Rapid molecular species identification of indigenous bats from Germany for surveillance purposes

Chiroptera form the second largest order of mammals and compromise >1200 species, of which only 51 species are abundant in Europe. As bats are important hosts involved in the emergence and spread of zoonotic infections, it is becoming more important to discriminate the different species of bats involved in the maintenance of causative agents. However, traditional taxonomic methods rely on morphological features and are challenging as they require long-lasting experience of an investigator and sometimes fail if the specimen is of poor condition. On the other hand, barcoding requires sequencing and is time consuming. Therefore, a versatile genetic approach for rapid species identification would be valuable. In this study, two mitochondrial loci, cytochrome b (cyt b) and cytochrome c oxidase subunit I (COI) were selected for the development of two multiplex qPCRs for differentiating four very abundant bat species in Germany using DNA extracted from the patagium or organ pools. Verification of the developed assays using a set of 1000 individual bat samples belonging to 20 different European species clearly showed that the multiplex qPCRs were able to determine the four most abundant species in this collection by a COI based qPCR. All other bat species which could not be covered by this approach could be easily identified by sequencing of the amplicon generated by broad-range qPCRs for cyt B and COI, respectively. Moreover, the double-check approach with cyt B and COI makes the identification of bats into species more reliable. The new multiplex PCRs allow a fast and easy genotyping of German bats and could be useful for screening approaches.