the most frequent species of dermatophytes are trichophyton 
 mentagrophytes , t. tonsurans , and t. rubrum , and these species cause a multiplicity of cutaneous disorders . they are keratinophilic fungi that attack keratinized tissue and cause a wide spectrum of clinical manifestations that vary from mild to severe , but infections are not life - threatening . owing to the high degree of phenotypic similarity among species , identification is difficult . conventional approaches for species - level identification in the diagnostic laboratory are based on morphological and physiological criteria , require several days or weeks to obtain results , and are frequently unspecific . to overcome these problems , molecular techniques have recently been developed to rapidly and precisely identify species of trichophyton that potentially cause human infection . numerous recent articles and reviews have exhaustively discussed the various molecular techniques available for dermatophyte species identification . molecular tools , such as sequencing of the internal transcribed spacer ( its ) region of ribosomal dna ( rdna ) , have shown promise for species identification in all genera of dermatophytes , and is relatively expensive ( 4 - 7 ) . moreover , its - based analyses have found that sequence variation is limited to only one or a few single nucleotide polymorphisms ( snps ) between certain species , e.g. , t. tonsurans and t. equinum or t. rubrum and t. soudanense ( 7 ) . this limited genetic variation suggests that the development of alternative molecular tools with sufficient specificity , reproducibility , and sensitivity is highly necessary ( 8) . rolling - circle amplification ( rca ) is a powerful and simple procedure for distinguishing closely related taxa at the species level ; it is based on the rolling replication of short single - stranded dna circles via specific dna polymerases under isothermal conditions . this enables the detection of target nucleic acid sequences , including snps , with high specificity ( 9 - 12 ) . the objective of the current study is to establish the use of rca based on its rdna to rapidly identify trichophyton species that potentially cause human and animal disorders . in the current study , all samples were obtained from different human sources that were referred to the mycology laboratory at tooba clinic , mazandaran university of medical sciences , iran and were suspected dermatophytosis cases . the study protocol was approved by the medical research ethics committee of mazandaran university of medical sciences ( ethical no . 90 - 2 - 28/90 - 14 ) and since the laboratory diagnosis was part of the patients routine care , informed consent for research purposes was not specifically obtained . a total of 61 isolates belonging to three species of trichophyton including the three reference strains t. rubrum ( centraalbureau voor schimmelcultures : cbs 130927 ) , t. mentagrophytes var . interdigitale ( national biological resource center : nbrc 5812 ) , and t. tonsurans ( nbrc 5928 ) were examined . all clinical samples from suspected patients were cultured on plates of sabouraud glucose agar ( merck , darmstadt , germany ) , with chloramphenicol and cycloheximide ( scc ) . the trichophyton species were identified based on microscopic morphology and in vitro tests including urease and hair perforating tests , as required . first , trichophyton species were grown on sabouraud dextrose agar ( sda , difco , usa ) for 10 days at 24c in dark conditions . a sterile blade was used to scrape off the hyphae from the surface of the plate , which were transferred to a 2-ml eppendorf tube containing 1 ml of lysis buffer ( 200 mm tris - hcl , ph 8.0 , with 25 mm edta , 0.5% [ wt / vol ] sodium dodecyl sulfate , and 250 mm nacl ) . cells were mechanically disrupted with a conical grinder for approximately 1 min , and then incubated at 100c for 15 min . next , 150 l of 3.0 m sodium acetate buffer was added , the mixture was vortexed and incubated for 10 min at -20c , and the solution was mixed and centrifuged for 5 min at 10,000 g . the supernatant was transferred to a new tube and phenol / chloroform ( 1:1 , v / v ) was used for extraction . dna was