The Development Of Real-time PCR For Detection Of Cryptococcus Neoformans And A Study On Genotype And Antibiotic Resistance Of Cryptococcus Neoformans Clinical Isolates

ObjectsThe purpose of this study is establishing a Real-time PCR method in detection of Cryptococcus neoformans in CSF and optimizing the DNA extraction methods of that.The aim is also to understand the seed,genotype and mating type,virulence and drug resistance characteristics, reflecting prevalence of C. neoformans in Guigang region the Guangxi Zhuang Autonomous Region and enrich Chinese epidemiological data,through molecular epidemiology investigation.MethodsDetermine the C. neoformans detection targets according to the literature, identified conserved gene sequence by sequence alignment, Real-time PCR primers and probes designed due to the primer design principles. With plasmid standards, establishing a standard curve and the detection limit of the detection system. To prepare the analog samples, we put clinically normal cerebrospinal fluid samples into different concentrations of C. neoformans, parallel detection using negative staining with ink,isolated culture and method of this study and comparing the results. In order to enhance the extraction of DNA in CSF samples of C. neoformans, various DNA extraction methods was referenced.Twenty clinical isolates of C. neoformans strains Guigang region in the Guangxi Zhuang Autonomous Region in 2009?2012 was collected. Molecular biology was verified by PCR amplified ITS sequence, serotypes, mating type was identified using PCR amplification primers. Identification and verification of genotypes was by M13-PCR fingerprinting and URA5-restriction fragment length polymorphism analysis. ST typ and relevance was detected by site sequence typing (MLST).Researches of virulence features include 37? growth test,melanin production test,urease test. Resistance study was through VITEK automatic detection and E-test method. C. neoformans clinical isolates and six kinds of common fungal drugs(Amphotericin B, Flucytosine, Fluconazole, Voriconazole, Posaconazole, Itraconazole)in vitro drug sensitivity test.Results1. Through the laboratory evaluation,the specificity and the sensitivity of the Real-time PCR detection system are both 100%.2. When the plasmid concentration in the range of 1.0×101-1.0×108 copies /?l, R2=0.998, there is a strong correlation between plasmid concentration and Ct value.Amplification efficiency (Eff) is 94.108%, Real-time PCR detection system amplification efficiency is higher.3. The detection capability of this Real-time PCR detection system is better than negative staining ink and cultured method, Real-time PCR can detected 3.25CFU /ml strains in CSF samples.4. Through molecular identification, among 20 clinical strains, 19 are C. neoformans var. grubii, blood type VG I . PCR fingerprinting and URA5-RFLP genotyping experimental results are consistent.5. MLST results showed Guigang region in the Guangxi Zhuang Autonomous Region advantages sequence typed was ST5, the new ST374 has close genetic relationship with ST5, they can be grouped into the same sequence group.6. The results of virulence test showed that all the strains grew well at 37? and positive in both urease test and melanin production test.7. Experimental strains drug susceptibility of six kinds of antifungal, Vitro Experimental results show that all strains were moderately sensitive to amphotericin B, 85% of the isolates to fluconazole is moderately sensitive, there are two strains of fluorocytosin performance moderately sensitive and showed sensitivity to other drugs, there is no resistant strains.Conclusions1. This study established a rapid Real-time PCR method to detect of C. neoformans in cerebrospinal fluid. According to laboratory evaluation, this method has high specificity and sensitivity, low detection limit. The advantages are quickly and accurately identifying of the cerebrospinal fluid in C. neoformans and Gert. By further clinical validation, this method can be used for rapid diagnosis of cerebrospinal fluid cryptococcal infection.2. This study shows that major pathogenic species of C. neoformans in Guigang area is C. neoformans var. grubii (95%), serotype type A, mating type a, genotype VN I type found C. gattii strains. ST5 is the mainly type (80%), the discovery of new MLST sequence type is ST374 which is closed to the ST5. C. neoformans clinical isolates and six kinds of common fungal drugs (Amphotericin B,Flucytosine, Fluconazole,Voriconazole,Posaconazole,Itraconazole) in vitro drug sensitivity test showed a sensitive or moderately sensitive, no resistant strains.