Identification of Cladosporium sp. Fungi by in- silico RFLP-PCR

Cladosporium sp. plays an important role in human health, it is one of the pathogenic fungi which cause allergy and asthma and most frequently isolated from airborne spores. In this study, a couple of universal PCR primers were designed to identify the pathogenic fungi Cladosporium sp. according to conserved region 5.8S, 18S and 28S subunit ribosomal RNA gene in Cladosporium species. In silico RFLPPCR were used to identify twenty-four Cladosporium strains. The results showed that the universal primer has the specificity to amplify the conserved region in 24 species as a band in virtual agarose gel. They also showed that the RFLP method is able to identify three Cladosporium species by specific and unique restriction enzymes for each one. These species are Cl. halotorenas by the two unique enzymes BsaXI and MobII, the other species is Cl. colrandse by two enzymes BccI and BtsCI, while the third species is Cl. aciculare by one enzyme BceAI. Each enzyme forms two bands in virtual agarose gel as a results of cutting the DNA by the enzyme, where the rest twenty – two species share more than one restriction enzymes. This method is active and rapid for identifying Cladosporium genus and three species by computational bases methods before applying it in the lab for more accuracy, efficiency, and specificity of designed primer to get good results in a short time.


Introduction:
Using the morphological methods for identifying fungal species needs a very long experience and skills to differentiate between a very wide range of fungal species. Molecular methods are more accurate from those classical methods especially when using specific primer for each species and genes (1).
The PCR techniques have been widely used in mycological study, the success of polymerase chain reaction depends on more than one factor; the most important and critical one is the ability of the primer to amplify the specific DNA template. To check this, the researcher needs to make the PCR reaction with all regents. In case the reaction failed to form a product in agarose gel, the reason behind this among more than one factor is needed to be known. The amplified DNA by using Thermocycler (PCR) is a basic tool in molecular biology and the most common technique used in laboratory researches and DNA studies.
The principle of this tool has been widely used and applied out of many other simple or complex nucleic acid amplification technologies (NAAT) beside laboratory which deals with experiments for amplification of nucleic acid technologies, in silico or virtual (bioinformatics) PCR have been developed, along within in-silico PCR analysis. In silico NAAT analysis is a very useful and efficient complementary method to ensure the specificity and efficient of primers or probes, (2).
For a long time, the internal transcript spacer (ITS) of the DNA has been used as a region for analyzing fungal diversity in many environmental sites, and has recently been selected as a biomarker for fungal DNA diagnosis, the ITS region has the ITS1 and ITS2 sequences separated by the 5.8s gene, and it is position between 18s (SSU) and 28s(LSU) genes, which is represented nrDNA repeated unit. Conserved regions in DNA or protein amino sequences are a good candidate for functional elements and thus conserved genes and sequences are similar or the same sequences in nucleic acids (DNA and RNA) or proteins among species or within a genome. Conserved regions indicated that sequences have been maintained by natural selection, a highly conserved sequence strongly remained relatively unchanged for keeping the phenotypic tree. Across many generations, nucleic acid sequences in the genome of an evolutionary steps are gradually changed over time due to the random changed like mutations and deletions and recombining of many sequences or may delete some of them as a results of chromosomes rearrangements. Conserved regions are sequences which resist any forces to change their sequences like mutation and another effect to conserve their position in the genome, (2). Ribosomal RNA specification is very important in the development and transformation, thus taxonomy and medicine. rRNA is one of only a few gene products present in all cells; for this reasons, a gene that encodes the rRNA (rDNA) is studied to identify an organism's classification group, estimating related and rates of species differences. Consequently, many thousands of rRNA sequences are known and stored in specialized databases NCBI (National Center for Biotechnology Information). rRNA is a target of numerous clinically relevant antibiotics and shown to be the most important tool in taxonomy and identification.
The Cladosporium is a very interested fungus in the environments as they are distributed in a wide level outdoor and indoor, (3). The in silico PCR is the best method to test the efficiency of the designed primers and many other reactions, in this study the in silico PCR was used to test the designed primer and to find and compare the specific restriction enzyme among twenty-four species of Cladosporium to used them as a marker for identification.

