BACKGROUND
members of the mycobacterium avium complex , which are responsible for opportunistic infections, particularly in aids patients, are the most important nontuberculous mycobacteria  in clinical or epidemiological contexts.
traditionally, the mac includes two species, m. avium and m. intracellulare .
in addition to these  <dig> species, recent advances in molecular taxonomy have fuelled the identification of novel species within the mac .
among these species, our group introduced the novel species mycobacterium yongonense, which is phylogenetically related to m. intracellulare and was isolated from a korean patient with pulmonary symptoms  <cit> .
notably, the rna polymerase β subunit gene  sequence of m. yongonense is identical to that of m. parascrofulaceum, a distantly related scotochromogen, suggesting acquisition of the rpob gene via a potential lateral gene transfer  event .
recently, tortoli et al.
reported pulmonary disease caused by m. yongonense strains isolated from patients in italy.
however, this strain notably harbors rpob sequences almost identical to those of m. intracellulare but not to those of m. parascrofulaceum, suggesting the possibility of the existence of another group of m. yongonense strains that were not subject to the lgt event involving the rpob gene from m. parascrofulaceum  <cit> .
furthermore, the potential of its misidentification has recently been proposed  <cit> .
therefore, the development of a novel diagnostic method for the precise identification of clinical strains of m. yongonense is necessary.
insertion sequence  elements have several traits of great interest in relation to epidemiological evaluations, taxonomic studies and diagnostic purposes.
depending on the degree of mobility and the copy number of is elements, dna fingerprints based on southern blotting and hybridization can be used to infer strain relatedness  <cit> .
for mycobacterial is elements, it has generally been accepted that genetic rearrangement due to their insertion may frequently be limited to the species or subspecies level .
this specificity has led to the use of is elements as markers for mycobacterial diagnosis, such as is <dig> for the detection of m. tuberculosis  <cit> , or is <dig> for the detection of m. paratuberculosis  <cit> .
the aim of the present study is to develop a novel real-time pcr method targeting is elements for the specific detection of m. yongonense.
to this end, we first sought to identify the most appropriate is element for use as a diagnostic target via comparison of the entire is elements of  <dig> strains of m. intracellulare  and  <dig> strains of m. yongonense , whose complete genome sequences were recently reported by our group .
firstly, the two m. yongonense strains, mott-h4y and mott-36y had been identified as m. intracellulare int  <dig> group  <cit> .
however, our complete genome based phylogenetic analysis proved they belonged into m. yongonense rather than m. intracellulare .
RESULTS
characterization of the ismyo <dig> is element specific to m. yongonense
to select is elements specific to m. yongonense strains for the diagnosis of m. yongonense, we compared the distributions of is elements/transposase sequences between 3 m. yongonense strains  and  <dig> other mac strains  via analysis of the seven retrieved mycobacterial genomes .
we identified a total of  <dig>   <dig> and  <dig> is elements in m. yongonense dsm 45126t, m. yongonense mott-36y, and m. yongonense mott-h4y, respectively, using the is finder program .
in the case of m. yongonense dsm 45126t,  <dig> types of is families  were identified in the genome.
through comparison of the distributions of is elements among the  <dig> retrieved mycobacterial genome sequences, seven is elements  were identified which found in only the genomes of m. yongonense strains.
among these is elements, we finally targeted an ismt2- like is element belonging to the is <dig> family, designated ismyo <dig>  which was found in only the 3 m. yongonense strains, and not in other examined strains .table  <dig> genome sequences used in this study
m. intracellulare atcc 13950t
m. yongonense dsm 45126t
as shown for other is elements of the is <dig> family  <cit> , the m. yongonense-specific is elements also exhibit two consecutive open reading frames: a long upstream frame, designated ista , and a shorter downstream frame, designated istb .
ista- and istb-like sequences overlap between the stop codon of ista and the start codon of istb.
additionally, upstream and downstream of the two overlapping orfs, there are three left inverted repeat  sequences  and two right inverted repeat  sequences  .fig.
