Beeheal: standardization of laboratory methods for sample processing, nucleic acids extraction and PCR for microsporidia and viruses analysis

Abstract

BEEHEAL is a project designed to determine the phenology and interaction of Nosema ceranae and viruses in four Mediterranean countries: Spain, France, Portugal and Israel, including some territories where Varroa destructor is not present (Azores and Ouessant islands). This will allow us to study and compare the interactions between pathogens in a wide range of hosts, beekeeping and climatic conditions. The honey bee samples collected along the year in the different countries will be analysed for pathogens in three laboratories. This requires a standardization of methods to compare the results in order to assign the effect of every variable in a reliable way. To that end, the participating laboratories have been working together to establish the sampling methodology, the conservation of the samples, the nucleic acids extraction and the PCR analysis. We analyzed the sample processing for nucleic acid extraction on TE buffer (with or without Proteinase K), CTAB buffer or commercial kits (Qiagen). The maceration of bees (either individually or in composite samples) in TE buffer and posterior incubation at 96ºC for 20 minutes showed a good sensibility level and good value for N. ceranae DNA extraction. This method also allowed the conservation of RNA at -80ºC for a month in the TE solution for later RNA extraction. A joint protocol for sample processing, DNA and RNA extraction and PCR analysis has been developed but adjusted to the particular conditions and equipment of each laboratory. The standardization of methods to be implemented by each participating laboratory will avoid the biases on conclusions based on the diverse methods applied. ; This work has been developed under the BEEHEAL project. BEEHEAL is funded through the ARIMNet2 2016 Call by the following funding agencies: INIA (Spain), MOARD (Israel), ANR (France), and FCT (Portugal). ARIMNet2 (ERA-NET) has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement no. 618127. ; info:eu-repo/semantics/publishedVersion