based on the results of molecular phylogenetic analysis , the mucorales , the core group of the traditional zygomycota , have been reclassified into the subphylum mucoromycotina of the new division , glomeromycota ( 1 ) . these fungi are ubiquitous saprophytes that are scattered widely in nature , food , soil , and air ( 2 ) . the mucorales are regarded as important animal and human pathogens , and they are responsible for mucormycosis ( formerly known as zygomycosis ) ( 3 , 4 ) , the third most common invasive fungal infection ( 5 - 7 ) . the mucorales are increasingly recognized as opportunistic pathogens in immunocompromised or immunosuppressed patients ( 8 , 9 ) . mucormycosis can be caused by any of the six families of mucorales , although the members of the mucoraceae including rhizopus , mucor , absidia ( lichtheimia ) , and rhizomucor are more frequently isolated in human infections ( 5 , 7 , 10 , 11 ) . members of the genus rhizopus are the most common isolates recovered in a clinical setting , with rhizopus arrhizus occurring the most frequently . members of the genus mucor come second to rhizopus with respect to frequency , while cunninghamella , apophysomyces , lichtheimia , saksenaea , rhizomucor , cokeromyces , and syncephalastrum each constitute a significantly lower percentage of clinical isolates ( 7 , 12 , 13 ) . due to the infection s rapidly progressive course and unexpectedness , many mucormycosis cases are only diagnosed postmortem . the number of cases of mucormycosis has increased over the last few decades , making the fast and accurate diagnosis of these infections imperative . unfortunately for patients who are affected by mucormycosis , no routine laboratory tests are available to diagnose this disease . clinical diagnosis instead relies on histopathology and the isolation of etiologic fungi from infected tissues ( 2 , 14 ) . the sporangia are characterized by the inclusion of a variously shaped columella ( 15 ) . the growth of the mucorales in media is rapid , with mycelia elements expanding to cover the entire plate in only a few ( one to seven ) days . however , culture - based identification is often difficult and time - consuming , and it relies entirely on the experience of the technician or expertise mycologist . conventional phenotypic methods usually identify isolates to only the genus level , and sometimes only as mucorales . if the diagnosis is not made early enough , dissemination often occurs ( 8 , 16 , 17 ) . based on the above , the development and validation of a new detection system , perhaps involving polymerase chain reaction ( pcr ) methods , for the fast and accurate diagnosis of mucormycosis pcr - restriction fragment length polymorphism ( rflp ) is a rapid and highly reliable method for the identification and differentiation of important common mucorales species ( 16 ) . this study aimed to identify the mucor and lichtheimia ( formerly absidia ) species among other genera of mucorales isolated in culture media using the pcr - rflp method . of the course of a year , 500 samples were taken from different city districts , parks , hospitals , greengrocers , food stores , laboratories , public restrooms , mosques , and cattle houses . the samples were cultured on sabouraud dextrose agar ( sda ; merck kgaa , darmstadt , germany ) and potato dextrose agar ( pda ; merck kgaa , darmstadt , germany ) supplemented with chloramphenicol . a diversity of different types of filamentous fungi and yeasts were identified in the media . finally , 120 pure cultures belonging to the mucorales were gained after the purification of the colonies . primary identification of the mucorales was performed on the basis of the macroscopic and microscopic features of the colonies . in the next step , a mixture of two genus - specific sense primers and one degenerate anti - sense primer from the 18s rrna gene region were selected to determine the two genera of lichtheimia and mucor within the mucorales . all of the pure colonies obtained were sub - cultured on 2% sda and pda and then incubated for one to seven days at 27c in stable conditions . the genomic dna was extracted and purified from each colony using the phenol - chloroform method as follows . briefly , hyphae ( without sporangia ) from fresh 48 hours