A multiplex PCR for the diagnostic detection of Coxiella burnetti in cow'smilk

A multiplex PCR based assay was developed for the highly sensitive and spec ific detection of Coxiella (C.) burnetii in cow's milk. The assay simultane ously amplifies a diagnostic target within the C. burnetii IS1111 sequence and a control target within the bovine CD18 gene. The internal PCR amplific ation control allows the discrimination of false negative results (single t ube reaction failures) from negative results due to true absence of target sequences. In order to maximize the sensitivity of the assay, a sample prep aration method including a centrifugation step to concentrate the bacterium was developed. In milk samples artificially contaminated with serial dilut ions of C. burnetii, about four particles per mi could reproducibly be dete cted. The sensitivities of both assays, multiplex PCR and PCR with only a s ingle pair of primers ('simplex' PCR), were observed to be similar.