High Resolution Melting Analysis coupled to real time PCR for detection and quantification of Nosema ceranae and Nosema apis in honey bees

Nosemosis is a gut disease of honey bee, Apis mellifera, caused by the microsporidia Nosema apis and N. ceranae. These two species are morphologically similar but differ in epidemiological pattern and virulence. Proper control strategies of the infection by beekeepers rely on correct species differentiation. N. apis and N. ceranae spores are not easily distinguishable under microscopic examination, requiring molecular analysis for species identification. High Resolution Melting Analysis (HRMA) is a molecular technique allowing single-nucleotide discrimination that is increasingly used in diagnostic microbiology and parasitology for species identification and genotyping (Reed GH et al., 2007, Pharmacogenomics 8:597\u2013608). AIM: This work aimed to design a qualitative and quantitative assay for the simultaneous detection and discrimination of N. ceranae and N. apis infection in honey bees, based on HRMA coupled to quantitative real time PCR (qPCR). MATERIALS AND METHODS: Samples of 25-30 adult honeybees as a balanced pool of individuals from each of 5-10 colonies were collected from apiaries during field investigation. Honey bee crushings underwent microscopic examination to assess the presence of Nosema spp. spores. For microscopically-positive samples, DNA extraction and Nosema DNA amplification through PCR for species identification were performed using standard protocols (Martin-Hernandez R et al., 2007, Appl Environ Microbiol 73:6331-6338). The primer pair for qPCR-HRMA amplification was designed in a region of Nosema 16S rRNA gene, where primer annealing sequences are conserved whereas the interposed sequences shows no intraspecific variability but interspecies variability at different positions. The assay protocol included a real time PCR reaction, a subsequent DNA high resolution melting process, and a normalization step for data analysis. The analysis was performed on the EcoTM Real-Time PCR System (Illumina, Inc., San Diego, CA, USA). The obtained amplicons were sequenced and similarity analysis was performed by using BLAST database search and GenBank. N.ceranae and N. apis amplicons were cloned into plasmids, and proper plasmid diluitions were prepared to serve as external reference curves for qPCR quantitation. RESULTS: each honey bee sample positive to microscopical evaluation was also positive to real time PCR-HRMA even after proper diluition with high amplification efficiency and sensitivity. The analysis of high resolution melting curves allowed clear discrimination of the two Nosema species, and mixed infection, according to the different melting temperature of the corresponding amplicons. CONCLUSIONS: This work validated a new qPCR-HRMA protocol assay for infection assessment of the two Nosema species affecting honey bees. This method could be particularly suitable for routine diagnostics applied to field investigation, as a quick and sensitive single step protocol using an unique primer pair and saturating dye without sequence specific probes. This allows a shorter analysis time, a reduced cost, and a comparable amplification efficiency for both targets, especially useful in case of simultaneous infection with the two Nosema species. This study was supported by Regione Lombardia, STRANOVA projec