Analysis Of Sporothrix Schenckii Of Mitochondrial DNA Type 4 And Population By Random Amplification Of Polymorphic DNA Assay

ObjectiveTo investigate the DNA polymorphism of Sporothrix schenckii of mitochondrial DNA type 4 and to study initially the relationshoip between mitochondrial DNA(mtDNA) typing and RAPD typing by Random Amplification of Polymorphic DNA(RAPD) Assay.Materials and Metheods1．Strains36 strains of Sporothrix schenckii confirmed as mtDNA type 4, which were collected from the northern China and from patients of differrent clinical types.24 strains sporothrix schenckii including 24 mtDNA types which were collected from different clinical types and various regions.2．Materials2．1 DNA extraction reagents: hexadecyltrimethylammonium bromide(CTAB) trisaminomethane-ethylenediaminetetraacetic acid (TE)2．2 PCR reagents 2．2．1 Random primer: OPBG011 5'-GTGGCTCTCC-3' OPBG14 5'-GACCAGCCCA-3' OPBG19 5'-GGTCTCGCTC-3' OPB07 5'-GGTGACGCAG-3' OPAA11 5 '-ACCCGACCTG-3' OPD18 5'-GAGAGCCAAC-3' OP02 5'-(GACA) 4' OP08 5'-TGCCGAGCTG-3' 2．2．2 5U/ul TaKaRa Taq3．Metheods3．1 DNA extractionThe genomic DNA was extracted with hexadecyltrimethylammonium bromide and diluted to the concerntration of 100ng/ul for PCR.3．2 PCR3．2．1 PCR reaction mixture contain :25ul total volumeprimer (100pmol/ul) 1ulTaKaRa Taq(5U/ul) 0．25ul10(?)buffer(include MgCl) 4uldNTP(dATP(?) dCTP(?) dGTPand dTTP 2．5mmol/L) 4ultemplate DNA(100ng/ul) 1ul3．2．2 amplification condition: Predenation 5min at 94(?), 45cycles of following procedure : 1min at 94 (?) (denation),1min at 36(?) (annealing), 1min at 72(?) (extention). Full extention 7min at 72 (?)3．2．3 The amplified products were subjected to electrophoresis in 1．5% agarose gels ,stained with ehidum bromide ,and visualized under UV light and taked photos by untralviolet light imaging system.3．3 Statistical AnalysisThe RAPD results of 36 strains with primers of OPB07, OPBG14 and OPB02 and 24 strains of type 1-24 of OPB 02 was analyzed by using NTSYS-PC 2．02 statistical package. Finally we got the tree diagram of clustering that showed the relative relation of the strains.Results1．36 strains of Sporothrix schenckii of mtDNA 4 type were divided into two genotypes by RAPD with primer 07, four genotypes with primer 14 and three genotypes with primer 02．2．24 strains of Sporothrix schenckii collected from different regions were amplified with primer 02．The results showed that the RAPD patterns of the Group B strains (mtDNA type 4-10,12,13,20-21 and24) have little difference between type 4 and 6, type7and 20, type 8,9,10,13 and 20 with the same RAPD patterns while type 5,12 and 24 with several different patterns from others. Then the difference between the RAPD patterns of the Group A strains (mtDNA type 1-3,11,14-19,22and23) is much bigger without the same patterns.3．Clustering assay results36 strains of mtDNA type 4 could be classified into 4 subtype at the level of SM=0．92 in the tree diagram with the range of the similarity from 0．87 to 1．0．24 strains of mtDNA 1-24 could be classified into 2 groups at the level of SM=0．57,which corresponded to Group A and B with the exeception of type 22．ConclusionsSporothrix schenckii of mtDNA type 4 could be divided into 4 subtypes by RAPD which is useful in molecular epidemiological investigations of S. schenckii; there maybe some consistency between RAPD typing and mtDNA typing.