Abstract
BackgroundSacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required.FindingsA one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid identification of SBV. The data demonstrated that, in a simple water bath, SBV RNA could be detected as early as 20 min at 65°C, and a positive amplification reaction was visible to the naked eye due to a color change brought on by the addition of nucleic acid stain SYBR Green.ConclusionsThe current study presents a method for the rapid and simple detection of SBV by RT-LAMP with high sensitivity and analytic specificity.
Highlights
Sacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required
The final, optimal reverse transcription loop-mediated isothermal amplification (RT-LAMP) protocol was determined to be for a time of between 30 and 50 min at a temperature in the range 63°C-65°C
The RT-LAMP product was detected using the naked eye by observing the white turbidity of the reaction mixture (Figure 3A) or color change of the solution when stained with SYBR Green I (Figure 3B)
Summary
Sacbrood virus (SBV) primarily infects honeybee broods, and in order to deal with the problem cost effective detection methods are required. The RT-LAMP assay successfully amplified the target sequence of the SBV-pol gene, as observed by 2% agarose gel electrophoresis. Amplified DNA in the LAMP reaction causes white turbidity due to the accumulation of magnesium pyrophosphate, a by-product of the reaction.
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