Study On Detection Of Three Kinds Of Quarantine Diseases By PCR In Bumblebees

Bumblebees(Bombus spp.)are important agricultural insect pollinators with huge potential.They are attacked by many pathogens and parasites in the process of breeding,such as Nosema bombi,Apicystis bombi,Crithidia bombi,Locustacarus buchneri and Aethina tumida et al.With the rise of modern ecological agriculture,the introduction of foreign bumblebees for the purpose of pollination is increasing day by day.The consequent risk of pest invasion is paid attention by various fields.However,the research about pathogens and parasites in bumblebees is less at home.At present condition of lacking corresponding quarantine measures against the entry of bumblebees to prevent from biological invasion,it is necessary to establish appropriate quarantine methods according to the inspection and quarantine requirements of entry bumblebees.In this experiment,a mitochondrial gene cytochrome oxidase subunit ?(CO ?)of Aethina tumida fragmented by synthesis and bumblebees collected at home were used as experimental materials.This thesis aimed at developing a detective system of disease diagnosis for imported bumblebees through screening a specific primer.The following results were achieved:1.PCR assays were developed and optimized for detecting Crithidia bombi,Nosema bombi and Aethina tumida.Specific primers Cri-F/R,Nos-F/R and Aet-F/R were designed and target bands were amplified,they were 447 bp,213 bp and 205 bp,respectively.In the PCR detective system of Crithidia bombi,the most optimal annealing temperature was at 59?,the final primer concentration was at 0.5 ?gmol/L,35 cycles were the best;The most optimal parameters for Nosema bombi were as follows,the optimal annealing temperature was at 57.2?,the final primer concentration was at 0.5 ?mol/L,45 cycles were the best;The optimal annealing temperature of Aethina tumida was at 52? the final primer concentration was at 0.5 ?mol/L,and 40 cycles finally.2.Specific primers of Cri-F/R,Nos-F/R and Aet-F/R can only specifically amplify target gene fragments of Crithidia bombi,Nosema bombi and Aethina tumida.There was no amplification to other pathogens and parasites which can infect bumblebees.The sequencing results of amplified products were consistent with the expected fragments by sequences alignment.It has proved that the sensitivities of Cri-F/R,Nos-F/R and Aet-F/R can reach to 1.32×10-6 ng/?L,2.86×10-4 ng/?L and 0.77×10-9 ng/?L,respectively.The minimum detectable concentrations of three primers were well repeated,indicating that three PCR detection methods were stable and reliable for the quarantine of entry bumblebees.3.When the established rapid PCR methods were used for detecting 340 samples of bumblebees,136 and 137 positive results of Nosema bombi and Crithidia bombi were obtained respectively,and the positive detective rates were 40.0% and 40.3%,respectively.