<dig> schematic representation of the sequence of the m. yongonense-specific is elements ismyo <dig>  for the ista-like and istb-like sequences, the stop and start codons overlap.
three left inverted repeat  sequences were found upstream of the ista-like sequence, and two right inverted repeat  sequences were found downstream of istb-like sequence
a blast database search at the protein level revealed that ista- and istb- like sequences from saccharopolyspora spinosa show sequence identities of  <dig>  %  and  <dig>  % , respectively, with those of ismyo <dig>  however, no mycobacterial is elements matching ismyo <dig> were found .
based on these findings, the is element identified in the m. yongonense strains was considered a putative novel is element belonging to the is <dig> family and was designated ismyo <dig> according to the nomenclature suggested by mahillon and chandler  <cit> , and its genbank accession no.
is kp <dig>
the copy numbers of ismyo <dig> in the genomes of the 3 m. yongonense strains, dsm 45126t, mott-36y, and mott-h4y, were  <dig>   <dig> and  <dig>  respectively .
exceptionally, in the genome of m. yongonense mott-36y, a copy of ismyo <dig>  was identified, but there was no stop codon between the ista- and istb-like sequences.
additionally, in the case of m. yongonense mott-h4y, a copy of ismyo <dig> included only an istb-like sequence , and no ista-like sequences .
for comparison with other is elements related to the is <dig> family,  <dig> additional is element sequences were retrieved from the genbank database and compared with the ista- and istb-like sequences of ismyo <dig>  ismyo <dig> clustered together with is <dig> from m. tuberculosis for two orfs, though they showed low sequence similarity at the amino acid level  .fig.
<dig> phylogenetic trees based on  ista-like sequences and  istb-like sequences from m. yongonense dsm 45126t and  <dig> other bacteria.
the trees were constructed using the neighbor-joining method.
bootstrap values were calculated from  <dig>  replications, and values of < <dig> are not indicated in the trees.
the bars indicate the numbers of substitutions per amino acid position
the phylogenetic tree based on the ista- and istb-like sequences from the m. yongonense strains showed the presence of four different groups .
in the case of m. yongonense dsm 45126t, the observed ismyo <dig> sequences were highly conserved in the genome.
however, those of m. yongonense mott-36y or mott-h4y showed variations in the genomes.
for the ista sequence , the sequence homologies between the  <dig> alleles of m. yongonense dsm 45126t, the  <dig> alleles of m. yongonense mott-36y, and the  <dig> alleles of m. yongonense mott-h4y ranged from  <dig>  to  <dig>  %, from  <dig>  to  <dig>  %, and from  <dig>  to  <dig>  %, respectively.
for the istb sequence , the sequence homologies between the  <dig> alleles of m. yongonense dsm 45126t, the  <dig> alleles of m. yongonense mott-36y, and the  <dig> alleles of m. yongonense mott-h4y were 100 %, from  <dig>  to  <dig>  %, and from  <dig>  to  <dig>  %, respectively.
development of a real-time pcr assay targeting ismyo <dig> for the detection of m. yongonense strains
to develop appropriate primer sets based on ismyo <dig> sequences for the specific amplification of m. yongonense strains, ismyo <dig> sequences from a total of  <dig> copies from the genomes of 3 m. yongonense strains were compared.
finally, we designed a primer set targeting sequences that are conserved in all  <dig> copies of ismyo <dig>  producing 338-bp amplicons  .fig.
<dig> primers designed for the identification of myocbacterium yongonense on the basis of ismyo <dig> sequence alignment for m. yongonense strains.
arrows indicate the primer positions.
the numbers indicate the nucleotide positions in the ista-like sequence of ismyo <dig> of m. yongonense.
boldface bases denote the bases that differ from those in the consensus sequence.
the strains included in this analysis were as follows: m. yongonense dsm 45126t; m. yongonense mott-36y; and m. yongonense mott-h4y
specificity of the diagnostic assays
the specificity of the real-time pcr assay developed for the identification of m. yongonenese was tested in  <dig> reference strains of mycobacteria via cq and melting curve analyses.
three m. yongonense strains  were specifically identified through measurement of cq and tm , whereas none of the  <dig> other reference strains showed any detectable cqs or melting temperatures .
the real-time pcr assay was then applied to  <dig> clinical isolates, including a number of mac species; again, only the 3 m. yongonense strains were identified, and not the  <dig> other clinical mac isolates , based on the measurement of cqs and specific tms  .
agarose gel electrophoresis analysis of the real-time pcr products of 6 m. yongonense strains  revealed a single electrophoretic band of the predicted size  .fig.
