Correlation between a commercial real-time PCR assay and Herrold's egg yolk medium culture for map in bovine faecal samples.

Disease control programmes for MAP rely on accurate and sensitive tools for the detection of infected animals. Culture based detection of MAP takes many weeks whereas PCR enables rapid detection. Several commercial and many user designed real-time PCR assays exist for the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in bovine faecal samples. We selected one commercial assay, the VetMAX TM MAP Real-Time PCR Screening Kit (Life Technologies), and calculated the correlation between real-time PCR threshold cycle (Ct) values and colony-forming units (CFU) on Herrold egg yolk medium (HEYM) culture, using different nucleic acid extraction kits. Results of HEYM culture of 40 faecal samples were negatively (inversely) correlated with their respective real-time PCR results. The Spearman's rank correlation between Ct and CFU ranged from good (0.67) to excellent (0.93), depending on which nucleic acid extraction kit was used. The MagMAX™ Total Nucleic Acid Isolation Kit (Life Technologies) and the InviMag ® Stool DNA Kit (Invitek) produced the best correlations with HEYM culture. These results suggest that this real-time PCR assay is a useful alternative to culture on HEYM. Introduction : Numerous studies have been conducted to evaluate the accuracy (sensitivity and specificity) of the tests available to detect MAP. 1 Culture on HEYM has for a long time been considered the gold standard test for MAP because it is highly specific. 1 However culture is a very slow process with results taking 6 weeks or more. Recent studies have shown that real time PCR is a highly accurate alternative to HEYM for the detection of MAP in bovine faeces. 2,3 The VetMAX™ MAP Real-Time PCR Screening Kit is a complete set of reagents for a simple real-time PCR assay. This assay is intended for use in the rapid, in vitro detection of MAP DNA purified from bovine feces. The assay targets a sequence element in the MAP genome to provide highly sensitive and specific results. This study looked at comparing the performance of different nucleic acid extraction kits when coupled with this screening kit to traditional HEYM culture for the detection of MAP in bovine faeces.