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Using substantial interspecific differences between the second internal transcribed spacer (ITS2) region within the rDNA gene of Dictyocaulus eckerti and Dictyocaulus viviparus a species-specific PCR was developed to distinguish between lungworm larvae of the two species from fallow deer and cattle. It was found that the method of DNA extraction was crucial for the sensitivity of the PCR. With serial dilutions of DNA extracted from 10,000 larvae the ITS2 fragment could be amplified from all dilutions down to a calculated amount of DNA equivalent to one larva. Using lower numbers of larvae, DNA from at least 100 larvae was necessary for a successful amplification. From this extraction a species-specific polymerase chain reaction (PCR) product was generated with a calculated amount of DNA equivalent to 33 larvae, whereas amplification of further diluted DNA was not successful. However, in a direct PCR single larvae could be detected after direct PCR amplification without preceding DNA extraction.