Lj. Hyman et al., IMPROVED PCR DETECTION SENSITIVITY OF ERWINIA-CAROTOVORA SUBSP ATROSEPTICA IN POTATO-TUBER PEEL EXTRACT BY PRIOR ENRICHMENT ON A SELECTIVE MEDIUM, Letters in applied microbiology, 25(2), 1997, pp. 143-147
A simple and sensitive method was developed to replace the need for co
mplex and laborious DNA extraction to remove inhibitory substances in
potato tuber peel extract before detection of Erwinia carotovora subsp
. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10(5) when
peel extract was inoculated onto a selective medium, CVP, and incubat
ed at 27 degrees C for 24 h. Bacterial micro-colonies which developed
were suspended in 500 mu l of water and the bacteria diluted in water
100-fold, or 10-fold followed by washing by centrifugation, before PCR
testing. The sensitivity of detection obtained with the former was ca
10(1)-10(2) cells ml(-1) and with the latter ca 10(1) cells ml(-1), w
hen different numbers of streptomycin-resistant Eca strain were added
to peel extract from three Eca-free potato cultivars. The method was v
alidated and the sensitivity confirmed relative to two different commo
nly used Eca detection methods using naturally contaminated tubers.