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. Additionally, the release of DNA is often poor due to cell walls or capsules that are not readily susceptible to lysis.
MycoSpin DTM spin Column kit is a method of DNA extraction which permits rapid lysis of cells and recovery of nucleic acids. To our knowledge, such rapid extraction procedures have not been applied to medically important fungi. We therefore investigated a new rapid DNA isolation method using MycoSpin DTM spin Column kit and incorporating chaotropic reagents and lysing matrices of protocols were better than standard phenol-chloroform (PC) extraction protocols for DNA isolation from three medically important fungi;Candida, yeast, and mushroom. The integrity and quality of the extracted genomic DNA were further validated by spectrophotometric detection (Ahn et al. 1996; Haqueet al. 2003) (Nicklas, 2003), determination of the maximum amplifiable dilution of DNA (Olexová et al. 2004), and semi-quantitative determination of the amount of DNA by staining with Ethidium bromide and evaluation on agarose gel.
1.3 Impact of microbe
Microbes are microscopic organisms which are found living all over the biosphere where ever water, soil, or air is present. Divers in nature, microorganisms include bacteria, fungi, Archaea and Protists and sometimes even viruses although none living. The impact of microorganisms on the environment and on humans may be advantageous or destructive. Microbes are extensively made use of by humans in genetic engineering and they also play a major role in balancing the environment. However disease causing pathogenic bacteria both in plants and animals are unfavorable.
Candida is a genus of yeasts. Many species are harmless commensals or endosymbionts of hosts including humans, but other species, or harmless species in the wrong location, can cause disease. Candida species are the most common cause of invasive fungal infections in humans, producing infections that range from non–life-threatening mucocutaneous disorders to invasive disease that can involve any organ. Candida albicans can cause infections (candidiasis or thrush) in humans and other animals, especially in immunocompromised patients (Ryan KJ et al, 2004). Many species are found in gut flora, including C. albicans in mammalian hosts, whereas others live as endosymbionts in insect hosts (Nguyen NH, Suh SO et al, 2007).
Candida yeast produces alcohol (ethanol) and acetaldehyde (chemical responsible for hangover symptoms) as the major products of its metabolism. A healthy body detoxification system can eliminate this problem, however an overgrowth of intestinal candida, means the levels of alcohol entering the bloodstream are greatly increased. If your body is constantly absorbing a high level of alcohol for years this will affect health as nutrients and valuable liver enzymes will become depleted resulting in oxidative stress, due to being constantly overworked trying to detoxify the yeast products. Direct damage to multiple body systems can also be caused by the yeast products. Effectively the whole body is poisoned by a candida overgrowth, resulting in an array of candida symptoms affecting the whole body.
Candida causes many different candida symptoms. This is because when the candida breaks through the gut and bowel walls it goes off round the body setting up colonies and infecting organs and muscle tissue. This often results in not only bloating but a wide range of unpleasant candida symptoms affecting different parts of the body in different severity for different people, hence making it difficult for doctors to diagnose. Candida symptoms include food intolerances, allergies, IBS, asthma, migraines, depression, arthritis, PMT and many other illnesses and chronic conditions.
