We applied quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) on DNA isolates from soft abdominal cuticle of 460 North American crayfish Orconectes limosus and Pacifastacus leniusculus, previously tested for Aphanomyces astaci presence by conventional semi-nested PCR. Both approaches target the internal transcribed spacers of the pathogen nuclear ribosomal DNA, but apply different specific sequence motifs and technologies. The real-time PCR approach seems to provide higher sensitivity; the number of crayfish that tested positive increased from 23 to 32%, and 10 additional crayfish populations were indicated as hosting the disease agent. However, the vast majority of newly recorded positives contained very low agent levels, from 5 to 50 PCR-forming units. An isolate producing a false positive result by the semi-nested PCR (apparently undescribed Aphanomyces related to A. astaci) remained negative using the real-time PCR. The present study shows that previous results based on the semi-nested PCR were not substantially influenced by false positives but might have suffered from some false negatives at low agent levels. Combining alternative methods may therefore provide more reliable conclusions on the pathogen's presence. Further, we found positive correlation between the prevalence of infection carriers in American crayfish populations and the average amounts of A. astaci DNA detected in infected local crayfish individuals.

Department of Ecology Faculty of Science Charles University Prague 12844 Prague 2 Czech Republic

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