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Detection of Neospora caninum DNA by the polymerase chain reaction

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Abstract

Neospora caninum is a cyst-forming coccidian parasite which is now recognised as a major cause of abortion and neonatal mortality in cattle and other livestock. This study describes the primary DNA structure of the transcribed spacer region of the rDNA of N. caninum. Of importance is that the sequence data generated have been used to develop a species-specific PCR test for N. caninum DNA, which will prove valuable in epidemiology studies on neosporosis.

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    Extracted DNA from all samples were resuspended in a final volume of 200 μL (Qiagen QIAamp) or 50 μL (MP – FastDNA Soil) and tested by PCR for the presence of N. caninum DNA using N. caninum-specific primers, Np6plus (5′-CTC GCC AGT CAA CCT ACG TCT TCT-3′) and Np21plus (5′-CCC AGT GCG TCC AAT CCT GTA AC-3′) based on the Nc5 region (Müller et al., 1996). Moreover, internal transcribed spacer 1 of the rDNA (ITS1) was amplified using JS4 (5′-CGA AAT GGG AAG TTT TGT GAA C-3′) and Tim11 (5′-CAC TGA AAC AGA CGT ACC-3′) (Payne and Ellis, 1996; Šlapeta et al., 2002a). Both regions were amplified using 2×MasterMix (Fermentas, USA) or 2×EconoTaq PLUS GREEN MasterMix (Lucigen, USA).

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