Saccharomyces cerevisiaeit isalso known as“sugar fungus”. That is what this yeast uses for food. They are found in the wild growing on the skins of grapes and other fruits.Saccharomyces cerevisiae has adapted in several important ways. One is the fact that they are able break down their food through both aerobic respiration and anaerobic fermentation. They can survive in an oxygen deficient environment for a period. Another adaptation they have is their ability to have both sexual and asexual reproduction. Very few other Ascomycota can do both processes. And very few organisms can do all four of these processes. This allows this species to live in many different environments (Madigan, 457). Saccharomyces cerevisiae has both asexual and sexual reproduction. In asexual reproduction the haploid of the yeast under goes mitosis and forms more haploid yeasts. There is a ά strain of these haploids. Then these haploid yeasts, one from each strain, can fuse together and become on cell. Then the nuclei of both cell fuses together and this cell is now the zygote. These diploid cells can go through mitosis, which they call budding, and four more zygotes or they can undergo meiosis and from an ascus which will split into four ascospores. These haploids can then undergo germination and become haploid yeast again. (Madigan, 457)
Saccharomyces cerevisiae is one of the most important fungi in the history of the world. This yeast is responsible for the production of ethanol in alcoholic drinks and is the reasons bread dough rises in the pan. That is where the names brewer’s and baker’s yeast come from. The process in which it produces ethanol is one way this yeast converts glucose into energy. There are two ways Saccharomyces cerevisiae breaks down glucose. One way is through aerobic respiration. This process requires the presence of oxygen. When oxygen is not present the yeast will then go through anaerobic fermentation. The net result of this is two ATP, and it also produces two by products; carbon dioxide and ethanol. So if this yeast is allowed to grow in a container lacking oxygen it will produce ethanol (alcohol). Humans have been isolating this process since the beginning of history. The yeast helps in the rising of bread with its other by-product carbon dioxide. The gas that is produce inside the dough causes it to rise and expand. Both of these processes use the haploid of this yeast for this process. In industry they isolate one strain, either a or ά, of the haploid to keep them from undergoing mating. (Madigan, 457) In the baker’s yeast they have a strain were the production of carbon dioxide is more prevalent then ethanol and vice versa for brewing. (Tomvolkfungi.net) Another importance is that “live yeast supplementation to early lactating dairy goats significantly increased milk production” (Stella, A.V.1).
Mushrooms have been recognized as most loved vegetarian food, rich in nutrition, particularly protein. With their flavor, texture, nutritional value, very high productivity per unit area and time, less dependence on land and ability to grow on a variety of residual agricultural wastes, mushrooms have rightly been identified as a food source to fight malnutrition in developing countries. A mushroom is the fleshy, spore-bearing fruiting body of a fungus, typically produced above ground on soil or on its food source. The standard for the name "mushroom" is the cultivated white button mushroom, Agaricusbisporus; hence the word "mushroom" is most often applied to those fungi (Basidiomycota, Agaricomycetes) that have a stem (stipe), a cap (pileus), and gills (lamellae, sing. lamella) or pores on the underside of the cap. Many mushroom species produce secondary metabolites that can be toxic, mind-altering, antibiotic, antiviral, or bioluminescent. Although there are only a small number of deadly species, several others can cause particularly severe and unpleasant symptoms. Many mushroom species produce secondary metabolites that can be toxic, mind-altering, antibiotic, antiviral, or bioluminescent. Although there are only a small number of deadly species, several others can cause particularly severe and unpleasant symptoms.Medicinal mushrooms are mushrooms or extracts from mushrooms used or studied as possible treatments for diseases. Some mushroom materials, including polysaccharides, glycoproteins and proteoglycans, modulate immune system responses and inhibit tumor growth. Some medicinal mushroom isolates that have been identified also show cardiovascular, antiviral, antibacterial, antiparasitic, anti-inflammatory, and antidiabeticproperties (Smith JE., 2000). Mushrooms can be used for dyeing wool and other natural fibers. The chromophores of mushroom dyes are organic compounds and produce strong and vivid colors, and all colors of the spectrum can be achieved with mushroom dyes. Before the invention of synthetic dyes, mushrooms were the source of many textile dyes (Mussak R., 2009)
1.4 Economival useful of fungi,
Medicinal use
Antibiotics such as penicillin (Penicilliumchrysogenum).
Immunosuppressantslike cyclosporine (Tolypocladiumnivenum).
Cholesterol-reducing lovastatin (Monascusruber, Aspergillusterreus).
Antifungal drug/agents like griseofulvin(Penicilliumgriseofulvum).
Oriental herbal medicines (Cordycepssinensis).
Recycling organic matter (plant debris) by saprophytic fungi
Mycorrhizalfungi:–
Associated with roots of 90% of all vascular plants such as arbuscularmycorrhizaein crops (Glomussp.,Gigasporasp.), and ectomycorrhizaein most woody plants (Lactarius sp., Laccaria sp.)