allowed to precipitate with an equal volume of isopropanol for 10 min at -20c and then centrifuged for 5 min at 10,000 rpm . the pellets were washed with cold 70% ethanol , dried at room temperature , resuspended in 97.5 ml of te - buffer with 2.5 ml of rnase ( 20 u / ml ) , and incubated for 5 min at 37c . the ribosomal dna internal transcribed spacers ( i.e. , the its region of rdna ) were amplified using the universal primers its1 ( 5-tcc - gtaggtgaacctgcgg-3 ) and its4 ( 5-tcctccgcttattgatatgc-3 ) ( 13 ) . pcr reactions were performed on a tc-312 thermal cycler ( techne , duxford , cambridge , united kingdom ) in 25-ml volumes containing 25 ng of template dna , 2.5 ml of reaction buffer ( 0.1 m tris - hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatin , and 1% triton x-100 ) , 0.2 mm of each dntp , and 2.0 u of taq dna polymerase . amplification was performed as follows : 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) , and 72c ( 120 s ) , with a final 7-min extension step at 72c . pcr products were visualized by 1.5% ( w / v ) agarose gel electrophoresis in tbe buffer , stained with ethidium bromide ( 0.5 g / ml ) , and photographed under uv transillumination . the approximate length of the three specific circular oligonucleotide probes for t. rubrum , t. mentagrophytes 
 var . interdigitale , and t. tonsurans used in this study was 96 to 102 bp , and comprised two target - complementary segments connected by a genetic linker sequence . ( 8) to minimize similarity between closely related strains and to allow primer binding during rca . however , specific padlock probes targeting the its1 and its2 regions were modified , and rca primers designed to specifically bind the linker region of the probes were synthesized by anaspec inc . purified pcr products ( 1 l ) were mixed with 2 u pfu ( 1 l ) dna ligase ( bioneer iso 13485 , alameda , ca , usa ) and 1 l of padlock probe in 500 mm tris - hcl ( ph 7.5 ) , 50 mm kcl , 100 mm mgcl2 , 2.5 l / ml bovine serum albumin ( ph 7.5 ) , 10 mm atp , and 50 mm dtt with a total reaction volume of 10 l . multiple cycle ligations were conducted with one cycle of denaturation at 94c of 5 min , followed by five cycles at 94c for 30 s and 4 min of ligation at 62c . rca reactions were performed in a 25-l volume containing 8 u of bst dna polymerase ( new england biolabs , ipswich , ma , usa ) , 200 m deoxynucleoside triphosphate mix , 1 l of each rca primer , and 2 l of ligation product . probe signals were amplified by incubation at 65c for 60 min and 85c for 2 min , and the accumulation of double - stranded dna products was detected by electrophoresis on 1% agarose containing ethidium bromide ( sigma , st . ladder - like patterns were interpreted as positive reactions , while negative reactions showed no illumination . in the current study , all samples were obtained from different human sources that were referred to the mycology laboratory at tooba clinic , mazandaran university of medical sciences , iran and were suspected dermatophytosis cases . the study protocol was approved by the medical research ethics committee of mazandaran university of medical sciences ( ethical no . 90 - 2 - 28/90 - 14 ) and since the laboratory diagnosis was part of the patients routine care , informed consent for research purposes was not specifically obtained . a total of 61 isolates belonging to three species of trichophyton including the three reference strains t. rubrum ( centraalbureau voor schimmelcultures : cbs 130927 ) , t. mentagrophytes var . interdigitale ( national biological resource center : nbrc 5812 ) , and t. tonsurans ( nbrc 5928 ) were examined . all clinical samples from suspected patients were cultured on plates of sabouraud glucose agar ( merck , darmstadt , germany ) , with chloramphenicol and cycloheximide ( scc ) . the trichophyton species were identified based on microscopic morphology and in vitro tests including urease and hair perforating tests , as required . first , trichophyton species were grown on sabouraud dextrose agar ( sda , difco , usa ) for 10 days at 24c in dark conditions . a sterile blade was used to scrape off the hyphae from the surface of the plate , which were transferred to a 2-ml eppendorf