Development of an immunocapture-polymerase chain reaction assay using IgY to detect Mycobacterium avium subsp. paratuberculosis.

A diagnostic assay using immunomagnetic separation was developed to capture Mycobacterium avium subsp. paratuberculosis (MAP) from bovine feces by means of IgY derived from chicken eggs. The antibody was coupled directly onto the surface of MagaCell cellulose/iron oxide beads or indirectly by being mixed with MagaBeads and a rabbit IgG linker against chicken antigen. Optimization parameters for the immunocapture included incubation time, temperature, volume, and type of immunocapture beads. Analytical sensitivity and specificity were determined by extracting DNA from the captured bacteria and amplifying it by polymerase chain reaction (PCR). The 2 bead preparations had the same analytical sensitivity, and the detection level of MAP cells in spiked bovine feces was 2 x 10(4) cells/g. No PCR inhibition was observed with DNA from the organisms captured with use of the MagaCell-IgY beads.

Immunological response to Mycobacterium avium subsp. paratuberculosis in chickens.

Enzyme-linked immunosorbent assay (ELISA) and culture are 2 common diagnostic tests for detecting Mycobacterium avium subsp. paratuberculosis (Map) in Johne's disease, but they are not as sensitive as polymerase chain reaction (PCR). However, inhibitors can coextract with the target DNA and cause interference in PCR. Development of an immune capture assay followed by PCR amplification can alleviate this problem. In this study, we were able to induce an immune response in chickens using heat or formalin inactivated Map. The purified immunoglobulin (Ig)Y has a molecular weight of 160 kDa. The titers were at 1:6400 and 1:12 800 at weeks 5 to 6 and 8 to 9, respectively, as determined by the IDEXX modified ELISA kit for Johne's disease. The IgY produced from inactivated bacterial cells had no effect on its ability to recognize live Map cells as illustrated by immunofluorescence assay and immune capture PCR results.

Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction.

Polymerase chain reaction (PCR) has been widely used due to its high specificity, sensitivity, and rapid turn-around time. However, inhibitory factors may be co-extracted with the target nucleic acid that will hinder the performance of PCR. In this study, DNA extraction methods for Mycobacterium avium subsp. paratuberculosis were evaluated including rapid lysis, organic extraction, silica-based and magnetic particle-based (MagaZorb) technologies on bacterial cells, and spiked bovine feces. Efficiency of the extraction was determined by PCR end point titration with primers targeting the insertion sequence, IS900. Results of the end point titrations are identical for bacterial cells and spiked feces. Inhibition was observed in PCR with DNA isolated from spiked feces, and a 1/100 dilution was able to alleviate this problem with DNA extracted by MagaZorb. A 1/1000 dilution was required for the other three methods. MagaZorb proved to be more efficient at removing inhibitory factors and required the least labor and completion time. Further evaluation is required for its utilization in other clinical specimens.