Aspergillus Fumigatus Identification and Molecular Character

genus genus genus Aspergillus Fumigatus Identification and Molecular CharacterIDENTIFICATION AND molecular(a) CHARACTERIZATION OF Aspergillus fumigatus FROM SOILR. V. Shalini, and Dr. K. AmuthaABSTRACT Soil was hoard, serially diluted and pure culture obtained slant was prepared in potato grape sugar agar and maintained through come to the fore the study. morphologic, smallscopical and macroscopically designation were carried out on the isolated organism. desoxyribonucleic acid was isolated from the 24 hour culture, for ITS-PCR addition. deoxyribonucleic acid was amplified by mixing the template deoxyribonucleic acid (50nm) with the polymerase reply buffer, dNTP mix, primers and Taq. Polymerase ambit chemical reaction (PCR) was performed in a total volume of 50L reaction mixture. The PCR product was blend with loading buffer (8L) containing 0.025% bromophenol blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplifi ed product was visualized under a UV trans illuminator for notwithstanding examination. The PCR products were at long last sequenced using the booster of an automated deoxyribonucleic acid sequenator at progen Ltd (Salem, India) and analyzed with the BLAST political platform provided by the National vegetable marrow for Bio-technology in changeion (NCBI) to confirm the fungal species. The current study demonstrates that desoxyribonucleic acid genome containing 18S rRNA has a high degree of analytical sensitivity and specificity (100%) for the detection of a all-inclusive range of kingdom Fungi.OBJECTIVE To isolate, identify and characterize Aspergillus fumigatus using molecular biological methods.MATERIALS AND METHODS The soil was self-collected from diametrical places, pooled together allowed to be dried at mode temperature. The morphology based realization of Aspergillus was through which includes the size, shape, colour, ornamentation of spore and mode of attachmen t. Unfortunately a lot of difficulties arose for phenotypical appellative of this fungus due(p) to its unstable characteristics. Comparatively a DNA sequence-based appellation format appeared to be the near promising in terms of its speed, ease, objectivity and dependableness for species identification.RESULTS The preliminary morphology based studies showed the isolated fungi as a species of aspergillus.However after the DNA closing off followed by sequencing it was concluded that the particular species set as Aspergillus was Aspergillus fumigatus.KEY WORDS Aspergillus, serial dilution, DNA, Sequenced. insane asylumThe presence of organic matter in the soil affects the quantity and musical note of microbes in the soil. The development of micro fungi in the soil is favoured by soils having acidic reaction and aerobic condition which is likely present in the soil. However the amount of de dance step in the soil is brought just about by the organisms present in the soil. 1The ra te at which the organic matter is decomposed is cover related with soil microbes. (Arunachalam et al., 1997). Microorganisms come in miscellaneous sizes and shapes and is determined by the soil ph., temperature, available moisture, degree of aeration, availability of nutrients in the soil and so forth The genus of spore forming fungi is found worldwide out of which Aspergillus is the most dominant allele species and is ubiquitoes.Out of that 95% is occupied by Aspergillus fumigatus. The otherwise unhealthful forms of Aspergillus species are Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus etc. This fungi exists only in mycelial form, and is thermo tolerant capable of growing at temperatures betwixt 15-53c.Being a spore producing fungi the spores gets dispersed by wind in the atmosphere.2Aspergillus fumigatus is the most common among all the airborne saprophytic fungal pathogens in repellent compromised patients mostly in developed countries (Lat ge, 1999). It is the main pathogenic agent of divers(a) diseases caused in humans including invasive pulmonary aspergillosis, aspergilloma and allergic bronchopulmonary aspergillosis (Tomee wagon train der Werf, 2001) the former is a frequent cause death in resistant compromised patients. The possession of dissimilar virulent traits gives A. fumigatus the ability to cause these diseases. It is a cognise fact that other members of the genus Aspergillus are either less pathogenic or non- pathogenic. Identification of the most common and important species remains baffling due to the variability in the phenotypic characters. However a clear and a careful approach of phenotypic classification (taxonomy) together with phylo contagious preaching of DNA sequence data is a prerequisite for a reliable and a rapid identification. In our investigation we used the molecular techniques (sequencing) for the reliable identification rather the identification based on their microscopical and few physiological features.MATERIALS METHODSCollection of soil samplesSoil samples were collected from different places (in and around Chengalpattu). The surface deposits were removed to a depth of about 10 cm and the exposed soil was collected to a depth of 2-3cm. The collected soil samples were stored in zip locked covers stored in refrigerator temperature for further analysis. The collected soil samples were passed through a sieve to remove the st iodins and other impurities. isolation of fungiThe glass wares were sterilized in an autoclave to a temperature of great hundredc for twenty minutes. The chemicals were of analytical grade (Himedia). The method used for the isolation of fungi from soil was serial dilution method. 