Molecular Identification And Molecular Diagnosis Of Trichosporon Spp.

ObjectiveInvasive infections caused by Trichosporon spp.are considered to be the second most common cause of mycosis in patients with hematological malignancies.Mortality is extremely high in patients with suppressed immune function,and different species have different resistance to antifungal drugs.Fast and accurate species identification helps early diagnosis and guides early clinical antifungal therapy.Methods 1.Molecular identification of Trichosporon spp..1)After 72 isolates of Trichosporon spp.were cultured,the internal transcription spacer(ITS)gene was amplified by PCR and sequenced,and the species was determined by BLAST;2)Combined with the reference sequence of Trichosporon spp.from NCBI,the N-J tree was constructed for phylogenetic analysis using MEGAsoftware.2.Establishment and evaluation of the high-resolution melting curve method of Trichosporon spp.1)Primers are designed by comparing the internal transcription spacer(ITS)sequences of Trichosporon spp..2)The reaction conditions were optimized;3)Specificity and sensitivity was validated by detecting Trichosporon spp.,common pathogenic fungi and human genomic DNA,the Trichosporon spp.nucleic acid was 10-fold diluted to measure the limit of detection(LOD)and construct a standard curve.3.Establishment and preliminary evaluation of the multiplex real-time PCR methodof Trichosporon spp.1)Primers and probes were designed by comparing the internal transcription spacer(ITS)and 28 S gene sequences of Trichosporon spp..2)The reaction conditions were optimized;3)Specificity and sensitivity was tested by detecting common pathogenic fungi and human genomic DNA,The Trichosporon spp.nucleic acid was 10-fold diluted to measure the limit of detection(LOD)and construct a standard curve.4)Under the same template amount and reaction conditions,comparing the Ct value between single fluorescence and multiplex PCR to evaluate the multiplex PCR system.Results1.72 strains of Trichosporon spp.were identified by DNA sequencing as 5 species,namely T.asahii,T.dermatis,T.inkin,T.dohaense,T.montevideense,and T.asahii is the main species;Phylogenetic analysis found that the interspecies relationship was consistent with theresults of sequencing,and there were obvious evolutionary differences between different species.2.The analysis results of the high-resolution melting curve showed that five kinds of Trichosporon spp.had melting peaks in different temperature ranges.The temperature range of T.montevideense was 75.41-78.05℃(Tm value was 77.23 ± 0.070℃),and T.dermatis was 76.16-78.52℃(Tm value was 77.70 ± 0.050℃),T.dohaense was 76.55-78.63℃(Tm value was 77.94 ± 0.070℃),T.asahii was 76.72-79.07℃(Tm value was 78.31 ± 0.025℃),T.inkin was 77.53-80.26℃(Tm value was 79.37 ± 0.045℃),which could be effectively distinguished by Tm value and melting curve distribution;Common pathogenic fungi and human DNA were used as negative controls,and the specificity was 100%;Compared with DNA sequencing results,the accuracy were 100%;From the results of the standard curve,the limit of detection of T.montevideense,T.dermatis,T.dohaense,T.dohaense,T.asahii,T.inkin were 160 copies.3.The multiplex real-time PCR was optimized experimentally,and double and triplex real-time PCR could be used to identify five species of Trichosporon spp.;Common pathogenic fungi and human DNA were used as negative controls,the specificity was 100%;Compared with DNA sequencing,the accuracy was 100%;And the standard curve showed that the limit of detection of five species of Trichosporon spp.were 40 copies.Under the same template volume and the same conditions,the changes in Ct values of T.asahii and T.montevideense underdifferent systems had statistics significantly in science(P<0.05),there was no statistically significant difference inT.dermatis,T.dohaense,and T.inkin(P>0.05).Conclusion1.Phylogenetic analysis are consistent with sequencing results,rDNA ITS sequencing can accurately identify Trichosporon spp.to species level.2.Real-time PCR combined with HRM analysis provides a simple,short-time,and low-cost method for the accurate and rapid identification of five kinds of Trichosporon.It realizes closed-tube analysis and can be combined with culture to assist clinical early diagnosis of invasive trichosporonosis.3.The multiplex real-time PCR based on ITS and 28 S sequence could identifyfive kinds of Trichosporon,and the accuracy and specificity are 100%,and the limit of detection is 40 copies.It is a simple,accurate,good specificity and high sensitivity analysis method.Quantitative analysis can be performed,which is conducive to early clinical diagnosis and guides early clinical antifungal therapy.