Edible fungi:–
Edible mushrooms, cultivated or wild (Boletus edulis, Morchellasp., Tuber sp., Lentinulaedodes, Agaricusbisporus)
Production of food in agriculture and the food-processing industry
Candida krusei,Geotrichum sp., Penicilliumcamemberti, Saccharomyces cerevisiae.
Use in biological control of insects, nematodes, pathogenic fungi, and weeds
Ampelomycesquisqualis, Cordycepssinensis, Nematophthoragynophila, Verticilliumchlamydosporium, Phlebiagigantea, Trichoderma sp., Fusarium sp.
Use in ecology as indicators of vegetationaloratmosphericalchanges
Lichens (association of a fungus and an alga)
Source of natural dyes -
Macromycetes (Boletus spp., Cortinarius spp., Hygrocybe spp.)
Lichens (Roccella spp. Ochrolechiatartarea, Parmeliasaxatilis)
Source of commercially important enzymes and natural products -
Trichodermareesei, Aspergillus sp., Mucor sp., Rhizopus sp. (Tadych, 1992).
Objective
Extraction of different fungal DNA from different types of fungi
Optimization of fungal DNA Extraction procedure to improve the sensitivity of the Extraction kit
Optimization of PCR program me for amplification of fungal DNA
METHODOLOGY
Materials
PCR
Gel electrophoresis
Method
First we prepared the sample and transfer into the eppendoff tube and centrifuged 5000 rpm for 5 minutes and discard the supernatant and take the pellet. Add 100 µl of Enzyme Buffer to the cell pellet and suspend the cells by either vortex mixing or pipetting the cells repeatedly using a 1 ml capacity pipette tip and add 5 µl of zymolase (50mg/ml) and incubate at 37 °c water bath for 30minutes. Centrifuge 5000 rpm for 5 minute, discard the supernatant. Add 200 µl of Lysis buffer (LB) and 20 µl of Protinase K to200 µl to the pellet. Mix briefly by vortexing and incubate at 70°c for 10 minutes.
Add 100 µl Protein Removal Buffer (PRB) and centrifuge 13000 rpm for 5 minutes and take the supernatant and add 400 µl of Binding mix. Mix briefly by vortexing. Transfer the mixture to a MycoSpin DTM column in a collection tube. Centrifuge at 8000 rpm for 1 minute. Discard the flow-through liquid from collection tube. Add 500µl of Wash Buffer 1 (WB-1) to the column. Centrifuge at 8000 rpm for 1 minute. Discard the flow-through liquid from collection tube. Add 500µl of Wash Buffer 2 (WB -2) to the column. Centrifuge at 8000 rpm for 1 minute. Discard the flow-through liquid from collection tube. Add 500µl of Wash Buffer 2 (WB-2) to the column. Centrifuge at 14000 rpm for 3 minute. Place the MycoSpin DTM column in a micro-centrifuge tube. Add 50 µl of Elution Buffer. Let it stand at room temperature for 5 minutes. Centrifuge at 8000 rpm for 1 minute.
PCR mixer already prepared and stored within refrigerator. So that extracted fungal DNA transfer into the PCR tubes and mixed with PCR mixer. After that kept into the PCR machine and cyclic parameter adjusted for one hour at 42° C as given below.
Initial denaturation at 94°C → 3 minutes
Denaturation at 94°C → 30 seconds
Annealing at 94°C → 30 seconds 40 cycles
Extentionat72°C → 30 seconds
Final extention at72°C → 10 minutes
Final hold at 20ºC
In agarose gel preparation, first TBE (70ml) and agarose powder (1.05g) added together in the conical flask. Then it covers with foil paper. We put the hold in the top of the foil paper, in order to decrease pressure. Then keep it in the oven. After that the loading buffer was mixed. Then the solution was poured into the gel tray without forming any air bubbles. Then the combs fixed into the two sides, and leave it room temperature until the gel become solid. After the samples taken out from PCR, 10µl taken from samples PCR tubes and treated with 2µl of loading dye and located into the well. Cover the gel plate and give the current to done this process. Finally the gel was taken out and excess liquid wiped out and placed under UV transilluminator observed the gel band in the computer monitor.