tube containing 1 ml of lysis buffer ( 200 mm tris - hcl , ph 8.0 , with 25 mm edta , 0.5% [ wt / vol ] sodium dodecyl sulfate , and 250 mm nacl ) . cells were mechanically disrupted with a conical grinder for approximately 1 min , and then incubated at 100c for 15 min . next , 150 l of 3.0 m sodium acetate buffer was added , the mixture was vortexed and incubated for 10 min at -20c , and the solution was mixed and centrifuged for 5 min at 10,000 g . the supernatant was transferred to a new tube and phenol / chloroform ( 1:1 , v / v ) was used for extraction . dna was allowed to precipitate with an equal volume of isopropanol for 10 min at -20c and then centrifuged for 5 min at 10,000 rpm . the pellets were washed with cold 70% ethanol , dried at room temperature , resuspended in 97.5 ml of te - buffer with 2.5 ml of rnase ( 20 u / ml ) , and incubated for 5 min at 37c . the ribosomal dna internal transcribed spacers ( i.e. , the its region of rdna ) were amplified using the universal primers its1 ( 5-tcc - gtaggtgaacctgcgg-3 ) and its4 ( 5-tcctccgcttattgatatgc-3 ) ( 13 ) . pcr reactions were performed on a tc-312 thermal cycler ( techne , duxford , cambridge , united kingdom ) in 25-ml volumes containing 25 ng of template dna , 2.5 ml of reaction buffer ( 0.1 m tris - hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatin , and 1% triton x-100 ) , 0.2 mm of each dntp , and 2.0 u of taq dna polymerase . amplification was performed as follows : 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) , and 72c ( 120 s ) , with a final 7-min extension step at 72c . pcr products were visualized by 1.5% ( w / v ) agarose gel electrophoresis in tbe buffer , stained with ethidium bromide ( 0.5 g / ml ) , and photographed under uv transillumination . the approximate length of the three specific circular oligonucleotide probes for t. rubrum , t. mentagrophytes 
 var . interdigitale , and t. tonsurans used in this study was 96 to 102 bp , and comprised two target - complementary segments connected by a genetic linker sequence . ( 8) to minimize similarity between closely related strains and to allow primer binding during rca . however , specific padlock probes targeting the its1 and its2 regions were modified , and rca primers designed to specifically bind the linker region of the probes were synthesized by anaspec inc . purified pcr products ( 1 l ) were mixed with 2 u pfu ( 1 l ) dna ligase ( bioneer iso 13485 , alameda , ca , usa ) and 1 l of padlock probe in 500 mm tris - hcl ( ph 7.5 ) , 50 mm kcl , 100 mm mgcl2 , 2.5 l / ml bovine serum albumin ( ph 7.5 ) , 10 mm atp , and 50 mm dtt with a total reaction volume of 10 l . multiple cycle ligations were conducted with one cycle of denaturation at 94c of 5 min , followed by five cycles at 94c for 30 s and 4 min of ligation at 62c . rca reactions were performed in a 25-l volume containing 8 u of bst dna polymerase ( new england biolabs , ipswich , ma , usa ) , 200 m deoxynucleoside triphosphate mix , 1 l of each rca primer , and 2 l of ligation product . probe signals were amplified by incubation at 65c for 60 min and 85c for 2 min , and the accumulation of double - stranded dna products was detected by electrophoresis on 1% agarose containing ethidium bromide ( sigma , st . ladder - like patterns were interpreted as positive reactions , while negative reactions showed no illumination . first , trichophyton species were grown on sabouraud dextrose agar ( sda , difco , usa ) for 10 days at 24c in dark conditions . a sterile blade was used to scrape off the hyphae from the surface of the plate , which were transferred to a 2-ml eppendorf tube containing 1 ml of lysis buffer ( 200 mm tris - hcl , ph 8.0 , with 25 mm edta , 0.5% [ wt / vol ] sodium dodecyl sulfate , and 250 mm nacl ) . cells were mechanically disrupted with a conical grinder for approximately 1 min , and then incubated at 100c for 15 min . next , 150 l of 3.0 m sodium acetate buffer was added , the mixture was vortexed and incubated for 10 min at -20c , and the solution was mixed and centrifuged for 5 min at 10,000 g . the supernatant was transferred to a new tube and phenol / chloroform ( 1:1 , v / v ) was used for extraction . dna was allowed to