Loop-mediated isothermal amplification for rapid detection of Chinese sacbrood virus

Abstract

Chinese sacbrood virus (CSBV) has emerged as an important etiologic agent of honeybee infections and is lethal for individual bees, even causing the collapse of entire colonies. Although diagnostic methods for CSBV have been established in many clinical laboratories, application of these methods is largely restricted by the apparatus needed to carry out the reaction and by cost, therefore a simpler and less expensive diagnostic method for CSBV infection is required. In this study a simple and inexpensive system is described that is based on the loop-mediated isothermal amplification (LAMP) assay. The LAMP and the polymerase chain reaction (PCR) methods were compared for their ability to detect CSBV in 31 clinical samples, in purified CSBV-LNQY strains or to be able to discriminate between cDNA samples from other viruses. The detection limit of the LAMP method was 1 pg, showing that LAMP is as sensitive as reverse transcriptase (RT)-PCR for CSBV detection. In addition, no DNA band from other related viruses samples was amplified by either method, suggesting that this LAMP assay is as specific as RT-PCR for CSBV detection. All 31 clinical samples that were LAMP assay-positive were also amplified by RT-PCR, however the LAMP assay was faster, more cost effective, and easier to perform as the target gene amplified rapidly, within 2 h, and only a standard laboratory water bath or heat block was required for the reaction. The results demonstrate clearly that this LAMP-based assay is a useful tool for the rapid and sensitive diagnosis of CSBV infection of bees.