Aspergillus Fumigatus Identification and Molecular Character

genus genus genus genus genus Aspergillus Fumigatus Identification and molecular CharacterIDENTIFICATION AND MOLECULAR CHARACTERIZATION OF Aspergillus fumigatus FROM SOILR. V. Shalini, and Dr. K. AmuthaABSTRACT Soil was collected, serially reduce and pure culture obtained slant was prep atomic number 18d in potato dextrose agar and maintained by dint ofout the make. Morphological, microscopical and macroscopically designation were carried out on the isolated organism. deoxyribonucleic acid was isolated from the 24 hour culture, for ITS-PCR intricacy. desoxyribonucleic acid was amplified by mixing the template desoxyribonucleic acid (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. The PCR product was mixed with loading buffer (8L) containing 0.025% bromophenol blue, 40% w/v sucrose in body of water and then loaded in 2% agarose gel with 0.1% of ethidium bromide and t he amplified product was visualized under a UV trans illuminator for further examination. The PCR products were finally sequenced victimisation the athletic supporter of an automated DNA sequencer at progen Ltd (Salem, India) and analyzed with the BLAST program provided by the National centralize for Bio-technology information (NCBI) to confirm the fungal species. The current study demonstrates that DNA genome containing 18S rRNA has a high degree of uninflected sensitivity and specificity (100%) for the detection of a wide range of fungi.OBJECTIVE To isolate, identify and characterize Aspergillus fumigatus using molecular biological methods.MATERIALS AND METHODS The ground was collected from different places, pooled together allowed to be dried at room temperature. The morphology based identification of Aspergillus was through with(p) which includes the size, shape, colour, ornamentation of spore and mode of attachment. Unfortunately a lot of difficulties arose for phenotypic al identification of this fungus due to its unstable characteristics. Comparatively a DNA sequence-based identification format appeared to be the most promising in terms of its zip up, ease, objectivity and reliability for species identification.RESULTS The preliminary morphology based studies showed the isolated fungi as a species of aspergillus.yet after the DNA isolation followed by sequencing it was concluded that the particular species identified as Aspergillus was Aspergillus fumigatus.KEY WORDS Aspergillus, serial dilution, DNA, Sequenced.INTRODUCTIONThe presence of constitutional way out in the speck affects the quantity and quality of microbes in the soil. The development of micro fungi in the soil is favoured by soils having acidic reaction and aerobic shape which is likely present in the soil. However the amount of degradation in the soil is brought about by the organisms present in the soil. 1The rate at which the organic matter is decomposed is inter related with s oil microbes. (Arunachalam et al., 1997). Microorganisms come in various sizes and shapes and is determined by the soil ph., temperature, available moisture, degree of aeration, availability of nutrients in the soil etc. The genus of spore forming fungi is found worldwide out of which Aspergillus is the most dominant species and is ubiquitoes.Out of that 95% is occupied by Aspergillus fumigatus. The other pathogenic 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 between 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 immune compromised patients mostly in developed countries (Latge, 1999). It is the main pathogenic agent of various diseases cause in humans including invasive pulmo nary aspergillosis, aspergilloma and allergic bronchopulmonary aspergillosis (Tomee van der Werf, 2001) the former is a betray cause death in immune compromised patients. The possession of different virulent traits gives A. fumigatus the ability to cause these diseases. It is a known fact that other members of the genus Aspergillus are either less pathogenic or non- pathogenic. Identification of the most common and important species remains problematic due to the variability in the phenotypic characters. However a validated and a careful approach of phenotypic classification (taxonomy) together with phylogenetic treatment of DNA sequence data is a necessary 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 microscopic 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 stones and other impurities.Isolation of fungiThe glass wares were sterilized in an autoclave to a temperature of 120c for twenty proceeding. 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 is olates were picked up based on apparent dissimilarity of cultural characteristics and purified. The purified isolates were identified according to the genera on the origination of cultural characteristics such as nature of growth, spore colour, and pigment production, and on morphological characteristics of mycelia and fruiting 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 tube. Simultaneously ground 1 microgram of dried (vacuum filter mycelium first) in a mortar and pestle treated with liquid nitrogen 5-6 times. 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 bath for incubation. Centrifuged at a speed of 3400 rpm for 5 minutes at room temperature an d aspirated out the top floor.Transfered the top aqueous layer into a fresh Eppendorf tube discarded the basis 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 well 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 resuspended in 100l of TE buffer (PH-8).After further purification DNA was quantified spectrophotpmetrically and the quality was analyzed in 0.9% agorose gel.Amp lification 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 containing primer (2M/L) 8.0L10X Buffer 5.0 L2mM dNTP Mix 5.0LTaq DNA polymerase (5U/L) 0.5LTemplate DNA (50ng) 2.0LSterile distilled water 29.5L count volume 50.0LPCR amplification conditionAmplification was carried out in a primus advanced gradient 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% bromophenol 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 align 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.RESULTSMacroscopic and Microscopic AnalysisAnalysis of the isolated Aspergillus species showed variation in the colonization colors, texture, and reverse lieu colours (table 1 and 2). The morphological microscopic and molecular characteristics showed that the isolate is Aspergillus fumigatus (details given in table 1and 2).Morphological characters of colony (table1)CharacteristicsAspergillus f umigatusSurface colourMargins revert sideGrowthGreen to dark greenEntireYellowRapidMicroscopic characteristics (table2)CharacteristicsAspergillus fumigatusHyphaeBranched septateConidiophorePresentVesicle garret shapedConidiaPresentPhialidesUniseriateFruiting bodyCleistotheciaFig A1 Fig A2Morphological characterization 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 internally transcribe spacer (ITS) region of rRNA gene. Segments of the entire ITS regions, including partial 5.8S rRNA and internal transcribe spacer 2, complete sequence, 28S rRNA,partial sequence were amplified using the primer PGF04 5-GGC ATC GGC C-3. Amplification of the ITS region of strain Aspergillus fumigatus had a size of 1703bp. It was submitted to the NCBI and the accession number KC 119199 was receive d.M 1 2Fig A3 represent the banding 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 concern 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 eminence 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 in like manner a time consu ming process and may not be accurate (Klich and Pitt, 1988 Samson 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 handling 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 study and published7 by several scientist (Henson and French, 1993 Marek et al., 2003 Haughland et al., 2004 Druzhinina etal., 2005).. Many 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. (20 10) 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 diversity in various Aspergillus niger isolates sourced from pigeon pea fields .Several molecular techniques have been tested to classify 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 subservient 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 prese nt study proves that the genomic DNA containing 18s rRNA based PCR is suitable for probing large range of medically significant fungi owing to its higher level of analytical sensitivity and specificity.CONCLUSION In this present study we had shown that molecular techniques are rapid and best for identification of fungi than the traditional morphological methods for early diagnosis and treatment of fungal infections. The finale 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 Morris-Jones,1 Roderick J. Hay3 and Andrew J. 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