Identification Of Sporothix Schenckii By Nested PCR Assay

ObjectTo set up the rapid molecular method to identify Sporothix schenckii from clinical samples.Methods1. strains1.1 38 stains of S. schenckii(including 24 mtDNA types) , from patients of different clinical types and various regions.1.2 10 strains of other clinical common fungi and 2 strains of the related to S. schenckii ( Ceratocystis minor) .2. animals5 male and 5 female, 8 ~ 10 - week - old BALB/c mice( average weight 22 ~24g) purchased from Animal Labortory Center of China Medical Universi-ty.3. Clinical samplesThe clinical biopsy specimens of 9 patients with sporothrichosis comfirmed by culture.4. DNA extractionThe genomic DNA was extracted from 38 stains of S. schenckii (including 24 mtDNA types) , which were collected from different clinical types and various regions , from the tail tissues of 8 experimentally infected mice, which were infected with 1 ATCC10268 strain of S. schenckii, from the clinical biopsy specimens of 9 patients with sporothrichosis comfirmed by culture, and from10 strainsof other clinical common fungi and 2 strains of the related to S. schenckii ( Cera-tocystis minor).5. Nested PCR5. 1 Amplify genomic DNA of which were collected from different clinical types and various regions , from the tail tissues of 8 experimentally infected mice, which were infected with 1 ATCC1G268 strain of S. schenckii, from the clinical biopsy specimens of 9 patients with sporothrichosis comfirmed by culture , and from 10 strains of other clinical common fungi and 2 strains of the related to S. schenckii ( Ceratocystis minor) with the the outer primers SS, and SS2.5. 1. 1 PCR reaction mixture contain: 50jjJ total volumetemplate DNA (100ng/|jd) 5jxl10 x PCR buffer(mg2+) 5jxleach of dATP, dTTP, dCTP, dGTP(2. 5mmol/|ii) ljxlSS]NSS2(20jjLmol/jxl) ljxlTaKaRa Taq (1. 25 U/|xl) 0. 3jxl5.1.2 amplification condition CD predenation 5min at 95 °C(2) 40 cycles of following procedure;1 min at 95 Xi ( denaturation ) , lmin at 56T1 (annealing) ,1 min at 72t (extention).(3) full extention 5 min at 72 Ti5. 1.3 Digested products were electrophoresised in 2% agarose gels 5.2 Nested PCR5.2.1 The reaction mixture of the nested PCR was identical , except that 5 ul of the first reaction prodouct and inner primer pair SS3 and SS4 were used.5.2.2 Digested products were electrophored in 2% agarose gels6. Animal model and experiment infection.A ATCC 10268 strain of S. schenckii was cultivated on sabouraud dextrose agar at 25T1 for 5 days, and theCFU counts of cultures were enumerated. Forexperimental infection, eight mice were injected subcutaneously at four points on their tails with 0. 05 ml of the yeast form suspension (7. 5x10 CFU/0. 05 ml) . Two mice injected with distilled water as the negative controls. The inflammatory tail skin tissues of the eight experimental mice, 35 days after inoculation, and those of the control group were subjected to DNA extraction.Results1. A 305 bp fragment was amplified from all the 38 strians of Sporothrix schenckii with the outer primers SS,andSS2. A 152bp fragment was obtained from 38 strians of sporothrix schenckii, 8 animal specimen and 9 clinical samples with the inner primers SS3 and SS4, but not from the other'species.2. The detection limit of S. schenckii target DNA with ethidium bromide staining by the first - round PCR was 5pg, whereas as little as 50 fg could be detected by the nested PCR. The sensitivity of the nested PCR was 100 times higher than that of the first - round PCR.3. A 152bp fragment was obtained from 8 animal specimen and 9 clinical samples with sporothrichosis comfirmed by culture.Conclusion1. Specificity of nested PCR for S. schenckii. A 152bp fragment was obtained from 38 strians of sporothrix schenckii, 8 animal specimen and 9 clinical samples , but not from the other species.2. Sensitivity of nested PCR for S. schenckii. The sensitivity of the nested PCR was 100 times higher than that of the first - round PCR.3. The capability of this technique to identify S. schenckii from clinical specimens makes it useful for rapid diagnosis of sporotrichosis with high sensitivity and specificity. This assay will not only allow a rapid diagnosis of sporotrichosis but will also provide a practical solution to the difficulties encountered in the identification of pathogens from histochemical and culture - negative sporotri-choid infection.