Optimization of duplex real-time PCR with melting-curve analysis for detecting the microsporidian parasites Nosema apis and Nosema ceranae in Apis mellifera

Abstract

Honey bees, Apis mellifera (L.) (Hymenoptera: Apidae), are parasitized by the microsporidians Nosema apis (Zander) and Nosema ceranae (Fries). Molecular techniques are commonly used to differentiate between these parasites because light microscopy is inadequate. Our objectives were to (i) adapt the previously published duplex polymerase chain reaction (PCR) targeting the 16S rRNA gene of N. apis (321APIS-FOR, 321APIS-REV) and N. ceranae (218MITOC-FOR, 218MITOC-REV) using qualitative real-time PCR assay with SYBR® Green I dye (R-T PCR) and DNA melting-curve analysis, and (ii) determine whether the two Nosema species can be detected simultaneously in honey bees. Total spore counts and purified total genomic DNA were obtained from 37 bee samples (19 individual workers and 18 pooled samples of 15 workers) collected in Nova Scotia, Prince Edward Island, and Newfoundland, Canada. Overall, the prevalence of Nosema species was 86.5% (32/37 samples of bee DNA), based on conventional PCR and the optimized R-T PCR assay. The melting-curve analysis showed three groups of curve profiles that could determine the prevalence of N. apis, N. ceranae, and co-infection (21.9%, 56.2%, and 21.9%, respectively). The duplex R-T PCR assay was efficient, specific, and more sensitive than duplex conventional PCR because co-infection was identified in 5.4% (n = 2) more samples. Sequencing of R-T PCR products confirmed the results of the melting-curve analysis. Duplex R-T PCR with melting-curve analysis is a sensitive and rapid method of detecting N. apis, N. ceranae, and co-infection in honey bees.

Detection of common dermatophytes in clinical specimens using a simple quantitative real-time TaqMan polymerase chain reaction assay

“…showed that a melting‐curve analysis step allows the detection of 11 species using four detection channels. However, some of the melting temperature ranges within this multiplex test differed by < 2 °C, but > 2 °C difference was found to be necessary for a clear discrimination of different PCR products . Multiplexing was avoided in our assay as this may reduce the sensitivity of the assay, as the increased number of oligonucleotides may compete for the targets .…”

Prevalence and infection intensity of Nosema in honey bee (Apis mellifera L.) colonies in Virginia

“…Bourgeois et al, 2010;Burgher-MacLellan et al, 2010), but most laboratories rely on spore counts or use conventional PCR for species determination. Interestingly, there have been no other reports to our knowledge that samples negative for spores can be infected with N. ceranae and we were interested in determining why this might have occurred.…”

Comparison of within hive sampling and seasonal activity of Nosema ceranae in honey bee colonies

“…N. apis was first detected molecularly in 2004 (Webster et al, 2004). Since then, standard PCR assays using species specific primers followed by separation on agarose gels (Chen et al, 2008;Klee et al, 2007;Martin-Hernandez et al, 2007) and real-time PCR (Bourgeois et al, 2010;Burgher-MacLellan et al, 2010;Chen et al, 2009a;Cox-Foster et al, 2007;Traver and Fell, 2011) have improved detection and species identification. Before the use of molecular assays for species determination, it is possible that N. ceranae infections prior to 1996 may have been improperly diagnosed as N. apis (Fries et al, 2006).…”