The fungi DNA sample was also taken to spectrophotometric machine for the analyzation ( absorbance)
RESULTS
1ST EXTRACTION (CANDIDA ALBICANS)
2ND EXTRACTION
1 2 3 4
CANDIDA ALBICANS
SACCHORAMYCIS SPECIES
TEA FUNGI SPECIES
MUSH ROOM SPECIES
1ST PCR( CandidaAlbicans)
1 2 3 4 5
1.0 µl sample
2.5 µl sample
5.0 µl sample
negative control
ladder
2nd PCR
1 2 3 4 5 6
Candida Albicans
SacchoromycisSpeies
Tea fungal Species
Mush room Species
Negative Control
Ladder
2.5 µl of DNA is used for each.
Figure- Spectrophotometric result sheet
DISCUSSION
Protocols for extraction of DNA of fungal cells either are very time-consuming or show poor yield of DNA compared to methods of extraction of DNA, e.g. of human cells. Other protocols require additional lysis steps like signification or mechanical disruption or harmful chemicals such as phenol-chloroform or guanidine thiocyanate. From these reasons, commercial nucleic acid extraction and purification kits are more and more popular. Nucleic acid samples with proper quality are important prerequisite for any further DNA associated identification method. This especially important for the dermatophytes providing minuscule samples from clinical sources and growing very slowly under laboratory conditions.
DNA isolation and purification are important steps for molecular biology studies. The quality and integrity of the isolated DNA directly affects the results of all subsequent scientific research. Like reagents, good quality DNA is essential to achieve good results in experiments. The major challenge for isolation of DNA of good quality and quantify from fungi lies in breaking the rigid cell walls, as they are often resistant to traditional DNA extraction procedures.DNA extractions from filamentous fungi possess difficulties because of its high polysaccharide contents. The use of liquid nitrogen in some extraction methods also effective against the breaking the fungal cell wall content, but in our present method the usage of such chemicals were completely eliminated. And also destroyed polysaccharide may interfere with extraction and purification of genomic DNA, which subsequently influence DNA restriction, amplification, and cloning.
For example; the presence of polysaccharides makes genomic DNA highly viscous with glue like texture that renders it unmanageable in pipetting and unsuitable for PCR by inhibiting Taq polymerase activity. For the future we would suggest further investigation and prevention of contamination of the DNA extraction as a main priority. In here we got band in negative control, so there was a contamination in the practical.
The extracted DNA was enough to perform PCR- based reactions and also used in spectrophotometric detection and semi-quantitative determination of the amount of DNA by staining with Ethidium bromide and evaluation on agarose gel.
The purity of a solution of DNA can be determined using a comparison of the optical
density values of the solution at various wavelengths. For pure DNA, the observed 260/280 nm
ratio will be near 1.8. Elevated ratios usually indicate the presence of RNA, which can be tested by running the sample, ~1µg, on an agarose gel. 260/280 ratios below 1.8 often signal the presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is indicated by 230/260 ratios greater than 0.5. Once you are sure your sample contains pure DNA, an accurate determination of the concentration of DNA can be made. The ratio was not similar as mention above, that have some difference in the amount. In this extraction we did not get proper band in gel picture, but the extracted DNA was confirmed by the spectrophotometric results.
Conclusion
In this research some of medically and environmentally important fungi DNA extracted. The spectrophotometric results and gel images shows that DNA has extracted. So the extraction method is correct using MycoSpin DTM column and specific buffers. There is no band observed in the post PCR gel image. So we can conclude that the PCR amplification has not happened properly. Therefore we can conclude that there is a problem with PCR primers. However Molecular Method is the most accurate and sensitive test for the identification of fungal diseases.
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