Isolation, PCR identification and real-time quantification of M. paratuberculosis

摘要

Sensitivity and specificity are of principal concern when testing for MAP (MAP). Inhibitors or a surplus of DNA in a sample can be deleterious using PCR methods because sensitivity is reduced or perhaps false negative results may arise. If these difficulties were removed would detection of MAP be more frequent? If the total assay time is very short, and if the isolation and detection process could be automated, wouldn't that be welcomed? Our solution to these issues is using immunomagnetic separation (IMS) whereby ferromagnetic beads bind the bacteria within 30 minutes. During washing the bacteria are permeabilized and the nucleic acids reside on the beads. Further washing removes non-specifically bound inhibitors and finally PCR-grade DNA is eluted from the beads. A revolutionary asymmetric real-time PCR using sequence specific primers for the amplification of MAP sequences and an internal control with monochromal multiplexing at SYBR Green wavelength allows the quantification of MAP sequences and the detection of the internal control with economical real-time cyclers. If a real-time cycler is not available or higher throughput is needed, the amplicons of product and internal control can be detected with a hybridization detection kit as a real-time read-out with hybridization sensitive to as low as 0,3 fg of MAP-DNA. Switching from conventional PCR to real-time PCR is thus made easy as the same amplification protocol can be used on all cyclers.