Development of multiplex RT-PCR assays containing an internal amplification control for the detection of dicistro-, iflaviruses and CBPV in honey bees. Part 1 - assays design and optimization

Viral infections can cause increased mortality of honey bee colonies. Because of an asymptomatic course of infections their detection in honey bees is only possible by the use of sensitive and specific diagnostic methods. Nowadays, the conventional RT-PCR assays are routinely employed for detection of honey bee viruses, but none of them have had their diagnostic usefulness confirmed through documented optimization and validation process. The aim of this study was to develop of the two in-house multiplex RT-PCR (mRT-PCR) assays for the detection of CBPV, iflaviruses (DWV-A, SBV) and dicistroviruses (ABPV, IAPV, BQCV) of bees. The sequences of the primers used in the developed Ifla-CBPV and Dicistro mRT-PCRs were refined to include point mutations observed in the respective genome fragments of honey bee viruses detected worldwide so far, including newly characterised virus strains from Poland. The optimization of the PCR mixtures composition and temperature-time profiles of the assays was performed. To avoid false negative results, an internal amplification control (IAC) was prepared and incorporated into the Ifla-CBPV and Dicistro mRT-PCR assays. The developed IAC-controlled mRT-PCR assays allow for simultaneous detection of mixed viral infections caused by six virus species commonly occurring in worldwide population of bees. The use of IAC during molecular detection enables monitoring of the assays correct performance.