Establishment And Optimization Of LAMP Detection Method For Mosquito-borne Japanese Encephalitis Virus And PCR-RLB Detection Method For Fly-borne Pathogenic Bacteria

Insect-borne diseases,a infectious disease carried by insects and spread to human and animals through direct or indirect contact,mainly include mosquito-borne,tick-borne and fly-borne diseases etc.The most widely popular and serious harmful pathogen,bring the main public health problems,are Japanese encephalitis virus?JEV?and bacteria in China.In this study,we focus on the mosquito-borne JEV and 7 species of fly-borne bacteria for developing a Loop-mediated isothermal amplification?RT-LAMP?and PCR-Reverse line blot?RLB?method respectively,aimed at providing technical support for investigation and risk assessment of insect-borne disease in Gansu province.1.According to Japanese encephalitis virus E gene sequence published on GenBank,four pairs of specific primers were designed,and the reaction system was optimized.RT-LAMP method for the detection of Japanese encephalitis virus was established,their sensitivity can reach 10^-2PFU,higher than that of RT-PCR method?10⁰PFU?.As-prepared ZnO nanomaterials with different morphologies?nanoflowers,nanosheets and nanoparticles?were used in the RT-LAMP method for the first time,the results showed that the effect of adding ZnO nanoflowers is the best,the reaction time and reaction temperature of RT-LAMP detection method were reduced from 50 min and 65?to 30 min and 60?,the field diagnosis value of this method can be greatly improved.2.16SrRNA gene sequence alignment of 7 species of bacteria?Staphylococcus aureus,Shigella flexneri,Aeromonas caviae,Vibrio parahaemolyticus,Salmonella enterica subsp entericaserovar typhimurium,Proteus vulgaris and Yersinia enterocolitica subsp enterocolitica?published on GenBank and the designs of universal primer,universal probe and species specificity oligonucleotide probes were performed using DNAStar.And the reaction conditions were optimized,the optimal concentration of universal primer,Staphylococcus aureus and Shigella flexneri probes and other 5 kinds of pathogenic probes were determined to be 25?M,50?M and 100?M,repectively.PCR-RLB detection method was established,the detection sensitivity of the above-mentioned 7 species of bacteria could reach 10^-610^-11ng/?L,much higher than that of PCR(10^-310^-8 ng/?L).3.Using as-established PCR-RLB method,the investigation of above-mentioned 7 species of pathogenic carried by houseflies was carried out.The number of houseflies collected from residential area,slaughter house,waste transfer station and around the hospital was 380,330,200,and 150,respectively in Lanzhou.It was found that the positive rate of Staphylococcus aureus,Shigella flexneri,Aeromonas caviae,Vibrio parahaemolyticus,Salmonella enterica subsp entericaserovar typhimurium,Proteus vulgaris and Yersinia enterocolitica subsp enterocolitica carried by houseflies in residential area,slaughter house,waste transfer station and around the hospital were 0%,33.93%,0%,0%,39.29%,0%and 17.86%;0%,37.50%,0%,0%,35.42%,0%and 14.58%;0%,20.00%,0%,0%,43.33%,0%and26.67%;7.14%,32.14%,0%,10.71%,14.29%,3.57%and 28.57%,respectively.That provides preliminary data for the evaluation the effect of city flies in the spread of bacterial disease.