Development And Preliminary Application Of Visual LAMP Method For Honeybee Sacbrood Virus And Black Queen Cell Virus

Black queen cell virus and Sacbrood virus are two common bee viruses,BQCV mainly infects the larvae and pupae of queen bee,with the walls of cells darkened areas after infection.SBV mainly infects 2 to 3-day-old larvae,for molting fluid accumulated on the skin,the body is formed cystic and look no luster finally,then the body becomes dark brown after death.Both viruses are currently developing rapidly with a high mortality,which brings great harm to beekeeping. Traditional methods were limited to concern reaction time,cost of test and accuracy rate,Loop-mediated isothermal amplification can overcome the above problems of traditional methods,due to its fast reaction、low cost and high accuracy.To improve the means of detection,this paper established a LAMP detection rechnique for BQCV and SBV,and conducted preliminary clinical detection of BQCV and SBV.According to BQCV JL1 strain(KP119603) capsid protein sequence,designed 5 sets BQCV LAMP primer for selection and monitored the whole process by Real-time Turbidimeter.Only BQCV showed specific amplification after constant temperature reactions at 63 ℃ for 60 min,while other viruses did not render the amplification reaction.After the reaction,all the judgement results of the turbidity of reaction tube, visualization colour added SYBR Green I,the color under UV light and agarose gel electrophoresis were consistent with that of the Real-time Turbidimeter.The sensitivity test showed that BQCV minimum detectable amount was 4.0×102copies/μL,100 times higher than the ordinary PCR.The results of repeatability and stability test showed that coefficient of variation of the maximum amplification rate was less than 10%,with a good repeatability and stability.The paper designed 5 sets SBV LAMP primer based on SBV-UK strain(AF092924) poly-protein sequence,selected the LAMP primers and monitored the reaction system and reaction conditions by LAMP real-time Turbidimeter.The result shows that only SBV efficiently amplified within 60 min at 63 ℃.The amplification results of other viruses were negative.At the end of the amplification,turbidity of the reaction tube,visualization color added SYBR Green I,the color under UV light and agarose gel electrophoresis matched with that of LAMP real-time Turbidimeter.The sensitivity test showed that SBV minimum detection limit was 3.2×101copies/μL,about 100 times higher than that of PCR.Repeatability and stability test results of SBV demostrated that the maximum amplification rate coefficient of variation of the same batch and different batches were less than 10%,with good repeatability and stability.This study successfully established a visual LAMP detection method for BQCV and SBV,and showed high specificity and sensitivity,while showed fast,efficient and visualization features,and provided a new way for rapidly detection of bee virus.The established methods were applied to detected BQCV and SBV in 217 clinical samples,the results of LAMP were completely consistent with that of TaqMan real-time RT-PCR,with the higher accuracy rate than RT-PCR,can be better applied to the initial clinical tests for BQCV and SBV.Thus,this established method has the potential for the detect,due to its simplicity,short reaction time and high accuracy and specificity.