PCR, ELISA, and DNA hybridization for the detection of Clavibacter michiganensis subsp. sepedonicus in field-grown potatoes.
Plant Disease, 80(5), 519-524.