1 gm of soil was weighed and mixed in 10ml of double distilled sterile water. This was used for preparing serial dilutions. 1 ml of the final dilution (10-6 ) was pipetted into the prepared potato dextrose agar media (PDA) amended with a suitable antibiotic Chloramphenicol (12mg/100ml). The plates were incubated at 30c for about seven days. Fungi that appeared on petriplates were isolated. The isolates were picked up based on likely dissimilarity of pagan characteristics and purified. The purified isolates were identified according to the genera on the basis of cultural characteristics such as nature of growth, spore colour, and pigment production, and on morphological characteristics of mycelia and fruit bodies (Domsch etal., 1980 Raper and Fennell 1965) and maintained in agar slants for future use3.Isolation of DNAGenomic DNA was extracted from 24 hour old culture. Measured 100 micro gram of mycelium into a sterile 1.5- micro centrifuge underpass-molded structure. Simultaneously plant 1 microgram of dried (vacuum filter mycelium first) in a daub and pestle treated with liquid nitrogen 5-6 clocks. Poured the frozen powder into the Eppendorf tube. Added 660 750 l of lysis buffer and 10 l of B-mercaptor.Vortexed the mixture for a few seconds. And Incubated at 65C for 1 hour. Used a water vat for incubation. Centrifuged at a speed of 3400 rpm for 5 minutes at room temperature and aspirated out the top layer.Transfered the top aqueous layer into a fresh Eppendorf tube discarded the bottom layer. Measured out 700 l of chloroform, isoamyl alcohol (241) into Eppendorf tube and adjusted the volume to meet a 11 ratio of aqueous phase.Vortexed the mixture for a few seconds. Centrifuged at a speed of 12000 rpm for 10 minutes at room temperature and aspirated out 550 600 l of the top layer. Transfered the top aqueous layer into Eppendorf tube and discarded the bottom layer. Added 0.1volume of 3m potassium acetate and 0.7 volume of isopropanol. Mixed wholesome by inverting the tube not by vortexing.Centrifuged for about 10 minutes and discarded the supernatant. Added 0.5 mL of ice cold ethanol (70% and inverted the tube gently, again it was centrifuged for about 5 minutes in a spinner) finally the pellets were res uspended in 100l of TE buffer (PH-8).After further purification DNA was quantified spectrophotpmetrically and the quality was analyzed in 0.9% agorose gel. elaboration of 18srRNA by PCRFor ITS-PCR amplification, DNA was amplified by mixing the template DNA (50nm) with the polymerase reaction buffer, dNTP mix, primers and Taq polymerase chain reaction (PCR) was performed in a total volume of 50L reaction mixture containingPrimer (2M/L) 8.0L10X Buffer 5.0 L2mM dNTP Mix 5.0LTaq DNA polymerase (5U/L) 0.5LTemplate DNA (50ng) 2.0LSterile distilled water 29.5LTotal volume 50.0LPCR amplification conditionAmplification was carried out in a primus advanced incline thermocycler. The PCR was programmed with an initial denaturing at 94C for 5 min, followed by 30 cycles of denaturation at 94c for 30 seconds, annealing at 61c for 30 seconds, and extension at 70c for 2 minutes and a final extension at 72c for 7 minutes.The PCR product was mixed with loading buffer (8L) containing 0.025% bromopheno l blue, 40% w/v sucrose in water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and the amplified product was visualized under a UV trans illuminator for further examination. (Sequencing)Sequencing of ITS region for identification of isolated fungi chosen Samples of the genomic DNA containing 18S rRNA were shortlisted for more specific species confirmation by using DNA sequencing. The sequenced PCR product was aligned with other isolate sequences from NCBI genbank for identification. The PCR products were finally sequenced using the help of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National Center for Bio-technology information (NCBI) to confirm the fungal species.RESULTS visible and microscopic AnalysisAnalysis of the isolated Aspergillus species showed variation in the colony colours, texture, and reverse placement colours (table 1 and 2). The morphological microscopic and molecular characteristi cs showed that the isolate is Aspergillus fumigatus (details given in table 1and 2).Morphological