Diagnosis Of The Main Bee Fungi By Loop-mediated Isothermal Amplification

Ascosphaera apis and Nosema spp. are the most serious fungal pathogen present in the honeybee, Ascosphaera apis results in the death of honeybee larvae. In addition, Nosema spp also affects the honey bees that affects growth and development of important gland, and shortens the span of life of the adult bee. Thus overall performance potential of honeybee colony will be reduced and make the colony decline or collapse, as a consequence, wintering bees can easily death over winter period.Currently, drug prevention and treatment of both diseases and the effectiveness of drug residues still present problems. While developing resistant traits by breeding queen in order to reduce the incidence of disease is also one measure of prevention, but also no ideal breeding techniques. At the same time in terms of pathogen detection, detection microscopy technology, immunology, molecular biology methods also have some limitation. The accuracy of first two methods is not high, especially in the study of immunology related proteins still have not been in-depth. There is a lack of theoretical support; increasing the accuracy of molecular biology can be helpful to identify pathogen species. However, these methods are problematicin case of instrument, specificity, simplicity, but cannot achieve accurate, sensitive and fast diagnosis. Therefore, the establishment of an accurate, rapid, high sensitivity and specific is necessary, without detection of A. apis practical and technical Nosema spp. Special equipment is necessary. Loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) technology as a molecular detection method under isothermal conditions can work better, the water bath to complete the reaction within about 1h, and the result is determined by observing the centrifugal reaction product and the color change after adding a fluorescent dye, can also be judged by agarose gel electrophoresis, and real-time detection of turbidity throughout the course of the reaction, the disease so as to achieve rapid, accurate and sensitive diagnosis.This study established and optimized LAMP detection system for A. apis and Nosema spp., And verify their specificity and sensitivity of by comparing with conventional PCR method, and finally clinical trials were done to verify the feasibility of the method.The main resulting:1) Four specific primers were designed based on the particular sequence of ITS of the A. apis using the soft of PrimerExplorer V4.0 online, the primers. A rapid and specific method for detction of A. apis was established. The optimum conditions for LAMP reaction were as follows:4 mmol?L-1 Mg2+,1.2 mmol·L-1 dNTPs,1.6 mmol·-L-1F3/B3, FIP/BIP,0.4 mol·L-1 betaine. The templates of A. apis, N. ceranae, N. apis, DWV, SBV, BQCV and IAPV were tested, the result showed that only the A. apis DNA template could produce typical ladder-like bands. The sensitivity test result showed that the PCR could detect the DNA templates as low as 0.2231×10-5μg·μL-1. While the LAMP could detect ten times lower of DNA templates 0.2231×10μg·μL-1. The LAMP is effective, simple and time saving for detecting the A apis infected honeybee.2) Four specific primers were designed based on the particular sequence of DNA-dependent RNA polymerase Ⅱ largest subunit (RPB1) of the N. ceranae using the soft of PrimerExplorer V4.0 online. We successfully established a rapid and specific method for detecting N. ceranae in honeybees. The optimum conditions for LAMP reaction were as follows:4 mmol-L-1 Mg2+,0 mol·L1 betaine,1.0 mmol·L-1 dNTPs,1.4 mmol·L1 FIP/BIP, 57℃ as reaction temperature and 70 min as reaction time. The templates of N. ceranae, N. apis, A. apis, M. pluton, DWV, SBV, BQCV and IAPV were Prepared and tested through this method, the results showed that the unique laddrdder-like bands can only amplified from N. ceranae DNA template. Results of the sensitivity test indicated that the minimum concentration for detecting DNA templates by PCR and LAMP are 0.136×10-5,0.136×10-6 μg·uL-1, respectively. The LAMP is effective, simple and time saving for detecting the N. ceranae infected honeybee.