Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

Accurate identification of mycobacterial species and subspecies is essential to evaluate their significance and to perform epidemiological studies. The subspecies ofMycobacterium avium have different attributes but coincide in their zoonotic potential. Our knowledge aboutM. avium subsp.silvaticum is limited, since its identification is uncertain.Mycobacterium avium subsp.avium andM. avium subsp.silvaticum can be discriminated from each other based only on phenotypic characteristics, as they have almost identical genome sequences. Here we describe the development of a diagnostic method which enables the molecular identification ofM. avium subsp.silvaticum and discrimination fromM. avium subsp.avium based on genomic differences in a duplex high-resolution melt andM. avium subsp.silvaticum -specific mismatch real-time PCR. The developed assay was tested on reference strains and 199 field isolates, which were analyzed by phenotypic methods previously. This assay not only identified all 63M. avium subsp.silvaticum and 138M. avium subsp.avium strains correctly but also enabled the detection of mixedM. avium subsp.avium -M. avium subsp.silvaticum cultures. This is the first time that such a large panel of strains has been analyzed, and we also report the first isolation ofM. avium subsp.silvaticum from red fox, red deer, wild boar, cattle, and badger. This assay is reliable, rapid, simple, inexpensive, and robust. It eliminates the long-existing problem of ambiguous phenotypic identification and opens up the possibility for detailed and comprehensive strain studies.