Screening Of Species-specific Probes And Primers For Diagnosis Of Sporotrichosis And Comparison Of DNA Typing Methods Of Sporothrix Schenckii

Sporothrix schenckii (S.schenckii) is the causative agent of sporotrichosis, which affects primarily the skin,subcutaneous tissue,lymph vessels, lymphnodes and rarely,visceral organs.Sporotrichosis spread widely in the world. Diagnosis of the disease is fairly difficult now. Species-specific primers and probes which has been reported in the literatures of sporotrichosis were identified and laid a foundation for molecular diagnosis. However, no information of various primers and probes carried out a systematic comparison.The conventional taxonomy of fungi has been maily based on morphology.A problem of S.schenckii morphotyping is that S.shenckii can not be sub-divided within the species. Use of molecular biological methods for gene typing shows certain advantages,such as random amplification of polymorphic DNA (RAPD) assay,mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) analysis and southern blotting analysis of ribosomal-DNA intergenic spacer regions (rDNA RFLP- Southern blotting). In this study,we attemped to use the same strain samples to compare the above three methods on the relationship between DNA patterns and geographic origins and clinical manifestations,to determine which is the best method on the genetic typing of S.schenckii. Also,we attempted to screen the highest specific and sensitive probe and primers from which had been reported of S.schenckii to lay the foudation for the rapid and correct diagnosis of sporotrichosis.Materials and Methods:1. Fifty clinical isolates within 1 ATCC 10268 strain of S.schenckii and 1 strain of Mucor,Aspergillus fumigatus,Candida albicans as the negativecontrols were used in this study. Four pairs of specific primers of S.shenckii have been so far reported.They are the primer set SS3-SS4 (targeting the 18SrRNA gene), the primer set R2-R2 (targeting the chitin synthase 1 gene), the primer set SSHF31-SSHR97 (targeting the DNA topoisomerase II gene) and the primer set ITS3-SSP (targeting the internal transcribed spacer region 2 of rDNA gene).The PCR products were electrophoresed in 1% agarose gel respectively.The DNA band which was at the same level with ATCC strain called positive band.The primers which using the lowest concentration of DNA template amplified the positive bands from S.schenckii strains,but not from negative controls were the highest specific and sensitive primers of S.schenckii.2. The strains were the same with 1. Screen the highest specific and sensitive probe from which has been reported of S.schenckii using the PCR-ELISA format.Five specific probes of S.shenckii have been so far reported.They are the probe U26852 , probe U26866,probe U26866',probe M85053 and probe AF117945. Based on color and the optical density to determine the sensitivity and specificity of the probes.3. On the basis of using "rDNA RFLP-Southern blotting" assay for the genotyping of S.schenckii in the past research,we used the same samples (32 clinical isolates within 1 ATCC 10268 strain of S.schenckii) to do mtDNA typing on RFLP pattern with HaeIII and DNA RAPD analysis with OPAA11. Analyzed the electrophoretic bands, and then compared the three methods.Result:1.With the same PCR conditions,three primer sets (S2-R2,ITS3-SSP,SSHF31-SSHR97) were of high specificity,and S2-R2 had the highest sensitivity. The primer set SS3-SS4 also amplified the positive band fromthe Candida albicans strain. With the same concentration of DNA templatesconditions,the primer set SSHF31-SSHR97 had the lower sensitivity.2. With the same hybridization and PCR-ELISA conditions,all 50 isolates of S.schenckii had a obvious colouration to probe U26852,butnot to other 4 probes.3. Thirty two strains of S.schenckii were composed of 5 mtDNA types, respectively were type 1,4,6,7,20 (correspond to the work of mtDNA anlysis of S.schenckii around the world by Ishizaki et al).The DNA patterns couldbe divided into 7 types by RAPD analysis with OPAA11,and in all the 32 strains a universal band at 0.6kb could be demonstrated.We don't found a obvious correlation between DNA patterns and different geographic areas and clinical manifestations.Conclusion:1.The primer set S2-R2 (targeting the chitin synthase 1 gene) of S.schenckii so far is highly specific and sensitive.2.The probe U26852 (targeting the 28SrRNA gene) of S.schenckii so far is highly specific and sensitive.3. rDNA RFLP-Southern blotting assay for DNA typing of S.schenckii provides a highly sensitive.It is also found that there is an obvious correlation between DNA patterns and different geographic distribution and clinical manifestations by this assay.