Development of a qPCR assay to identify and differentiate insect-associated strains of the complex.

The complex contains important opportunistic pathogens of humans and vertebrate animals, as well as insects and other invertebrates. To date, the methods used for the identification of species within the genus , including PCR assays, have poor discriminatory power and may require further molecular typing or genomic sequence analysis to determine clinical relevance. We developed a duplex TaqMan probe-based quantitative real-time PCR (qPCR) assay targeting the gene, which is involved in chitin degradation and transport, and the gene, which is involved in urease production. This allowed us to simultaneously identify all members of the complex ( positive) and differentiate those most likely to act as insect pathogens ( and positive). We applied our assay to identify potentially entomopathogenic members of the complex in the context of a conservation program for the critically endangered insect and found it to be a useful aid for rapid and accurate detection of infection with complex strains in insects and determination of their clinical relevance. By targeting 2 genes likely to be virulence factors, this assay may also be of use for research investigating the pathogenesis of entomopathogenic spp.