Relationship Of Mitochondrial DNA Of Sporothrix Schenckii And Clinical Patterns Of Sporotrichosis

ObjectiveTo compare the mycological characteristic of S.schenckii from different clinical patterns of sporotrichosis by routine mycological morphology and understand mitochondrial DNA(mtDNA)of S.schenckii in Liaoning China.At the same time,to explore the relationship of mtDNA and clinical patterns of sporotrichosis.Materials and Metheods1.Strains47 strains S.schenckii which were collected from different clinical types in Liaoning province and in which,23 strains collected from the regional lymphangitic sporotrichosis and others from fixed cutaneous.2.Materials(1)Sabouraud liquid medium(2)EDTA buffer(3)Zymolyase 100T(4)β-mercaptoethanol(5)20%SDS(6)TE buffer(7)HaeⅢenzyme(8)phenol(9)Agarose3.Metheods (1)The sporothrix strains causing cutaneous sporotrichosis were compared by routine mycological morphology.(2)Preparation of mtDNA:mtDNA from S.schenckii was prepared by the modified method.In brief,S.schenckii cells cultured in Sabouraud liquid medium were disrupted by sonication after treatment with Zymolyase-100T in saline EDTA buffer. The mitochondrial fraction was obtained by centrifugation at 3,600rpm for 10 minutes, followed by centrifugation of the supernatant at 13,000 rpm for 10 minutes.The mtDNA was isolated from the mitochondria by phenol extraction and ethanol precipitation.(3)Restriction analysis:Each mtDNA was digested with HaeⅢand electrophoresed on 0.8%agarose gels.(4)Statistical Analysis①The relationship of mtDNA of 47 strains and clinical patterns of Sporotrichosis was analyzed by using SPSS statistical package.②Analyzed the relationship of gender and clinical patterns using SPSS statistical package.Results1.The feature of the clinical isolated sporothrix strains are same as each others and couldn't discriminate.2.47 isolates of S.schenckii comprised 5 mtDNA types:(1)Type 4:bands at 10kb,4.5kb,3.6kb,1.9kb,1.88kb,1.25kb,0.86kb和0.75kb(2)Type 5:bands at 10kb,4.5kb,3.6kb,1.9kb,1.25kb,0.96kb,0.86kb和0.75kb(3)Type 6:bands at 10kb,8.2kb,4.5kb,3.6kb,1.9kb,1.25kb,0.86kb和0.75kb(4)Type 20:bands at 10kb,4.5kb,3.6kb,2.4kb,1.9kb,1.25kb,0.86kb和 0.75kb(5)Type24:bands at 10kb,6.4kb,4.5kb,3.6kb,1.9kb,1.25kb,0.86kb和0.75kb3.In the 5 mtDNA types:40 isolates of Type 4,2 of Type 6,lof Type 5,1 of Type 20 and 2 of Type 24;18 isolates of Type 4 in the regional lymphangitic sporotrichosis,22 of Type 4 in fixed cutaneous sporotrichosis.Conclusions1.Use routine mycological morphology methods do not distinguish between different clinical patterns isolated sporothrix strains.2.Isolates of S.schenckii in Liaoning comprised of Types 4,5,6,20,24 and Type 4 is predominant in Liaoning province.3.Clinical patterns of sporotrichosis(type of lymphangitic and fixed)is related to mtDNA Type 4,and has nothing to do with gender.