cultures on sda or pda were suspended in a 200 l lysis buffer ( 100 mm tris - hcl , 10 mm edta ( ph = 8) , 2% triton x-100 , 1% sds , 100 mm nacl ) ( merck kgaa , darmstadt , germany ) in 2 ml eppendorf tubes and grinded well . then , the suspension ( without the hyphal elements ) of each tube was transferred to a new eppendorf tube . next , 200 l phenol - chloroform ( 1:1 ) was added and vortexed ( or shaken by hand for 5 minutes ) rigorously with 200 l of glass beads ( 0.5 mm in diameter ) to release the dna . after centrifugation for 5 minutes at 5,000 rpm , the supernatant was mixed with an equal volume of 2-propanol and 0.1 volume of 3 m sodium acetate ( ph = 5.2 ) , vortexed , and incubated at -20c for 10 min . all of the solution was gently removed and then 100 l of 70% ethanol was gently added to the tube and centrifuged for 5 minutes at 5000 rpm . the ethanol was then removed from the tube , dried , and resuspended in 50 l of distilled deionized water , and it was kept at -20c as the purified dna until use . selected primers targeted an 830 bp sequence in the 18s fungal ribosomal gene , which excludes the amplification of human dna and other filamentous fungi . the specific sense primers were mucl1 : 5 tgatctacgtgacatatttct 3 and absl1 : 5 tga tctacacggcatcaaat 3 ( bioneer , daejeon , south korea ) , which corresponded to the sequences of mucor sp . and absidia ( lichtheimia ) , respectively . a degenerate anti - sense primer ( mr1 : 5 agtagtttgtcttcggtcaa 3 ) was applied for the mucorales . the sense primers annealed to the region of the template starting at position 75 , while the antisense primer annealed to the region at position 901 ( 18 ) . each amplification reaction included 12.5 l of premix ( containing 2.5 u taq dna polymerase , pcr buffer , 1.5 mm mgcl2 , and 200 m dntp ) ( ampliqon , odense , denmark ) , 2 l ( about 10 ng ) of template dna , 0.5 l of each primer ( 3  0.5 = 1.5 l ) , and 9 l of distilled deionized water in a final volume of 25 l . amplification was performed on a 2700 thermocycler ( applied biosystems , singapore ) as follows . one cycle of 1 minute at 94c ( primary denaturation ) , 30 cycles of 1 minute at 94c ( denaturation ) , 1 minute at 60c ( annealing ) , 1 minute at 72c ( extension ) , and finally one cycle of 5 minute at 72c . negative controls ( no dna template ) were included in each run to detect the presence of any dna contamination in the reagents and reaction mixtures . the pcr products of 120 mucorales were individually digested with selected restriction enzymes including afiii , xmni ( thermo fisher scientific , vilnius , lithuania ) , and acii ( new england biolabs , usa ) , which were specified to the mucor sp . : mucor circinelloides , m. racemosus , m. ramosissimus , m. plumbeus , l. corymbifera , and l. blakesleeana . eighteen s amplified fragments of the 120 mucorales were separately digested by the three abovementioned enzymes . the amplicons were digested for 1 hour at 37c in a total volume of 25 l ( containing 2 l of the enzyme , 2.5 l of related buffer , 10 l of pcr product , and 10.5 l of distilled water ) . the digested amplification products were subjected to electrophoresis , and the sizes of the restriction fragments were determined by comparison with a 100 bp ladder standard dna molecular weight marker ( fermentas , vilnius , lithuania ) . the restriction site , specificity , and fragment size of each enzyme are detailed in table 1 . agarose gel in a tbe buffer ( 90 mm tris , 90 mm boric acid , and 2 mm edta ) at 100 v for 45 - 120 minutes in a 1% , 1.5% , and 2% gel was used for the electrophoresis of the extracted dna , pcr products , and rflp products , respectively . the mucor sp . typically exhibits rapid growth , producing globose sporangia on sporangiophores that are either solitary or branched . these features distinguish the mucor spp . from the other producers of globose sporangia ( 8) . the characteristic features of some mucor and lichtheimia species are summarized in table 2 ( 8 , 15 , 19 - 23 ) . of the course of a year , 500 samples were taken from different city districts , parks , hospitals , greengrocers , food stores , laboratories , public restrooms , mosques , and cattle houses . the samples were cultured on sabouraud dextrose agar ( sda ; merck