The Establishment Of Loop-mediated Isothermal Amplification For Echinococcus Granulosus Of Dogs

Hydatid disease is a serious zoonotic parasitic disease,which is caused by larva of Echinococcus. In China,the hydatid disease is mainly caused by Echinococcus granulosus(Eg) and Echinococcus multilocular(Em). Despite decades of prevention and treatment, the disease is still very serious in pastoral areas in Xinjiang.The prevalence rate of sheep hydatid disease and the canine parasite rate was over 60%and 45% respectively in Bavaria,Altay,Ili and Kashgar.With the development of urbanization, companion animals provide a new condition for the occurrence of hydatid disease,and the hydatid disease occurs increasingly in cities. Using traditional methods can not distinguish Eggs of Eg and Em from dog feces, therefore the establishment of a rapid,simple and sensitive method for epidemiological survey and diagnosis of Eg could be very important significance.Firstly, according to the eight epidemiological survey and diagnosis of Eg mitochondrial genes which are cox1,cox2,cox3,nad1,nad2,nad4,nad5,nad6,two outer primers F3 and B3 and two inner primers FIP and BIP were designed by Primer Explorer V4 respectively.Specific genes of Eg were screened through conventional PCR with outer primers of eight genes. And then,for selected specific gene,6 temperatures which contain 60 ℃,61 ℃,62℃,63 ℃,64 ℃,65 ℃and 6reaction time which contain 10 min,20 min,30min,40 min,50 min,60 min were opitimized to,establish the LAMP reaction conditions,and detect the specificity and sensitivity.Finally,the LAMP was applied for dog feces detection.The results showed that only cox1,cox2,nad4,nad6 genes were amplified specific fragments in the Eg cyst fluid,not in Em,Cysticercus tenuicollis,Moniezia expansa,Thysaniezia,A. centripunctata,flagella nematodes,fascioliasis hepatica,H.contortus. The cox2 gene was selected as specific gene of Eg. The most optimal reaction temperature and time of the LAMP assay for the cox2 gene were 63℃ and40 min,respectively. Turbid precipitation was only in the tube of Eg cyst fluid and showed green,color,while,the tube of Em,Cysticercus tenuicollis,Moniezia expansa,Thysaniezia,Acentripunctata,flagella nematodes,fascioliasis hepatica,H.contortus were clear,and showed brown color. Only LAMP product of Eg cyst fluid can displayed specific characteristic ladder-like pattern of electrophoresis. Using Eg cox2 standard recombinant plasmid as template, the sensitivity of this LAMP assay(0.016fg/μL) was 1000 times,higher than conventional PCR(16.09fg/μL).In addition,DNA extraction of 50 dog feces were detected by conventional PCR and LAMP, feces antigen were detected with ELISA, no differences were found by comparing the LAMP, conventional PCR and ELISA, only four samples were positive,and the coincidence rate was 100%.A speed LAMP assay for Eg mitochondrial cox2 genes was established. The established LAMP assay is an effective and low-cost procedure with high specificity and sensitivity that requires no specialized equipments and amplification results were visualized. The rapid and simple assay is a potential useful technique forepidemiological survey and diagnosis of Eg in field condition.