Development and Application of a Real Time PCR Detection Kit Specific for Mycobactium paratuberculosis

Abstract:

The real time PCR method based on TaqMan fluorescent DNA probe that specific for Mycobacterium paratuberculosis detection was established and developed into testing kit for rapid clinical diagnosis. The kit provided reagents for real time PCR and DNA extraction. The whole detection procedure included sample treatment and real time amplification could complete within 1 d. The testing kit could specifically identify 8 reference strains of M. paratuberculosis among various environmental existing microorganisms and 12 strains of other mycobacteria including M. tuberculosis, M. bovis and M. avium. Tests on M. paratuberculosis culture samples and serial ten fold dilution of recombinant plasmid containing target template showed that, the assay could detect single bacterium and 15 copies of target gene, which was 100 fold increase of sensitivity than the gel-based PCR using primers of the same sequences. And the test on 20 mimic infected milk sample containing 50~100 bacteria of M. paratuberculosis all yielded positive results. The inter-assay CV% and intra-assay CV% of the kit was 1.41%, 2.42% respectively. We used the testing kit to investigate infection of M. paratuberculosis on domestic and imported animals. Nature samples including 250 fecal and milk samples from 7 cattle farms, 143 serum samples from 10 pig farms within Guangdong province, and 100 serum samples from three shipments of imported monkey, were collected and tested. The positive rate of M. paratuberculosis was 7.7% in cattle serum, 3.7% in cattle fecal, 8.2% in pig serum and 3.0% in monkey serum respectively.

Key words:M. paratuberculosis, Real time PCR, Testing kits, Animal infection investigation