Introduction.

The separation of PCR-product using electrophoresis and ethidium bromide staining is the most usual method for detection of PCR-products. However, in the cases of low amount of PCR-product this method is not sensitive enough. In the article, the method of PCR-ELISA that can be used in the molecular diagnostics of paratuberculosis in order to increase the sensitivity is described.

Material and methods.

Suspensions of Mycobacterium avium subsp. paratuberculosis in 10 different concentrations were added to 10 faecal paratuberculosis-negative samples to compare the sensitivities of different methods: culture method, PCR with detection of PCR-products with electrophoresis and PCR-ELISA. For comparison of sensitivity of PCR with detection of PCR-product whit electrophoresis, nested PCR, dot-blot hybridisation and PCR-ELISA 20 samples of Mycobacterium avium subsp. paratuberculosis DNA isolated from intestines and lymph nodes tissue of serologically positive sheep and goats were used. For the PCR-ELISA procedure we have designed the 'ELISA 900' DNA-probe (5' CTC CGT AAC CGT CAT TGT CCA GAT CAA CCC AGC 3').

Results and discussion.

We have observed the lowest sensitivity in the culture test method, which was 20% positive when containing 300 bacteria/ml. The most sensitive method was PCR-ELISA method where 10% of samples with 6 bacteria/ml were positive. The comparison of the sensitivity of PCR with electrophoresis and ethidium bromide staining, nested PCR, dot-blot hybridisation and PCR-ELISA we have obtained the following results: in PCR 40% samples were positive, in dot-blot hybridisation 45%, in nested-PCR 50% and in PCR-ELISA 65% samples were positive.

Conclusions.

PCR-ELISA is highly sensitive and specific method for detection of M. avium subsp. paratuberculosis DNA in faecal and tissue samples. The method enables the processing of the numerous samples and automation of the procedure.