The Molecular Epidemiology Of E. Suis In The Northeast And The Development Of Rapid Assay By Loop Mediated Isothermal Amplification Method(LAMP)

Eperythrozoonosis which caused by Eperythrozoon is parasitic in the surface of erythrocyte, plasma and bone marrow of animal and human. The main clinical features is hemolytic anemia,jaundice and fever.We identified 600 blood samples come from the three provinces in northeast of china and made the nucleotide sequencing of Eperythrozoon suis 16S rRNA gene.Conservative gene order was selected,amplificated and compared. The results showed that the positive rate of Eperythrozoon suis was 93.5%(187/200) in HeiLongJiang,89.5%(179/200) in LiaoNing and 91.5%(183/200) in JiLin. The homology among HeiLongJiang, LiaoNing and GuangDong strain was up to 100%; JiLin and American strain was up to 97%. Genetic relationship between HeiLongJiang and LiaoNing strain was the nearest, but further with JiLin strain, indicating that the prevalence was severe in the three provinces and the sequences of 16S rRNA gene were different in the three provinces in northeast of china.Loop-mediated isothermal amplification (LAMP) was a new type of detection technology in recent years which has become a new alternative to PCR to be widely used in nucleic acid research, disease diagnosis and prevention, sex determination of animal embryos,tested of genetically modified food and other areas with the advantage of specificity,high sensitiy,accurate and simple operation. The detection of Eperythrozoon suis was used for the first time at home and abroad. Primers were designed using online software according to the conserved regions of 16S rRNA gene. In the following step, several important parameters of LAMP were optimized including outer and inner primers, Mg2+ concentration, the temperature and reaction time of the reaction and so on.LAMP assay was established and then sensitivity, specificity and stability was tested.The result showed that Eperythrozoon suis gene was detected by the LAMP. The sensitivity of detection concentration was 23.7 fg/μl, which was 10 times higher than PCR.The specificity of LAMP assay was powerful and no amplification was found with the samples of Neospora, Toxoplasma gondii, Sergenti.The detection of the 5 different templates of the same templates and the same template was amplified 5 times show hat the method has good stability;The prevalence rate of 24 pig blood samples from Yanbian in Jilin Province with LAMP was 95.9%, it shows that LAMP can be applied to the rapid detection of Eperythrozoonosis.This test provided a scientific theoretical basis for the condition of popularity of Eperythrozoon suis in the three northeastern provinces and provided a specific, sensitive and effective detection method of Eperythrozoonosis.