The Application Of Molecular Biologic Method In The Early Diagnosis Of Deep Fungal Infection

With the wide use of antibiotics, glucocorticoid and immunosuppressive agents, deep fungal infection had been reported in both immunocompromised and immunocompetent individuals. Research showed that a strain of Candida fungal infection had become the first place. According to invasive aspergillosis (IA) patients, data showed that the period between dagnosis and death was generally less than 14 days, therefore, there was an urgent call for us to find new methods to realize the early diagnosis of deep fungal disease, and identified pathogens sensitive to the choice of species to the level of effective drug treatment in the shortest time as pssible. Traditional pathogen identification of fungi was built on the basis of positive clinical specimen culture. It suggested that we might wait for a few days even a few weeks to make a further species identification. According to the clinical statistics, blood cultures of 45%-75% disseminated candidiasis or IA patients were negative, which limited the scope of the application, for the fungal culture-negative samples could not be used to identify the species.Therefore, we tried to establish the application of new methods to solve these problems and provide better service to the clinical. We used rapid PCR method to test the urine, alveolar lavage fluid, peritoneal fluid and bile from the clinical patients in vitro. This research applied PCR and real-time PCR in the meantime, they could analyze the samples both qualitative and quantitive, it's the first step try of applying molecular bilolgy method into the clinical fungi testing directly.PART 1 The study on rapid PCR test of pathogenic fungi from the simulated body fluid (SBF)Objective: Test pathogenic fungi from simulated body fluid by the rapid PCR. Methods: Applying fungal ITS4 and ITS86 universal primers to amplify pathogenic fungi genes in the SBF with method of rapid PCR, PCR products corresponding strain -- with the control group using traditional methods to extract DNA fragments after the scan results contrast.Result:No significant difference of the length of amplicons was found between the fungal suspension and control. The whole process took only 7h.Conclusion: The application of ITS fungal universal primers and PCR to test the fungal in the SBF might provide an accurate, specific, sensitive, and rapid approach to detect the pathogenic fungi.PART 2 The study on rapid PCR test of pathogenic fungi from humoral clinical specimensObjective: Test pathogenic fungi from humoral clinical specimens by the rapid PCR. Methods: The pathogenic fungi in the clinical humoral specimens were detected by rapid PCR with fungal ITS4 and ITS86 universal primers. PCR products corresponding strain -- with the control group using traditional methods to extract DNA fragments after the scan results contrast.Result:Humoral direct clinical specimens by PCR amplification of DNA fragments with the scan results were similar. The difference was not significant. Conclusion: The combination of humoral clinical specimens PCR with ITS fungal universal primers might provide an accurate, specific, sensitive, and rapid approach to detect the pathogenic fungi.PART 3 Prospective study of PCR test with humoral clinical specimensObjective: To evaluate the sensitivity and specificity of fast PCR diagnostic method for the deep fungal infectionMethods: Application of fungal ITS4 and ITS86 universal primers on 83 cases of humoral clinical samples for PCR test to detect pathogenic fungi, the results were compared with traditional methods and analyze statistically.Result:The positive rate of PCR test with clinical body fluid samples and the traditional method was similar. There was no significant statistical difference. Conclusion: To detect the humoral clinical body fluid samples with ITS fungal universal primers and prospective PCR test might provide an accurate, specific, sensitive, and rapid approach to detect the pathogenic fungi.PART 4 The application of Real-time PCR to detect the content of fungi in clinical specimensObjective: Detect the content of fungi from clinical specimens by the method of Real-time PCR.Methods: The clinical specimens were amplified by real-time PCR with specific primers of Candida albicans, which were labeled with FAM. The changes in the fluorescence signal through PCR process were recorded as Ct volumes, then the initial template quantity were calculated.Result:To detect the clinical specimens directly with real-time PCR could accurately show the original copies of fungi in the sample, which might be quantitatively analyzed. Conclusion: With the application of Candida albicans specific primers and fluorescence probe body, the method of real-time PCR could detect the content of fungi in clinical samples, which might be an accurate, rapid and quantitatively approach.