<dig> specificity test for the real-time pcr assay developed for the identification of m. yongonense with using  <dig> reference strains of mycobacterium species.
m. yongonense was specifically identified based on cq and melting temperature measurements.
the tested strains are the same as those listed in additional file  <dig> and were tested in duplicate via sybr green i real-time pcr.
a, amplification curves; b, melting curve analysis
sensitivity of the diagnostic assays
to determine the detection limits of the real-time pcr assay for the detection of m. yongonense strains, serially diluted dna from all  <dig> strains of the tested m. yongonense  was subject to real-time pcr.
the detection limit of the real-time pcr assay for m. yongonense species was 10 fg of genomic dna in one strain  and  <dig>  pg in all of the other tested strains .fig.
<dig> analysis of the detection limits of the real-time pcr assay for the identification of m. yongonense species.
all of the strains of m. yongonense species tested were detected, using as little as  <dig>  pg of their genomic dna
discussion
among the members of the mac complex, for the diagnosis and epidemiology of 4 m. avium subspecies , at least four major is elements have been widely used, namely is <dig>  is <dig>  is <dig> and is <dig>  <cit> .
however, despite the increases in global clinical cases and in the species diversity of m. intracellulare-related strains, the description of is elements for their diagnosis and epidemiology has rarely been reported thus far.
in this study, we first introduced a novel is element, ismyo <dig>  specific to m. yongonense strains, which belong to the m. intracellulare-related species.
this element can be used for the diagnosis and epidemiology of these strains, particularly to resolve the current taxonomic need of selectively discriminating m. yongonense from other m. intracellulare related strains.
in this study, genome analysis using is finder revealed the presence of a total of  <dig> is elements and is-like elements, consisting of at least  <dig> types of is families, in the genome of m. yongonense dsm 45126t.
of these is elements, a total of  <dig> were shown to be present in only the genomes of 3 m. yongonense strains, and were absent in the genomes of 3 m. intracellulare strains .
however, considering the copy numbers, blast search results, and sequence conservation between is alleles in a genome, we finally selected ismyo <dig>  belonging to the is <dig> family, as a target is element for the diagnosis of m. yongonense strains.
it is worth noting several characteristic features of ismyo <dig> that make it a useful diagnostic marker for m. yongonense.
the first is the high specificity of ismyo <dig> for m. yongonense.
to date,  <dig> types of is <dig> family members have been described in the genus mycobacterium, three in m. tuberculosis   <cit>  and one in m. avium   <cit> .
despite belonging to the is <dig> family, the ismyo <dig> element identified in this study shows a very low level of sequence homology with other is <dig> members from mycobacteria, particularly in terms of dna sequences, suggesting the feasibility of using ismyo <dig> as a diagnostic marker for m. yongonense.
furthermore, the application of a real-time pcr assay targeting ismyo <dig> in reference mycobacterial strains demonstrated that the assay was specific to only m. yongonense strains .
furthermore, it did not produce any amplicons in any of the  <dig> examined m. intracellulare clinical isolates, which included diverse genotypes such as int- <dig>  int- <dig> and int- <dig>  which are closely related to m. yongonense .
second, the ismyo2- targeting assay was able to detect  <dig> types of m. yongonense variants.
our genome analysis revealed that ismyo <dig> is also present in the genomes of  <dig> strains of m. yongonense, mott-36y and mott-h4y, which were previously designated as the m. intracellulare int- <dig> genotype, as well as in genome of m. yongonense dsm 45126t.
although two int- <dig> strains exhibit rpob sequences identical to that of m. intracellulare but not to that of m. parascrofulaceum, our phylogenetic analysis based on complete genome sequences showed that these strains and m. yongonense dsm 45126t are tightly clustered and are separated from other m. intracellulare strains , suggesting that they may be members of the same species, m. yongonense.
our results also strongly supported the hypothesis previously put forth by tortoli et al.
