| Background and purposeEpidemiological studies on Trichosporon infection have shown that Trichosporon species has become the second most common yeast pathogen in patients with hematological malignancies and fungemia.Among the invasive yeast infections,the pathogenic rate of Trichosporon species is second only to that of Candida and Cryptococcus.Trichosporon asahii is the most commonly isolated from patients with trichosporonosis.The mortality from disseminated trichosporonosis is as high as 50%to 80%,and even 100%in patients with malignant hematologic diseases and persistent neutropenia.Due to the rapid deterioration of patients with invasive or disseminated trichosporonosis,rapid and accurate diagnosis method is vital for the timely and rational application of antifungal drugs and reversing the situation of high mortality.Nucleic acid amplification technology(NAAT)has been widely used in the diagnosis of Trichosporon infection because it can rapidly and efficiently amplify the characteristic nucleotide sequences of pathogens.However,in the past,NAAT mostly relied on pure culture of pathogens,DNA extraction and subsequent product sequencing to obtain test results,which seriously weakened the advantage of high timeliness of NAAT.In order to improve the timeliness of NAAT in the diagnosis of Trichosporon infection,we chose colony PCR technology which does not require complex DNA extraction steps and recombinase polymerase amplification(RPA)requiring only 10-20 minutes for amplification to establish 2 rapid diagnosis methods for T.asahii infection suitable for well-equipped and poorly equipped medical institutions,respectively.The first part of this study aimed to establish a DNA releasing method of T.asahii colony that can replace the DNA extraction before nucleic acid amplification,and to initially form rapid processing strategy for clinically accessible samples infected by T.asahii.The second and third parts of this study aimed to achieve rapid specific detection of T.asahii by colony PCR and colony RPA based on the results of the first part,and to observe the applicability of these 2techniques to clinical samples.The fourth part of this study aimed to test the utility of 2rapid detection techniques using a mouse model of T.asahii disseminated infection.MethodsPart I:PCR based on universal primers of intergenic spacer(IGS)was used to evaluate the DNA releasing efficiency of T.asahii by glass beads method,freeze-thaw method,direct method,enzymatic hydrolysis method and conventional DNA extraction method.Then the performance of the optimal method for the processing of blood samples was observed.1.Sensitivity evaluation was performed by PCR which used decimal concentration gradient of suspensions of T.asahii CBS2479 processed by 5 methods as the template;2.Positive rate evaluation was performed by PCR which used suspensions with a concentration of 10~3CFU/mL of 15 T.asahii strains processed by 5 methods as the template;3.The DNA releasing efficiency of T.asahii colony by four methods was indirectly evaluated by sensitivity and positive rate,while being compared with those of conventional extraction method.Finally,an optimal method was selected;4.Response surface method was used to optimize the selected method,and the improved DNA releasing efficiency was verified by the sensitivity changes of PCR;5.After processing of whole blood,urine and bronchoalveolar lavage fluid(BALF)sample mimicking T.asahii infection with the optimal method,the DNA releasing efficiency of T.asahii in the mimicking sample was evaluated indirectly by PCR results,and the processing method of the mimicking sample was improved accordingly.Part II:1.The species-specific primers of T.asahii were manually designed based on the comparison results of the 15 IGS genotypes of T.asahii and the sequence of IGS of closely related species;2.In small sample tests,primers were screened according to their specific performance in PCR,and then the specificity of optimal primer was verified by a large sample test based on blind design;3.The sensitivity of colony PCR to serial dilution of T.asahii suspension and DNA samples was observed;4.The colony PCR reaction system was optimized by orthogonal design.The improved performance of colony PCR after optimization was verified by the change of sensitivity detection level,and the specificity of colony PCR detection was re-checked;5.The sensitivity and detection rate of colony PCR to sample mimicking T.asahii infection were observed by gel electrophoresis image and direct fluorescence staining.Part III:1.Based on the IGS sequence differences between T.asahii and its closely related species used in the primer design of the part II,the species-specific primers of T.asahii used in this part was designed manually according to the design requirements of RPA primer;2.The specificity of primers was observed in small sample tests and optimized by temperature gradient amplification;3.Based on blind design,the specificity of primers was verified by large