Mycobacterium avium subsp. paratuberculosis detected and quantified using different DNA extraction and real-time amplification methods in artificially inoculated fecal samples from cattle

摘要: The aim of this study was to identify the best combinations DNA extraction and real-time amplification methods available using different genomic targets for a sensitive specific PCR detection Mycobacterium avium subsp. paratuberculosis (Map). Homogenized fecal samples from paratuberculosis-free cow were spiked with 10 107 bacteria per gram as assessed by direct microscopic count in Neubauer Improved chamber two Map cultures (K10 reference strain 764 field isolate). Serial dilutions plated assess viable cells inocula. Six commercially kits employed on triplicate each level inoculation both strains. extracts all submitted triplex assays an internal control rule out inhibition. Two included their own method that used respective kit only. these multi-copy insertion elements IS900, ISMav2, ISMAP02 single copy element F57. In addition, quantitative based sequence F57 quantify mycobacterial present extracts. Culture inoculated feces compare isolation sensitivities is also progress. have been discarded because low efficiency. appeared highest sensitivity according quantification results. showed excellent correlation between estimated number copies actual cell concentration. Most reliably detected 10,000 counts or, 1,240 CFUs 223.75 plate