An efficient DNA extraction method for polymerase chain reaction-based detection of Mycobacterium avium subspecies paratuberculosis in bovine fecal samples.

摘要

Due to the lipid rich cell wall of Mycobacterium avium subspecies paratuberculosis (MAP), the complex nature of bovine feces, and intermittent organism shedding by infected cattle, it is difficult to recover a sufficient amount of high-quality MAP DNA from fecal samples, directly affecting the sensitivity of downstream polymerase chain reaction (PCR) tests. In the current study, a DNA extraction method, designated the Mississippi Veterinary Research and Diagnostic Laboratory (MVRDL) method, was developed for PCR-based detection of MAP in bovine fecal samples. The MVRDL method combined multiple procedures, including chemical pretreatment, 1-tube cell lysis and extraction, chelex matrix absorption, and mini-column purification. The DNA yield and purity, as measured by spectrophotometry, was 3.36 fg per colony forming unit (CFU) MAP and A260/280 absorbance ratio of 2, respectively. This method was further evaluated by real-time PCR. A linear correlation was found between cycle-threshold (Ct) and log input CFU (ranging from 7.2 to 7.2 x 10(7) CFU per ml or CFU per g). The detection limit of the real-time PCR assay was 3 CFU per ml of MAP culture or per g of MAP-spiked feces. In addition, the MVRDL method was validated by performing 7 Johne's direct fecal PCR proficiency tests administered by the National Veterinary Service Laboratories. Based on culture results as the "gold standard," the specificity of MVRDL PCR was 100%, and the sensitivity was 98.46% for samples containing more than 1.5 CFU per tube of fecal cultures. To the authors' knowledge, this is the most efficient MAP DNA extraction method in comparison with all previously published protocols.