Materials and Methods:
The rRNA ribosomal subunit of twenty-five Cladosporium strains sequences was obtained from NCBI web site, A set of primer was designed depending on conserved regions of twenty-five of Cadosporium sp. sequences by using Clustal W tools to assess the fungal conserved regions, (4).
A set of a primer depending on conserved regions of ribosomal RNA subunit (18S, 5.8S, and 28S) of twenty-five Cladosporium sp. was designed by using the Geneious tool, (version 10.1). This program is able to diagnose, detect and compare the similarity region in relative species, the primer design is a very critical step to any successful PCR amplification. The designed primers specificity and sufficiently to amplification for all Cladosporium sp. studded species was tested by using NCBI primer blast, the DNA amplified by the designed primers was tested by using virtual agarose gel electrophoresis and the size of bands formed by amplification was measured, (5).

The restriction fragment length polymorphisms (RFLP):
The RFLP is a technique that can divide the DNA in-to more than two fragments in special sites according to the type of the restriction enzymes and sequences. The DNA, which is cut to different size segments, can be detected by using agarose gel electrophoresis. All the amplified DNA of Cladosporium sp. by universal primers were subjected to the restriction enzymes with the help of NEBcutter tool, (6). The enzymes which digested and cut the DNA to segments and unique for specific Cladosporium sp. have been chosen.

Results and Discussion: Universal primer efficiency test:
According to twenty-five Cladosporium. sp.ribosomal RNA (rRNA) conserved region; two sets of universal primer were designed using in silico programs; Geneious tools, (v. 11.1.5), (Fig. 1 and 2) and tested by SnapGene and Amplifix tools, (Table 1) (7). The specific primer is able to amplify twenty-four species out of twenty-five.  The band size of the amplified region of all species was 363 bp; (Fig. 3), the species Cl. penidielloides have not been detected by the designed primer and this may be due to the short fungal sequences in NCBI. The sequences are shorter than the other species which made the primer out of its beginning and end sequences. It's not possible to make the primer start point compatible with the shorter sequences; this can make the amplified band size very small in agarose gel, (8).

Identification of Cladosporium species by RFLP
The results of using RFLP technique to identify Cladosporium sp. showed that there are three species of Cladosporium fungi among the 24 amplified DNA species that can be identified by using unique restriction enzymes for each species and not sharing them with other species. The remaining species are shared with one or more RE and can't be used for identification, while the RE ApekI and AseI were found in all Cladosporium species and can be used for the differentiation between Cladosporium genus and other fungi genus like Aspergillus in which these enzymes have no sites in seven Aspergillus species sequences, ( Table  3). The species Cl. aciculare have a one unique RE; BceAI , ( Fig. 4), cutting the DNA into two segments in sizes 310 and 54 bp, Fig. (5).   The virtual agarose gel electrophoresis showed the cutting segments, one by BsaXI which forms two segments one in size 315 bp and the other in size 48 bp, while the second RE forms two segments in size 200 and 163 bp respectively, (Fig. 7). 225 And the species Cl. colrandse have three unique RE; BccI , BtsCI and FoKI (Fig. 8). The first one forms two segments in size 47 and 316 bp, the second enzyme forms segments in size 58 and 305 bp and the last one forms segments in size 65 and 298 bp (Fig. 9). The results showed the possibility of using RFLP technique for identifying the fungi species which have a variation in sequences and far away in phylogenetic tree, as shown in Fig. 10. The very close species have shearing most restriction enzymes and cannot be used as a tool in identification methods; but with those are far away in genetic. The species which have a unique restriction enzyme have differences in their genetic characteristics, (9).