Establishment And Preliminary Application Of LAMP Detection Method Based On ?-Enolase Gene Of Mycoplasma Suis

Swine eperythrozoonosis is a multiple zoonotic diseases that the pathogen(Mycoplasma suis,M.suis)parasitises in the pig's red blood cells,plasma,tissue fluid and bone marrow etc.Eperythrozoonosis has a wide range of infected hosts,such as pigs,cattle,goats,dogs,cats,etc.,including humans.Swine eperythrozoonosis mostly shows recessive infection pattern and injures the body immune ability,which led to complication with other infectious diseases or secondary infection,seriously impeding the healthy development of China's pig industry,aquaculture and public health.Early diagnosis is the key of the prevention agaist porcine erythrocyte disease.Routine diagnostic methods include smear microscopy,ELISA,PCR,etc.,but because these diagnostic methods require special equipment and reagents,and the user should have a certain professional operation skills,it's hard for the grassroots farms to promote those methods easily.Loop-mediated isothermal amplification method(LAMP)is a novel nucleic acid amplification method established by Notomi et al.LAMP can accurately identify 4-6 specific regions of target sequence by BstDNA polymerase under constant temperature conditions.Rapid growth of the target gene will be confirmed with trapezoidal bands by electrophoresis or SYBR Green I dye staining directly after the reactions.LAMP has many advantages like fast responsiveness,high efficiency,no need for special equipments,and direct observation of the results,making it popular in the majority of farms to diagnose animal infectious diseases.In recent years,M.suis was identified as Mycoplasma and its whole genome has just been sequenced by scholars.The a-enolase gene(en-enolase)is a newly reported Eperythrozoon suis surface functional protein involving adhesion and is often used as a target gene for molecular diagnostic methods.Therefore,a pair of specific primers were designed according to the published M.suis genome(NC-015153.1)from GenBank.The a-enolase gene of M.suis was amplified and sequenced by PCR and TA cloning.According to the correctly sequenced a-Enzyme gene,we designed a LAMP specific primers at the conserved region of the gene.LAMP pre-test was carried out to optimize the reaction temperature,time and Mg2+ concentration,and the sensitivity,specificity,repeatability tests of the LAMP were conducted.Finaly the LAMP method was established for the clinical diagnostic method of swine eperythrozoonosis.The results showed that the partial a-enolase gene of M.suis was cloned in length 864bp.The optimal reaction temperature of the established LAMP reaction was 63 ?;the optimum concentration of Mg2+ was 6mmol/L;the optimum concentration ratio of inside and outside primer was 1:6;the whole reaction could be completed within 50 min;and after dyeing with SYBR Green I dye,the results could be directly observed by naked eyes.The specificity test showed that there was no any amplifications of the genomes of Toxoplasma gondii,Streptococcus suis type ?,Escherichia coli and Cynanchum paniculata,proving the good specificity of LAMP.The sensitivity test showed that the lowest dilution factor is 10-6,i.e.30 copies/?L,100 times higher than the sensitivity of common PCR method,proving the high sensitivity of LAMP.The repeatability test showed that the 7 reactions in each 5 different batch templates all resulted in the same pattern,proving the good reproducibility of LAMP.Using this LAMP method,we tested 84 blood samples from one pig farm located in Longjing City,Jilin Province.The positive rate was 41%,which was identical with the results of the higher than PCR.The results showed that the established LAMP diagnostic method based on the M.suis alpha-enolase gene was highly specific,highly sensitive,reproducible and easy to operate.No special instrument was needed.The direct color observation results could be used for clinical diagnosis of M.suis causing disease in grassroots pig farms.