<cit>  that there may be at least two variants in m. yongonense strains, one of which was subjected to the rpob lgt event from m. parascrofulaceum, including strains such as m. yongonense dsm 45126t, and another, phylogenetically older variant, that was not subject to the rpob lgt, including strains such as the int- <dig> strains mott-36y and mott-h4y.
third, the ismyo <dig> targeting assay exhibited high sensitivity in detecting m. yongonense strains.
our genome analysis showed that more than  <dig> copies of ismyo <dig> are present in m. yongonense genomes .
furthermore, the sequence conservation between the ismyo <dig> alleles found in the m. yongonense genomes facilitated the development of a common primer set capable of pcr amplification of all of these alleles .
thus, we successfully developed a real-time pcr assay capable of pcr amplification of all of the alleles via performing multiple sequence alignments of  <dig> ismyo <dig> alleles from the genomes of 3 m. yongonense strains.
our real-time pcr assay can detect pcr amplicons at a dna level of 100 fg in all 6 m. yongonense strains , suggesting its feasibility as a diagnostic method for m. yongonense strains.
CONCLUSIONS
in conclusion, we identified a novel ismyo <dig> is element belonging to the is <dig> family that is specific to m. yongonense strains via genome analysis and developed a real-time pcr method based on its sequences.

 members of the mycobacterium avium complex, which are responsible for opportunistic infections, particularly in aids patients, are the most important nontuberculous mycobacteria in clinical or epidemiological contexts. traditionally, the mac includes two species, m. avium and m. intracellulare. in addition to these species, recent advances in molecular taxonomy have fuelled the identification of novel species within the mac. among these species, our group introduced the novel species mycobacterium yongonense, which is phylogenetically related to m. intracellulare and was isolated from a korean patient with pulmonary symptoms. notably, the rna polymerase β subunit gene sequence of m. yongonense is identical to that of m. parascrofulaceum, a distantly related scotochromogen, suggesting acquisition of the rpob gene via a potential lateral gene transfer event. recently, tortoli et al. reported pulmonary disease caused by m. yongonense strains isolated from patients in italy. however, this strain notably harbors rpob sequences almost identical to those of m. intracellulare but not to those of m. parascrofulaceum, suggesting the possibility of the existence of another group of m. yongonense strains that were not subject to the lgt event involving the rpob gene from m. parascrofulaceum. furthermore, the potential of its misidentification has recently been proposed. therefore, the development of a novel diagnostic method for the precise identification of clinical strains of m. yongonense is necessary. insertion sequence elements have several traits of great interest in relation to epidemiological evaluations, taxonomic studies and diagnostic purposes. depending on the degree of mobility and the copy number of is elements, dna fingerprints based on southern blotting and hybridization can be used to infer strain relatedness. for mycobacterial is elements, it has generally been accepted that genetic rearrangement due to their insertion may frequently be limited to the species or subspecies level. this specificity has led to the use of is elements as markers for mycobacterial diagnosis, such as is for the detection of m. tuberculosis, or is for the detection of m. paratuberculosis. the aim of the present study is to develop a novel real-time pcr method targeting is elements for the specific detection of m. yongonense. to this end, we first sought to identify the most appropriate is element for use as a diagnostic target via comparison of the entire is elements of strains of m. intracellulare and strains of m. yongonense, whose complete genome sequences were recently reported by our group. firstly, the two m. yongonense strains, mott-h4y and mott-36y had been identified as m. intracellulare int group. however, our complete genome based phylogenetic analysis proved they belonged into m. yongonense rather than m. intracellulare.  