Optimization of DNA Extraction from Medically and Environmentally important Fungi for Polymerase Chain Reaction

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Optimization of DNA Extraction from     Medically and Environmentally important Fungi for Polymerase Chain Reaction

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Acknowledgment

My sincere thanks to Prof. R. S. Dassanayake, Dr. O. V. D. J. Weerasena, (ceygenbiotecdurdans hospital) for granting me a place to do my research.

My thanks to Mr. MohanGeekiyanage, Miss. Pushpamali Silva they helped in each and every practical.

Finally my thanks to Dr.Sajani( lecture of BMS), Mr. NisamRasak ( Director of BMS), for helping me in finding a research project.

Abstract

Fungi are eukaryotes that have cell walls composed of chitin with or without cellulose. They are ubiquitous and comprise an estimated 250,000 species, of which only about 150 have been shown to cause disease in humans. PCR-based fungus specific protocols have been worked out for the medically and environmentally important fungi responsible for the detection using DNA extraction. Following PCR procedures, gel loading was done to observe the band. Spectrophotometric calculation was also carried out to find the amount of extracted quantity of DNA.

Introduction

Fungi are organisms which are neither plants, nor animals. They are one of the most important group of organisms on this planet, some of the world's largest and possibly oldest individuals, silent killers with deadly poisons, vital ingredient in beer and bread, decomposers, essential for natural recycling, helping to guarantee life on earth, miracle cures for disease and indispensible partners for many plants.

The fungi (singular, fungus) include several thousand species of nonphotosynthetic eukaryotic, spore bearing organisms that obtain simple organic compounds by absorption. The organisms have no chlorophyll and reproduce by both sexual and asexual means. The sexual lifecycle of fungi is different from all other eukaryotic in the zygote is the only diploid cell. When two mating types fuse, the two nuclei do not fuse but coexist until the right condition are present. Fusion presents in the diploid zygote, which immediately undergoes meiosis to return to the haploid. The coexistence of two different mating types of nuclei is the dikaryotic stage, which is unique to fungi. The fungi are usually filamentous, and their cell walls have chitin.

Fungi can be recognized by the following five characteristics:

The cells of fungi contain nuclei with chromosomes (like plants and animals, but unlike bacteria).

Fungi cannot photosynthesize (they are heterotrophic, like animals)

Fungi absorb their food (they are osmotrophic)

They mostly develop very diffuse bodies made up of a spreading network of very narrow, tubular, branching filaments called hyphae. These filaments exude enzymes, and absorb food, at their growing tips. Although these filaments are very narrow, they are collectively very long, and can explore and exploit food substrates very efficiently.

They usually reproduce by means of spores, which develop on, and are released by, a range of unique structures (such as mushrooms, cup fungi, and many other kinds of microscopically small fruiting bodies).

Most fungi are filamentous, many grow as unicellular yeasts and some primitive fungi, such as the chytridomycetes, grow as individual rounded cells or dichotomous branched chains of cells with root-like rhizoids for attachment to a nutrient resource.

1.1 Medically important fungi

About 200 fungal species are known to cause human infections. Generally speaking, fungal infections of humans are most common in tropical regions of the world. In recent years, however the number of individuals infected with fungi has been increasing drastically in all regions of the world. This is due primarily to the fact that there are more individuals who are predisposed to fungal infections than ever before. The use of antibacterial and immunosuppressive agents contributes the increase in the number of fungal diseases. Acquired immunodeficiency syndrome (AIDS) patients, for example, are commonly attacked by fungi as are cancer patients, burn victims and organ transplant patients who have been given immunosuppressive drugs. In some cases, the fungi that attack these individuals are species that are recognized for their abilities to cause infections in humans, but in other cases the species involved are ordinary saprobes not normally regarded as threats to human health.

Kwon- Chung and Bennett (1992) provided a list of about 30 of the important mycoses, the sources of the organisms that cause them and the portals by which they enter the body. These diseases vary from superficial mycoses (the skin, nails, etc.) to deeper infections that involve the muscles, blood system and internal organs, examples of some of the most significant mycoses found in different parts of the world include Blastomycoses, Cccidomycoses, Histoplasmosis, Aspergillosis, Crytococcosis and candidiasis. Any of these infections can be fatal under certain conditions.

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1.2 Diagnosis of fungi

                                        Figure (1) Diagnosis of fungi

The rapid availability of genomic DNA from medically important fungi is becoming increasingly important in reference clinical molecular laboratories for accurate molecular epidemiologic subtyping and diagnostic PCR. Direct sequencing systems which will rely upon the rapid availability of genomic DNA are currently under development for clinical molecular laboratories. Previously described methods for genomic DNA extraction from fungal pathogens require hours to days to complete and often incorporate toxic chemicals. ...

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