precipitate with an equal volume of isopropanol for 10 min at -20c and then centrifuged for 5 min at 10,000 rpm . the pellets were washed with cold 70% ethanol , dried at room temperature , resuspended in 97.5 ml of te - buffer with 2.5 ml of rnase ( 20 u / ml ) , and incubated for 5 min at 37c . the ribosomal dna internal transcribed spacers ( i.e. , the its region of rdna ) were amplified using the universal primers its1 ( 5-tcc - gtaggtgaacctgcgg-3 ) and its4 ( 5-tcctccgcttattgatatgc-3 ) ( 13 ) . pcr reactions were performed on a tc-312 thermal cycler ( techne , duxford , cambridge , united kingdom ) in 25-ml volumes containing 25 ng of template dna , 2.5 ml of reaction buffer ( 0.1 m tris - hcl , ph 8.0 , 0.5 m kcl , 15 mm mgcl2 , 0.1% gelatin , and 1% triton x-100 ) , 0.2 mm of each dntp , and 2.0 u of taq dna polymerase . amplification was performed as follows : 2 min at 94c for primary denaturation , followed by 35 cycles at 94c ( 45 s ) , 52c ( 30 s ) , and 72c ( 120 s ) , with a final 7-min extension step at 72c . pcr products were visualized by 1.5% ( w / v ) agarose gel electrophoresis in tbe buffer , stained with ethidium bromide ( 0.5 g / ml ) , and photographed under uv transillumination . the approximate length of the three specific circular oligonucleotide probes for t. rubrum , t. mentagrophytes 
 var . interdigitale , and t. tonsurans used in this study was 96 to 102 bp , and comprised two target - complementary segments connected by a genetic linker sequence . ( 8) to minimize similarity between closely related strains and to allow primer binding during rca . however , specific padlock probes targeting the its1 and its2 regions were modified , and rca primers designed to specifically bind the linker region of the probes were synthesized by anaspec inc . purified pcr products ( 1 l ) were mixed with 2 u pfu ( 1 l ) dna ligase ( bioneer iso 13485 , alameda , ca , usa ) and 1 l of padlock probe in 500 mm tris - hcl ( ph 7.5 ) , 50 mm kcl , 100 mm mgcl2 , 2.5 l / ml bovine serum albumin ( ph 7.5 ) , 10 mm atp , and 50 mm dtt with a total reaction volume of 10 l . multiple cycle ligations were conducted with one cycle of denaturation at 94c of 5 min , followed by five cycles at 94c for 30 s and 4 min of ligation at 62c . rca reactions were performed in a 25-l volume containing 8 u of bst dna polymerase ( new england biolabs , ipswich , ma , usa ) , 200 m deoxynucleoside triphosphate mix , 1 l of each rca primer , and 2 l of ligation product . probe signals were amplified by incubation at 65c for 60 min and 85c for 2 min , and the accumulation of double - stranded dna products was detected by electrophoresis on 1% agarose containing ethidium bromide ( sigma , st . ladder - like patterns were interpreted as positive reactions , while negative reactions showed no illumination . the trichophyton species were not easily differentiated from each other based on mycological criteria . of the 61 clinically isolated samples , 31 isolates ( 50.8% ) were identified as t. mentagrophytes var interdigitale , 11 isolates ( 18.2% ) as t. rubrum , 9 isolates ( 14.7% ) as t. tonsurans , and 1 isolate as ( 1.8% ) t. violaceum . rca and subsequent detection by gel electrophoresis yielded positive results , and the test proved to be 100% specific for each species . neither cross - reaction between the examined species of trichophyton nor false positive or false negative results were observed ( figure 1 ) . positive rca reactions were visualized by uv irradiation as ladder - like , strongly illuminated smears on 1.2% agarose gels , while the background remained clean for negative reactions ( figure 1 ) . rca reactions were performed for all isolates ; 45 isolates ( 73.3% ) were positive for the probe specific to t. mentagrophytes , 8 isolates ( 13.1% ) for the probe specific to t. rubrum , and 4 isolates ( 6.6% ) for the probe specific to t. tonsurans ( figure 1 ) . however , four isolates ( 8.6% ) did not test positive using any of the three species - specific padlock probes and probably represented separate dermatophyte species . members of the genus trichophyton are the most common agents of dermatophytosis in humans and other animals . identification of trichophyton species