sample test s;4.Observe the sensitivity of RPA to serial dilution of T.asahii DNA samples;5.Orthogonal design was applied to optimize the RPA reaction,and the improved performance of RPA after optimization was verified by the change of sensitivity level,and the specificity of RPA detection was re-checked;6.Observe the sensitivity of glass beads method combined with RPA for detecting serial dilution of T.asahii suspension;7.The sensitivity and detection rate of colony RPA to sample mimicking T.asahii infection were observed by gel electrophoresis image and direct fluorescence staining.Part IV:1.Immunosuppression in mice was performed by cyclophosphamide and dexamethasone,and the model of T.asahii disseminated infection was established by tail vein inoculation;2.Through fungal microscopy,fungal culture of tissue homogenate,histopathological examination and PCR product sequencing assay,to confirm the infectious strain and the success of model establishment,and calculate the fungal burden of each organ;3.Samples of blood,urine and BALF at 6 time points were detected by colony PCR and colony RPA respectively.ResultsPart I:The glass beads method can efficiently release DNA of T.asahii colony and is suitable for the processing whole blood,urine and BALF.1.The sensitivity of PCR based on glass beads method and freeze-thaw method was3×10~2CFU/mL,and the sensitivity of direct method,enzymatic method and conventional DNA extraction method was 3×10~3 CFU/mL;2.The positive rate of PCR based on glass beads method was 100%,which was significantly higher than the other four methods;3.The procedure time of glass beads method was shorter than other methods except direct method;4.The sensitivity of colony PCR was improved to 30 CFU/mL after the glass beads method was optimized;5.The sensitivity of PCR to the three clinical samples processed by glass beads was 30CFU/mL,and the sensitivity of whole blood depended on the addition of concentration and purification steps after glass beads processing.Part II:The colony PCR is highly sensitive and specific to T.asahii,and is suitable for directly detecting T.asahii in whole blood,urine,and BALF.1.Colony PCR based on primers TA4F and TA4R was highly specific to T.asahii;2.The sensitivity of colony PCR for T.asahii suspension and DNA sample was 10CFU/mL and 1fg/mL,respectively;3.The results of gel electrophoresis and direct fluorescence staining were consistent.The sensitivity of colony PCR to detecting whole blood sample mimicking T.asahii infection was 30 CFU/mL,the sensitivity to urine and BALF was 10 CFU/mL,and the detection rate to mimicking samples with spore concentration of 300 CFU/mL was 100%.Part III:The colony RPA allows rapid and accurate detection of T.asahii in whole blood,urine,and BLAF within a short time.1.Colony RPA based on primers TA4RPAF and TA4RPAR4 meets the requirements for specific detection of T.asahii at reaction temperature of 47.5℃;2.The sensitivity of colony RPA for T.asahii DNA sample and suspension was 1fg/mL and 30 CFU/mL,respectively;3.The sensitivity of colony RPA to detecting whole blood,urine and BALF samples mimicking T.asahii infection was all 30 CFU/mL,the detection rate of mimicking infectious samples was 100%,and the results of gel electrophoresis and direct fluorescence staining were consistent.Part IV:Both colony PCR and colony RPA can detect T.asahii in the blood,urine,and BALF of mouse model with confirmed disseminated T.asahii infection.1.According to the results of tissue culture and sequencing,T.asahii disseminated infection was observed in all the 18 experimental mice,and the model was successfully established;2.The blood,urine and BALF of mice in the experimental group were tested by colony PCR and colony RPA,and the results were all positive,while those of the control group were all negative;3.The results can be accurately judged by gel electrophoresis and direct fluorescence staining;4.Colony PCR and colony RPA can detect T.asahii from blood,urine and BALF of mice 6 hours after hematogenous dissemination.Conclusions1.The glass beads method can serve as an efficient technology for releasing DNA of T.asahii in whole blood,urine,and BALF prior to application of NAAT,and the amplification results can be read directly by fluorescence staining;2.The method for rapid detection and identification of T.asahii by colony PCR,which can be applied directly to whole blood,urine and BALF,has been successfully established for immediate diagnosis of T.asahii infection;3.The method for rapid detection and identification of T.asahii by colony PRA,which can be applied directly to whole blood,urine and BALF,has been successfully established,this method is simple and time-saving,and can be used as a tool for immediate diagnosis of T.asahii infection in poorly equipped regions or institutions;4.Blood,urine and BALF can be selected as samples for the early diagnosis of T.asahii disseminated infection,and the two methods can be used for the early diagnosis of T.asahii disseminated infection. |
