Microsatellite Of Trichinella Spiralis And Its Application In Identification Of Insects

Trichinellosis is a serious zoonotic disease caused by Trichinella spiralis.T richinella genus is divided into nine species and three genotypes,which are T.spiralis,T1;T.nativa,T2;T.britovi,T3;T.pseudospiralis,T4;T.murrelli,T5;T6;T.nelsoni,T7;T8;T9;T.papuae,T10;T.zimbabwensis,T11;T.patagoniensis,T12.Trichinella characterized by an wide host range and geographical distribution,which can infect people and hundreds of species of ainimol,reptiles and birds.Human are mainly caused by eating raw or half-raw meat infected with Trichinella spiralis larvae.In China,economic losses are nearly 1.8 billion yuan each year,while it is up to 1 billion dollars and 570 million dollars each year in the United States and the EU.At the same time,the zoonotic disease is seriously harm for the health of the people in the epidemic area.Therefore,the precise identification of Trichinella species not only provide the basic and premise for the study of Trichinella,but also have great significance for prevention and treatment of trichinosis.For a long time,Trichinella species is based on adult morphology,epidemiological characteristics classificating and identificating.However,the different species of individual is not obvious,the limitations of the traditional classification method has become more prominent,the urgent need to develop new classification methods.The development of molecular biology,the progress of sequencing technology has brought new ideas to Trichinella classification technology.In recent years,scientists have been trying to explore the genetic relationship analysis,classification and identification of Trichinella spiralis from the molecular level.SSR is one of the many advantages of classification and identification methods.SSR has a wide range of distribution,high polymorphism,many species,Mendelian co-dominant genetic and other advantages.SSR marker method is very suitable for the identification of parasite classification method.According to the genome sequence of Trichinella spiralis(T1)published on the Genbank,we lookuped the SSR of the genome by use of program MISA,then 50 pairs of primers were designed on program of primer3-1.1.4 WINXP and synthesized by company.In this study,3 % agarose gel electrophoresis were used to seperate the SSRPCR product,10 pairs of primers were selected,which can distinguish four widely different trichinella species(T.spiralis(T1),T.native(T2),T.britovi(T3),T.pseudospiralis(T4)).Clear bands,high polymorphism and good reproducibility was found in the SSR-PCR product of this 10 pairs of primers.Then the SSR-PCR reaction system and reaction procedure was optimized and confirmed.The optimized SSR-PCR reaction system consisted of 0.5 U of Ex-Taq enzyme,0.1 pmol / ?L at the final concentration of common primers,and 7.5 ng / ?L of DNA in 20 ?L reaction system.The SSR-PCR reaction procedure was as follows: 98 ? pre-denaturation 5min;98 ? 10 s,(54??56??58?)30s,72 ? 30 s,35 cycles;72 ? extension 7min.Then,8 % denaturing polyacrylamide gelelectrophoresis were used to seperate the SSR-PCR product,two pairs of SSR core primers(Nos.8 and 10)were screened,which can be completely used for the identification of 12 Trichinella species and genotypes.After that,the SSR-PCR method(the selected No.10 primer)and the multiplex PCR technique were used to identify Trichinella isolate from the pigs and dogs.The results of identification by these two methods are consistent.It is demonstrated that the SSR-PCR method for Trichinella species identification established in this research is feasible and reliable.In this study,SSR-PCR method has been establish for Trichinella species classification and identification,in which single pair of core primer was able to completely distinguish 12 species and genotypes.This study provided the base for research of etiology,epidemiology and prevention and treatment of trichinosis.