Population Genetics And Molecular Diagnosis Of Sporothrix In Some Areas,of China

BackgroundSporothrix complex is a dimorphic fungi and consists of several species with different geographic distributions,virulence,and antifungal susceptibilities.Among the genus,S.brasiliensis,S.globosa,S.schenckii s.str and S.luriei make up the ‘pathogenic clade'.These pathogens could cause chronic infections of human and other animals by invading skin,subcutaneous tissue and lymph,called sporotrichosis.The clinical manifestations of sporotrichosis are diverse and vary in severity.The diagnosis of this disease is mainly based on fungal culture and histopathological examination.With the development of molecular biology,an increasing number of methods based on nucleic acid detection have been applied for the rapid diagnosis of infectious disease.However,there are still shortcomings such as long time-consuming,low sensitivity and complicated operation steps.The incidence of sporotrichosis is increasing,which has attracted the attention of researchers.Northeast China is a epidemic area of sporotrichosis,almost all cases are infected by S.globosa.The genetic diversity of S.globosa was thought to be very low;thus,genotyping and population genetics studies would be difficult.In order to analyze the population genetic diversity of S.globosa in China and make up for the shortcomings of clinical rapid diagnosis of sporotrichosis.In this study,microsatellite length polymorphism(MLP)was used to analyze the population genetics of S.globosa,and real-time PCR assays for sporotrichosis were established.It is hoped to provide a powerful tool for epidemiological monitoring and rapid diagnosis of Sporothrix and sporotrichosis.Methods:1.Population genetic of S.globosa.(1)One hundred and twenty clinical isolates from different regions of China were collected and identified by morphological analysis and sequencing of calmodulin(CAL)sequences.(2)Microsatellites in the S.globosa genome were identify by SSR Identification Tool.And primers were designed using Primer Premier 5.0.(3)Annealing temperatures were optimised with a gradient PCR.Polymorphic microsatellite loci were selected for population genetic analysis of S.globosa.(4)PCR was performed on 120 clinical isolates,and the data were analyzed by bioinformatics software.2.Establishment and evaluation of a real-time PCR assay for S.globosa.(1)Primers and probes were designed by comparing internal transcribed spacer(ITS)sequences of Sporothrix genus in NCBI database.(2)Optimize the reaction system.(3)Sensitivity and specificity was validated by detecting 30 S.globosa,33 other common pathogens and the human genome.The S.globosa nucleic acid was 10-fold diluted(from 2 ng/?l to 0.2 fg/?l for eight gradient concentrations)to measure the limit of detection(LOD).(4)The detection ability was evaluated by clinical samples of S.globosa infection.3.Fast diagnosis of sporotrichosis caused by S.globosa,S.schenckii s.str.,and S.brasiliensis based on real-time PCR.(1)Primers and probes were designed by comparing CAL sequences of Sporothrix genus in NCBI database.(2)Optimize the reaction system.(3)The analytical specificity of the assays was tested by analysing 33 DNA samples derived from other pathogenic fungi,bacteria and tissues from human.(4)The analytical sensitivity,standard curves and LODs of the assay were determined by using three 10-fold dilutions of the previously constructed plasmids,ranging from 2.0×105 copies/?l to 0.2 copies/?l.S.globosa,S.schenckii s.str and S.brasiliensis were detected by FAM,VIC,and CY5 fluorescence,respectively.(5)Comparison of single fluorescence and multiple fluorescences,single sample and mixed samples;clinical samples and simulated infection samples were used to evaluate the multiplex real-time PCR detection system.Results:1.A total of 200 microsatellite locis were found,only 10 locis were amplified stably.The number of alleles per loci ranged from 3 to 13 with an average of 5.50,presenting a moderate PIC value of 0.441.The cumulative discriminatory power of these 10 locis was 1.0000.STRUCTURE analyses showed the clustering level K=3 yielded the largest delta-K value.At K=3,the strains from Changchun were assigned to all three clusters,while the populations from Dalian and Chongqing fell into two clusters.Only cluster II could be found in all three geographic areas.The Fst values among the clusters were notably larger and more significant.PCo A supported the result of the STRUCTURE analyses,suggesting that the population of S.globosa was divided into three clusters.The AMOVA results also indicated that genetic variation was more significant among the three clusters than the groups divided by clinical type.2.The sensitivity and specificity of the real-time PCR for S.globosa were both 100%,the LOD was 10 fg.For the 30 clinical samples,the positive detection rate was 100%,Ct value were between 34.3 and 39.24.3.The sensitivity and specificity of the multiplex real-time PCR were both 100%,LODs were 10,10,and 100 copies for S.globosa,S.schenckii s.str.,S.brasiliensis,respectively.The Ct values of the single fluorescence real-time PCR for S.globosa,S.schenckii s.str.,and S.brasiliensis were 21.67±0.15,24.64±0.15,and 27.00±0.11,while under the same templates and condition,the Ct values obtained from the multiplex real-time PCR were 21.80±0.07,24.77±0.07,and 27.32±0.08.There was no significant difference in Ct values between multiplex and single fluorescence real-time PCR except for S.brasiliensis(T-test,p = 0.03).For the mixed templates,the multiplex real-time PCR assay could detect the corresponding fluorescent signals in the same tube without nonspecific amplification.Of the 40 clinical specimens,7 were excluded.Of the 33 selected samples,the positive detection rates of the culture,species-specific PCR and multiplex real-time PCR assays were 87.9%(29/33),39.4%(13/33),and 93.9%(31/33),respectively.The positive detection rates of the culture and multiplex real-time PCR assays were not significantly different(?2,p = 0.4142).Differences were observed between the multiplex real-time PCR assay and species-specific PCR(?2,p<0.0001).The positive detection rates of the simulated samples were both 100%,and the Ct values were 33.03-38.57(S.schenckii s.str.)and 30.23-34.84(S.brasiliensis),respectively.Conclusion:1.The genetic diversity of S.globosa was low.In this study,we developed a set of microsatellite markers that have a cumulative discriminatory power of 1.000.And S.globosa in this study can be separated into three clusters.2.The population structure diversity of S.globosa in Changchun is higher;the genetic diversity of Cluster II is the highest among three clusters.3.At present,no correlation between the three clusters and clinical types of S.globosa has been found by MLP.4.The sensitivity and specificity of the real-time PCR for S.globosa were both 100%,LOD was 10 fg,and the positive detection rate of clinical samples was 100%.5.The sensitivity and specificity of the multiplex real-time PCR for S.globosa,S.schenckii s.str.and S.brasiliensis were both 100%,LODs were 10,10 and 100 copies,respectively.The assay had a lower reaction cost and sample volume requirements,could detect co-infections and allowed for standardized operation and easier interpretation of results.