Development of a single tube nested polymerase chain reaction assay for the detection of Neospora caninum DNA

Sensitive detection techniques are required to study the life cycle of Neospora caninum and to diagnose infections. In this study, we describe the development of a PCR assay for N. caninum based on two successive amplification steps within a single tube. This technique, called single tube nested PCR, was sensitive to a single copy of target sequence, and able to amplify parasite DNA from biological specimens such as formalin-fixed, paraffin-embedded tissues of naturally infected dogs and cattle. An internal standard (or PCR MIMIC) is also described. This assay should prove useful in the study of the biology of N. caninum.