The Establishment Of Three Common Bee Virus Loop-mediated Isothermal Amplification Method

The bees are important pollinators in natural ecological system. Black queen bee viruses (BQCV), deformed wing virus (DWV), and sacbrood virus (SBV) are common pathogens and harmful to honey bee. The common methods such as molecular biology, symptoms, immunological detection were developed to detection these virus. But these methods have some shortcoming such as special requirements on instruments and technology, and the detection specificity was not idea.. Therefore, establishing a rapid, high sensitivity, strong specificity, and without special equipment are desperately needed for practical detection of the three viruses. The loop-mediated isothermal amplification techniques can be performed under isothermal condition, such as in water-bath water, with the potential to be used in the three bee virus detection.This study destined to set up and optimize the LAMP reaction for specially detection the BQCV, DWV and SBV in honeybee, and the sensitivity of LAMP was compared with conventional PCRmethod. Lastly, the practical detection of clinical samples was evaluated by comparing with common PCR method.The main results are as follows:1) The BQCV-LAMP, DWV-LAMP, SBV-LAMP reaction system were successfully established and optimized, the optimized parameter for BQCV-LAMP were as follows the outer and inner primers ratio was 1:3, betaine concentration was 0.8 mmol/L, magnesium concentration was 2.0 mmol/L, and the reaction was kept at 63℃ for 45 min; The optimized parameter for SBV-LAMP were as follows, the outer and inner primers was 1:2, betaine concentration was 0.6 mmol/L, magnesium concentration was 2.0 mmol/L, and the reaction was kept at 63 ℃ for 45 min. The optimized parameter for DWV-LAMP were as follows, the outer and inner primers ratio was 1:4, betaine concentration was 0.6 mmol/L, magnesium concentration was 2.0 mmol/L, and the reaction was kept 63℃for 60 min.2) The detection specificity of the developed BQCV-LAMP, DWV-LAMP, SBV-LAMP were certificated. Furthermore, the sensitivity of BQCV-LAMP could detect as low as 86 fg of RNA template, while the sensitivity of conventional PCR was 8.6 pg of RNA template, illustrating the sensitivity of BQCV-LAMP is 100 times higher than -common PCR method; The sensitivity of SBV-LAMP could detect as low as 93 fg of RNA template, while the sensitivity of conventional PCR was 0.93 pg of RNA template, illustrating the sensitivity of SBV-LAMP was 10 times higher than the common PCR method; the sensitivity of DWV-LAMP could detect as low as 8.9 fg of RNA template, while the sensitivity of conventional PCR was 8.9 pg of RNA template, illustrating the sensitivity of DWV-LAMP was 100 times higher than the common PCR method.3) The developed LAMP methods were used to detect the clinical sample from Italian bee (Apis mellifera) and eastern bee (Apis cerana cerana). For BQCV-LAMP detection,5 samples from Apis cerana cerana and 15 samples from Apis mellifera were positive. The positive detection rate was 25% and 75% respectively; only three 3 and 11 samples were positive detected from the same Apis mellifera and Apis cerana cerana samples by common PCR method respectively. The positive detection rates were 15% and 55% respectively. For SBV-LAMP detection,12 samples from 20 Apis cerana cerana samples were positive. The positive detection rate was 60%. Only 9 samples were positive detected from the same 20 samples by common PCR. For DWV-LAMP detection,11 from 20 Apis mellifera samples were positive, the positive detection rate of 55%; while only 7 from the same 20 Apis mellifera samples were positive detected when using the common PCR method, the positive detection rate was 35%. All the three developed BQCV-LAMP, DWV-LAMP, SBV-LAMP detection methods were more sensitive than the corresponding conventional PCR methods.