is essentially based on macroscopic and microscopic observations of their morphological features ; however , the identification is complicated and laborious owing to the morphological similarity , polymorphism and cultural variability of trichophyton species . thus , accurate identification is time - consuming and requires a significant level of knowledge and technological expertise ( 4 ) . its - based analyses have found that sequence variation is limited to only one or a few snps between certain species , such as between t. tonsurans and t. equinum , and between t. rubrum and t. soudanense ( 7 ) . although sequencing of the its region of rdna is currently the gold standard for the identification of trichophyton species and relatives , the technique is relatively expensive and time - consuming , and is impractical for the analysis of large numbers of isolates for epidemiological studies . to overcome these problems , recently , isothermal techniques , such as loop - mediated amplification and rca , have been applied for rapid identification . the use of circularizable oligonucleotides or padlock probes is based on the rolling replication of short single - stranded dna circles by certain dna polymerases under isothermal conditions to detect target nucleic acid sequences , including snps , with high specificity ( 9 - 16 ) . such probes comprise two sequences that are complementary to the 5 and 3 termini of the target sequence joined by a genetic linker region . upon hybridization to the target , the two probe ends become juxtaposed and are joined by dna ligase to form a closed molecule . the intensity of the signal generated by the probe is then increased exponentially by hyper branching or rca , and 109-fold signal amplification of each circle can be achieved within 90 min ( 8 , 13 , 17 ) . the rca technique was initially established by fire and xu ( 18 ) and liu et al . it is one of a series of robust and simple techniques for distinguishing closely related taxa at the species as well as the strain level . rca is a rapid ( requiring less than 1 working day ) , specific ( to the single - nucleotide level ) , and economical ( requiring minimal equipment ) tool for fungal screening ( 20 , 21 ) . previous studies have shown that trichophyton species have several unique nucleotide positions suitable for the development of specific padlock probes for species characterization ; these can be used to distinguishing closely related species ( 8) . in this study , species - specific padlock probes were used to distinguish three species of trichophyton , t. mentagrophytes , t. rubrum , and t. tonsurans . using species - specific probes , we correctly identified all clinical isolates . three standard species of trichophyton , t. rubrum ( cbs 130927 ) , t. mentagrophytes var . interdigitale ( nbrc 5812 ) , and t. tonsurans ( nbrc 5928 ) were also positively identified using only the species - specific padlock probes . the sequencing results for the its regions of rdna showed 100% concordance with the rca results . additionally , these results were perfectly concordant with phenotypic identification . the rca procedure required less than one working day , including dna extraction , pcr amplification , hybridization , ligation of padlock probes , and rca amplification , rather than sequencing . while the results of this study can be applied generally ( 8) , the specificity of the t. tonsurans probe ( tt - its2 ) is ambiguous because t. equinum is highly closely related . the two species differ by only a single base in the its1 region ( 7 , 22 ) ; therefore , the its region does not have sufficient discrimination ability . designing padlock probes that target other genes , such as -tubulin ( bt2 ) and translation elongation factor 1- ( tef1 ) , may be beneficial . in conclusion , species identification of trichophyton is important for epidemiological and phylogenetic purposes and for genotype delineation . despite the shortcomings of current molecular identification systems , there is a strong stimulus for the continued use of its polymorphisms to generate identification barcodes . therefore , the rca - based assay is an alternative to dna sequencing for species identification .