Establishment And Application Of Loop-mediated Isothermal Amplification For Detection Of Trichinella Spiralis

Trichinosis is an important zoonotic parasitic disease.Humans and animals were infected by ingestion of raw or undercooked meat.Once infected by this parasite,symptoms as abdominal pain,diarrhea,edema around eyes,fever,would be occurred in humans.This disease was regarded as one of the greatest threat for human health.Developing of rapid diagnostic technology for Trichinella spiralis would be helpful for prevention and control of this disease.At present,to inspect T.spiralis in animal meat,microscopic method and digestionmethod were widely used.The advantage of these techniques was easy to be operated.However,to hand this work,professional knowledge,plenteous experience,and intense labor would be required.PCR greatly improved the sensitivity in the inspection.But the application of PCR is limited because of the expensive equipment.In this paper,Loop-mediated isothermal amplification(LAMP)would be established.With the advantages of rapid,sensitive,specific and easy to perform,this method would be applied in the detection of T.spiralis in meat products.1.Total DNA was extracted from T.spiralis and internal transcribed spacer 2(ITS 2)gene that between 18s and 28s region of the ribosomal DNA was amplified by PCR.This gene was used in the establishment of LAMP.2.Primers for LAMP were designed based on ITS2 gene.The LAMP was performed using the genome of T.spiralis as template.Followed by DNA electrophoresis and visualization with addition of SYBR Green I fluorescent dye.To test the sensitivity of this method,T.spiralis genome was diluted by 10 times as a series,followed by detection by LAMP and PCR.Results showed that the sensitivity of LAMP method is 1000 times higher than that of PCR.To get mock samples,different amounts of muscle larvae were added to the same weight of mice muscles(without infection of T.spiralis).Then the DNA was extracted from these samples and detected by LAMP method,separately.The results showed that one larva in 2 gram of meat could be detected by LAMP method.The experiment was repeated three times and the stable result was obtained.To study the specificity of this method,genomes from T.spiralis,Toxoplasma gondii,Haemonchus contortus,Eimeria tenella,Cysticercus cellulosae and Escherichia coli were used as templates,respectively.The results indicated that the LAMP method was specific for T.spiralis.3.110 of tongue samples from pig,chicken,duck,lamb and cattle were tested by LAMP.None of samples showed infection of T.Spiralis.In this study,the LAMP method for detection of T.spiralis was established.With the advantages of rapid,sensitive,specific and easy to perform,this method would be widely applied in the detection of T.spiralis in meat products.