Updating Sacbrood Virus Quantification PCR Method Using a TaqMan-MGB Probe.

Wei-Fone Huang,Zhiguo Li,Yakun Zhang,Shahid Mehmood,Chunsheng Hou,Zhengwei Wang

doi:10.3390/vetsci8040063

Abstract

Sacbrood virus (SBV) is a common honey bee virus disease. SBV variants and strains identified in Asian honey bees, Apis cerana, have created confusion in identifications. Although the regional names indicated the expansions of the virus in new regions, pathogenesis, and genomes of these variants are not distinct enough to be a separate virus species. However, current SBV qPCR methods may not detect newly identified A. cerana SBV variants (Ac SBV) according to the genome sequences. Since these Ac SBV can naturally infect A. mellifera and possibly other hymenopterans, ignorance of Ac SBV variants in detection methods is simply unwise. In this report, we updated the qPCR method based on Blanchard’s design that used conserved regions of VP1 to design a TaqMan method with an MGB (minor groove binder) probe. We tested the method in bees and hornets, including A. mellifera, A. cerana, and Vespa velutina. The updated primers and the probe can match published SBV and Ac SBV genomes in databases, and this updated method has reasonable sensitivity and flexibility to be applied as a detection and quantification method before the discovery of variants with more mutated VP1 gene.

Highlights

Sacbrood virus (SBV) is a common honey bee virus that causes obvious symptoms in larvae
The updated qPCR primers resulted in sufficient PCR efficiencies (90–110%) within the temperature range of 56–61 ◦C in the SYBR Green qPCR method; unspecific amplicons, were noted in no-template controls (NTC) after 35 cycles
We tested the MGB probe in 250 and 300 nm, and both concentrations generated good PCR efficiencies (90–110%). Both A. cerana SBV variants (Ac SBV) clones from Chongqing and Fuzhou generated strong and low Cq values in the TaqMan qPCR, and we used the Fuzhou clone as standards in the following tests

Summary

Introduction

Sacbrood virus (SBV) is a common honey bee virus that causes obvious symptoms in larvae. Asymptomatic infections can be found in adult worker bees [1] that still performed nursing or foraging duties, even in colonies that have no significant larva losses due to sacbrood disease. Apis mellifera, usually cope with sacbrood disease better than Asian honey bees, A. cerana. A. cerana populations can decrease dramatically after the introduction of SBV into the regions [3], whereas A. mellifera colony losses caused by SBV were rarely reported in the same environment. A Chinese sacbrood virus variant (CSBV) was found in South China in 1972 [4] with observations of severe A. cerana colony losses, and related variants were later reported in North and East China, similar but not identical in sequences [5,6]. Bailey described the Thai sacbrood strain [7] that was later found in the regions across Southeast Asia [8], which resulted in more than 90% mortalities of local A. cerana populations

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Results

Conclusion