kgaa , darmstadt , germany ) and potato dextrose agar ( pda ; merck kgaa , darmstadt , germany ) supplemented with chloramphenicol . a diversity of different types of filamentous fungi and yeasts were identified in the media . finally , 120 pure cultures belonging to the mucorales were gained after the purification of the colonies . primary identification of the mucorales was performed on the basis of the macroscopic and microscopic features of the colonies . in the next step , a mixture of two genus - specific sense primers and one degenerate anti - sense primer from the 18s rrna gene region were selected to determine the two genera of lichtheimia and mucor within the mucorales . all of the pure colonies obtained were sub - cultured on 2% sda and pda and then incubated for one to seven days at 27c in stable conditions . the genomic dna was extracted and purified from each colony using the phenol - chloroform method as follows . briefly , hyphae ( without sporangia ) from fresh 48 hours cultures on sda or pda were suspended in a 200 l lysis buffer ( 100 mm tris - hcl , 10 mm edta ( ph = 8) , 2% triton x-100 , 1% sds , 100 mm nacl ) ( merck kgaa , darmstadt , germany ) in 2 ml eppendorf tubes and grinded well . then , the suspension ( without the hyphal elements ) of each tube was transferred to a new eppendorf tube . next , 200 l phenol - chloroform ( 1:1 ) was added and vortexed ( or shaken by hand for 5 minutes ) rigorously with 200 l of glass beads ( 0.5 mm in diameter ) to release the dna . after centrifugation for 5 minutes at 5,000 rpm , the supernatant was mixed with an equal volume of 2-propanol and 0.1 volume of 3 m sodium acetate ( ph = 5.2 ) , vortexed , and incubated at -20c for 10 min . all of the solution was gently removed and then 100 l of 70% ethanol was gently added to the tube and centrifuged for 5 minutes at 5000 rpm . the ethanol was then removed from the tube , dried , and resuspended in 50 l of distilled deionized water , and it was kept at -20c as the purified dna until use . selected primers targeted an 830 bp sequence in the 18s fungal ribosomal gene , which excludes the amplification of human dna and other filamentous fungi . the specific sense primers were mucl1 : 5 tgatctacgtgacatatttct 3 and absl1 : 5 tga tctacacggcatcaaat 3 ( bioneer , daejeon , south korea ) , which corresponded to the sequences of mucor sp . and absidia ( lichtheimia ) , respectively . a degenerate anti - sense primer ( mr1 : 5 agtagtttgtcttcggtcaa 3 ) was applied for the mucorales . the sense primers annealed to the region of the template starting at position 75 , while the antisense primer annealed to the region at position 901 ( 18 ) . each amplification reaction included 12.5 l of premix ( containing 2.5 u taq dna polymerase , pcr buffer , 1.5 mm mgcl2 , and 200 m dntp ) ( ampliqon , odense , denmark ) , 2 l ( about 10 ng ) of template dna , 0.5 l of each primer ( 3  0.5 = 1.5 l ) , and 9 l of distilled deionized water in a final volume of 25 l . amplification was performed on a 2700 thermocycler ( applied biosystems , singapore ) as follows . one cycle of 1 minute at 94c ( primary denaturation ) , 30 cycles of 1 minute at 94c ( denaturation ) , 1 minute at 60c ( annealing ) , 1 minute at 72c ( extension ) , and finally one cycle of 5 minute at 72c . negative controls ( no dna template ) were included in each run to detect the presence of any dna contamination in the reagents and reaction mixtures . the pcr products of 120 mucorales were individually digested with selected restriction enzymes including afiii , xmni ( thermo fisher scientific , vilnius , lithuania ) , and acii ( new england biolabs , usa ) , which were specified to the mucor sp . : mucor circinelloides , m. racemosus , m. ramosissimus , m. plumbeus , l. corymbifera , and l. blakesleeana . eighteen s amplified fragments of the 120 mucorales were separately digested by the three abovementioned enzymes . the amplicons were digested for 1 hour at 37c in a total volume of 25 l ( containing 2 l of the enzyme , 2.5 l of related buffer , 10 l of pcr product , and 10.5 l of distilled water ) . the digested amplification products were subjected to electrophoresis , and the sizes of the restriction fragments were determined by comparison with a 100 bp ladder standard dna