characters of colony (table1)CharacteristicsAspergillus fumigatusSurface colourMarginsReverse side step-upGreen to dark greenEntireYellowRapidMicroscopic characteristics (table2)CharacteristicsAspergillus fumigatusHyphaeBranched septateConidiophorePresentVesicleDome shapedConidiaPresentPhialidesUniseriateFruiting bodyCleistotheciaFig A1 Fig A2Morphological depicting of Aspergillus species on potato dextrose agar A1-Aspergillus fumigatus surface colour, A2-Reverse side of the colony.DNA sequencing of ITS region for identification of speciesThe species of fungi from the PCR sample was identified by DNA sequencing of the immanently transcribed spacer (ITS) region of rRNA gene. Segments of the entire ITS regions, including partial 5.8S rRNA and internal transcribed spacer 2, complete sequence, 28S rRNA,partial sequence were amplified using the primer PGF04 5-GGC ATC GGC C-3. Amplificati on of the ITS region of strain Aspergillus fumigatus had a size of 1703bp. It was submitted to the NCBI and the accession snatch KC 119199 was received.M 1 2Fig A3 represent the stria pattern of Aspergillus fumigatus from PCR reactions Lane M= Marker, Lane 1= Aspergillus fumigatus, lane 2=Aspergillus fumigatus.DISCUSSIONDetection of A. fumigatus is of great consult because it is a dangerous allergen associated with aspergillosis 5(Abraca et al., 1994 Schuster et al., 2002 Noonimabcet al.2009 Edwin et al., 2010 Gautam et al., 2011). This highlights the importance of correct identification and taxonomical differentiation between different species of Aspergillus.The taxonomy of Aspergillus has always been complex due to its great number of species (nearly 250), which have very few differences. The identification of different Aspergillus species, on the basis of their morphological characters (example, colony colours, and reverse side) is one of the oldest and most adopted methods. Some of the species of Aspergillus have the same morphological features which make it difficult to distinguish between them it is also a time consuming process and may not be accurate (Klich and Pitt, 1988 cop et al., 2004)6. This shows that morphological and microscopical characters are not enough for fungal identification and it renders the need of molecular techniques for correct species identification.Molecular characterization on the other hand, is a rapid and a quick procedure which requires minimal treatment of pathogens. It also helps in distinguishing morphologically, similar fungal species. Several similar studies on the application of PCR technology were used for the identification and detection of fungi, by using internal transcribed spacer (ITS) were already been studied and published7 by several scientist (Henson and French, 1993 Marek et al., 2003 Haughland et al., 2004 Druzhinina etal., 2005).. legion(predicate) more such studies were also carried out very recently by God et and Munaut (2010) in the differentiation of Aspergillus flavus, A.parasiticus, A.tamarii and A.nomius by PCR-RAPD markers. Similarly, Leema et al. (2010) confirmed the species A. flavus by verifying using the molecular methods that is, by amplification of the internally transcribed spacer regions. By using the help of RAPD-PCR, 8Khan et al. (2007) studied change in various Aspergillus niger isolates sourced from pigeon pea fields .Several molecular techniques have been tested to tell apart different Aspergillus species like random amplification of polymorphic DNA (RAPD) (Yuan et al., 1995), the internal transcribed spacer (ITS) region (Kumeda and Asao, 1996 Henry et al., 2000 Kumeda and Asao, 2001 Rigo et al., 2002) and the aflatoxin gene cluster (Chang et al., 1995 Watson et al., 1999 Tominaga et al., 2006).In this study care was taken to choose the genomic DNA containing 18sRNA specifics primers that were helpful in amplifying medically important fungi. The genomic DNA containing 18s rRNA was the right candidate for detection of fungus as it is a mutli-copy gene which evolves slowly and is conserved among fungi. The present study proves that the genomic DNA containing 18s rRNA based PCR is suitable for probing large range of medically authoritative fungi owing to its higher level of analytical sensitivity and specificity. goal In this present study we had shown that molecular techniques are rapid and top hat for identification of fungi than the traditional morphological methods for early diagnosis and treatment of fungal infections. The goal of our study was to identify a practical, quick, cheap, method for the identification of A. fumigatus, the most common of the Aspergillus pathogens.REFERENCESArunachalam K, Arunachalam A, Tripathi RS, Pandey HN. 1997 Dynamics of microbial population during the gradation phase of a selectively logged subtropical humid forest in north east India. Tropical Ecology 38, 333341.Sirida Youngchim,1,2 Rachael Morri s-Jones,1 Roderick J. Hay3 and Andrew J. 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