Studies On Molecurlar Typing Methods Of Cryptococcus Neoformans

Cryptococcus neoformans is an encapsulate yeastlike fungus that causes the disease cryptococcosis in immuno-compromised persons. C. neoformans have been recognized,originally distinguished in five serotypes: serotypes A, D AD, B and C. In order to discriminate between individual isolates in a population which is morphologically and physiologically indistinguishable, a stable and sensitive genetic marker is needed. Such a marker would enable the identification of different serotypes and allow discrimination between a relapse and a reinfection.The aim of this paper is to research the molecular typing methods of C.neoformans, incuding (1) Studies on protoplasts formation and methods of DNA extraction from C.neoformans (a) Studies on experimental conditions for production of C.neoformans protoplasts :The isolation of protoplasts was a very useful procedure in prepareing DNA! RNA and organelles such as mitochondria for biochemical studies. To establish conditions for the protoplast formation of C.neoformans, Various enzymes (Lywallzyme and NovoZyme234) and concentration of enzyme (1,3,5mg/mi) and pH values (p1-14,5,6,7,8) and osmotic stabilizers (sucrose,mannitol and sorbitol) were examined. The result of our experiment showed that: The early exponential phase cells were easy to form protoplast, under the action of pH 6.0 and 5mM DTT, 0.~ M sorbitol used as osmotic stabilizer, NovoZyme 234 at the concentration of 3mg/mi was the most effective for the protoplast yield . The ratio of protoplast formation were 98% in 75minutes. The results of Lywallzyme were similar to NovoZyme 234. (b) Rapid Extraction of mitochondrial DNA from C.neoformans and examined by electronic microscopy: To establish rapid and effective method for the extraction of mitochondrial DNA from C.neoformans and assay their morphological characteristics. The method is based on :C. neoformans may be grown to the exponential phase,are broken by a combination of lysis of the cellular membrane with NovoZym234 and mechanical means,and mitochondrial DNA was extracted from DNaseI-treated 3 t~Z mitrochondrial preparetion by differential centrifugation. Three pellets,mcluding yeast cell , protoplasts, mitochondrial, were examined by transmission electronic microscopy 20kg mtDNA were obtained from C. neoformans, and its purity was enough. A rapid and simple method for the preparation of the mitochondrial DNA of C. neoformans is established. (c) Development of rapid methods and comparison of different methods to extract DNA from C.neoformans: DNA is an important fundamental prerequisite to further downstream molecular analyses including nucleic acid amplification techniques. In this study ,Two simple and rapid methods for extracting genomic DNA were established and used as template for PCR. Comparative studies of four different DNA extraction methods were evaluated in our study in order to ascertain which method would be the most suitable to isolate DNA ,which could be used further in a molecular analysis, from C.neoformans. These methods investigated were as follows:3%SDS lysis, classical lytic enzyme treatment,glass beads and the benzyl chloride methods. In glass beads method ,cells were broken by vigorously mixing with glass beads on a votorex;Jn 3%SDS method,cells were heated in 3%SDS extraction buffer containing 10mM DTT for 20 minutes .The lysate was extracted with 5 M KAc and isopropanol. Yield of DNA was calculated from the A260 for c...