molecular weight marker ( fermentas , vilnius , lithuania ) . the restriction site , specificity , and fragment size of each enzyme are detailed in table 1 . agarose gel in a tbe buffer ( 90 mm tris , 90 mm boric acid , and 2 mm edta ) at 100 v for 45 - 120 minutes in a 1% , 1.5% , and 2% gel was used for the electrophoresis of the extracted dna , pcr products , and rflp products , respectively . the mucor sp . typically exhibits rapid growth , producing globose sporangia on sporangiophores that are either solitary or branched . these features distinguish the mucor spp . from the other producers of globose sporangia ( 8) . the characteristic features of some mucor and lichtheimia species are summarized in table 2 ( 8 , 15 , 19 - 23 ) . the genomic dna belonging to the mucorales was successfully amplified with the selected primers and a product of approximately 830 base pair ( bp ) was amplified for all mucorales . the pcr products were electrophoresed in 1.5% agarose gels in the presence of ethidium bromide and then visualized under uv light ( figures 1 and 2 ) . the obtained sizes of the rflp products were exactly comparable with the sizes detailed in table 1 . all samples yielded a single band of approximately 830 bp in lanes 1 to 30 . lanes n , negative control ; m , 100 bp molecular size marker . lanes 1 to 13 , mucor sp . ; n , negative control ; m , 100 bp molecular size marker . two fragments ( 750 + 78 ) were visualized following the afiii restriction . the specific restriction pattern of m. circinelloides , m. racemosus , m. ramosissimus , and m. plumbeus was established using xmni ( 613 + 224 ) . additionally , two fragments ( 518 + 306 ) were visualized following digestion with acii , which represented the specific pattern for lichtheimia corymbifera and l. blakesleeana ( figures 3 and 4 ) . lanes 12 , 11 , 10 , 9 , 8 , 2 and 13 , m. circinelloides , m. racemosus , m. ramosissimus or m. plumbeus ; lanes 3 , 1 , 7 , 6 , 5 , 4 , and 14 , l. corymbifera or l. blakesleeana ; n , negative control ; m , 100 bp molecular size marker . a , colony of m. plumbeus ; b , microscopic morphology of m. plumbeus ; globos sporangia with spinolus walls and pyriform columella ; c , colony of m. circinelloides ; d , microscopic morphology of m. circinelloides with globos sporangia and spherical columella ; e , colony of l. corymbifera ; f , microscopic morphology of l. corymbifera with pyriform sporangia and flask like apophysis ; g , colony of l. blackesleenana ; h , microscopic morphology of l. blackesleenana with pyriform sporangia and flask like apophysis ; i , colony of m. ramosissimus ; j , microscopic morphology of m. ramosissimus with globos sporangia and round columella ; k , colony of m. racemosus ; l , microscopic morphology of m. racemosus with globos , light brown and encrusted walls sporangia , and ellipsoidal columella . the discrimination of these species was conducted based on the macroscopic and microscopic features as well as the growth ability in different temperatures ( figure 4 ) . , namely m. circinelloides ( 9.17% ) , m. racemosus ( 5.83% ) , m. plumbeus ( 10.83% ) , and m. ramosissimus ( 3.33% ) . twelve colonies ( 10% ) were identified as lichtheimia , which belonged to l. corymbifera ( 9.17% ) and l. blakesleeana ( 0.83% ) . the identification of zygomycetes is mainly based on macroscopic and microscopic characteristics , which is a difficult and time - consuming process that sometimes needs the expertise of a reference laboratory ( 14 ) . additionally , the precise identification of the mucorales down to a species level may hold great importance for further research on antifungal effectiveness ( 18 , 24 , 25 ) . molecular techniques have showed enormous potential for rapidly and accurately identifying the ecological agents of mucormycosis , which helps in conducting epidemiologic investigations . molecular detection assays for the mucorales are , however , not yet widely available ( 18 , 26 ) . different regions of the rrna operon have most frequently been the targets for the detection of zygomycetes , with previously reported pcrs for zygomycosis . several prior reports have described the utilization of universal fungal primers from the 18s , 28s , or its rrna gene regions for pcr amplification followed by the sequencing or hybridization of the product to specific probes ( 2 , 13 , 27 , 28 ) . pcr - rflp is a reliable and easy to perform technique that can be used in epidemiological and research studies . pcr - rflp - based methods target the 18s ribosomal gene of zygomycetes on dna extracted from human specimens and may therefore provide clinicians with a rapid and definitive diagnosis of mucormycosis ( 29 ) . in the present study , a pcr - rflp method based on the 18s ribosomal gene of zygomycetes , which had been previously developed by machouart et al . the identification of this region by pcr amplification with selective primers has proved to be reliable for the identification of zygomycetes ( 18 ) . ( 30 ) developed a pcr - based method targeting the 18s rrna gene for the identification of mucormycosis and aspergillosis agents in paraffin wax embedded tissue . ( 31 ) identified l. corymbifera among other fungi with pcr - rflp using the its region as a target sequence and the acii restriction enzyme . in our study , afiii was used for mucor identification to the genus level and , after that , the xmni restriction enzyme was applied to distinguish m. circinelloides , m. racemosus , m. ramosissimus , and m. plumbeus . digestion with afiii , xmni , and acii generated two fragments of 750 + 87 , 613 + 224 , and 518 + 306 bp , respectively . therefore , following rflp , the differentiation of the four abovementioned species was performed based on comparing the macroscopic , microscopic , and other features . ( 19 ) identified m. circinelloides as a cause of primary zygomycosis using a sequence analysis of the its region as well as phenotyping methods . the mucor species are considered to be a distant third behind the rhizopus species and absidia corymbifera in terms of causing zygomycosis . these include the thermotolerant species m. racemosus , which either does not grow or else grows poorly at 37c . the presence of fungal species has been considered to be an environmental microbiological indicator , and some of the fungi have been found to cause fungal infection ( 26 ) . a considerable number of mucormycosis cases have been associated with m. circinelloides , which appears to be the most common cause of the disease . however , no mycosis cases associated with m. plumbeus have yet been reported ( 32 ) . in this study , the genera of mucor and lichtheimia were represented in 29.17% and 10% of the 120 pure mucorales cultures , respectively . alvarez et al . ( 33 ) studied 190 isolates morphologically identified as zygomycetes using sequencing of the its region of the rdna , which revealed that m. circinelloides , l. corymbifera , and m. indicus represented approximately 9.5% , 5.3% , and 2.6% of these isolates , respectively . among the absidia species , the most important species associated with mucormycosis , it was proposed that three absidia species , namely a. corymbifera , a. blakesleeana , and a. hyalospora , should be reclassified as a separate family , the lichteimiaceae fam . , and the three species renamed as l. corymbifera , l. blakesleeana , and l. hylospora . l. blakesleeana was subsequently reduced to a synonym of l. hyalospora ( 34 , 35 ) . in dannaoui ( 24 ) study , almost all of the pcr results for m. circinelloides were negative . however , in the present study , pcr amplification of all the pure cultures using specific primers was carried out successfully , and the obtained bands were fully sharp . finally , our findings revealed that molecular methods can be used for the rapid detection and differentiation of species that are responsible for infection , and they can hence help in conducting epidemiologic investigations . in contrast to other methods , a pcr - based approach has the potential to be time efficient , highly specific , and endowed with a good sensitivity ( 36 ) . in summary , the present study maintains that the diagnosis of zygomycetes to a species level based on macroscopic and microscopic features is very difficult . at the same time therefore , it seems that more research is needed to modify the present molecular approaches . in a future study , we intend to design a pcr - rflp method that needs to lower specific primers and enzymes in order to identify the genera and species belonging to zygomycetes .