characterization of the ismyo is element specific to m. yongonense to select is elements specific to m. yongonense strains for the diagnosis of m. yongonense, we compared the distributions of is elements/transposase sequences between 3 m. yongonense strains and other mac strains via analysis of the seven retrieved mycobacterial genomes. we identified a total of and is elements in m. yongonense dsm 45126t, m. yongonense mott-36y, and m. yongonense mott-h4y, respectively, using the is finder program. in the case of m. yongonense dsm 45126t, types of is families were identified in the genome. through comparison of the distributions of is elements among the retrieved mycobacterial genome sequences, seven is elements were identified which found in only the genomes of m. yongonense strains. among these is elements, we finally targeted an ismt2- like is element belonging to the is family, designated ismyo which was found in only the 3 m. yongonense strains, and not in other examined strains.table genome sequences used in this study m. intracellulare atcc 13950t m. yongonense dsm 45126t as shown for other is elements of the is family, the m. yongonense-specific is elements also exhibit two consecutive open reading frames: a long upstream frame, designated ista, and a shorter downstream frame, designated istb. ista- and istb-like sequences overlap between the stop codon of ista and the start codon of istb. additionally, upstream and downstream of the two overlapping orfs, there are three left inverted repeat sequences and two right inverted repeat sequences.fig. schematic representation of the sequence of the m. yongonense-specific is elements ismyo for the ista-like and istb-like sequences, the stop and start codons overlap. three left inverted repeat sequences were found upstream of the ista-like sequence, and two right inverted repeat sequences were found downstream of istb-like sequence a blast database search at the protein level revealed that ista- and istb- like sequences from saccharopolyspora spinosa show sequence identities of  % and  %, respectively, with those of ismyo however, no mycobacterial is elements matching ismyo were found. based on these findings, the is element identified in the m. yongonense strains was considered a putative novel is element belonging to the is family and was designated ismyo according to the nomenclature suggested by mahillon and chandler, and its genbank accession no. is kp the copy numbers of ismyo in the genomes of the 3 m. yongonense strains, dsm 45126t, mott-36y, and mott-h4y, were and respectively. exceptionally, in the genome of m. yongonense mott-36y, a copy of ismyo was identified, but there was no stop codon between the ista- and istb-like sequences. additionally, in the case of m. yongonense mott-h4y, a copy of ismyo included only an istb-like sequence, and no ista-like sequences. for comparison with other is elements related to the is family, additional is element sequences were retrieved from the genbank database and compared with the ista- and istb-like sequences of ismyo ismyo clustered together with is from m. tuberculosis for two orfs, though they showed low sequence similarity at the amino acid level.fig. phylogenetic trees based on ista-like sequences and istb-like sequences from m. yongonense dsm 45126t and other bacteria. the trees were constructed using the neighbor-joining method. bootstrap values were calculated from replications, and values of < are not indicated in the trees. the bars indicate the numbers of substitutions per amino acid position the phylogenetic tree based on the ista- and istb-like sequences from the m. yongonense strains showed the presence of four different groups. in the case of m. yongonense dsm 45126t, the observed ismyo sequences were highly conserved in the genome. however, those of m. yongonense mott-36y or mott-h4y showed variations in the genomes. for the ista sequence, the sequence homologies between the alleles of m. yongonense dsm 45126t, the alleles of m. yongonense mott-36y, and the alleles of m. yongonense mott-h4y ranged from to  %, from to  %, and from to  %, respectively. for the istb sequence, the sequence homologies between the alleles of m. yongonense dsm 45126t, the alleles of m. yongonense mott-36y, and the alleles of m. yongonense mott-h4y were 100 %, from to  %, and from to  %, respectively. development of a real-time pcr assay targeting ismyo for the detection of m. yongonense strains to develop appropriate primer sets based on ismyo sequences for the specific amplification of m. yongonense strains, ismyo sequences from a total of copies from the genomes of 3 m. yongonense strains were compared. finally, we designed a primer set targeting sequences that are conserved in all copies of ismyo producing 338-bp amplicons.fig. primers designed for the identification of myocbacterium yongonense on the basis of ismyo sequence alignment for m. yongonense strains. arrows indicate the primer positions. the numbers indicate the nucleotide positions in the ista-like sequence of ismyo of m. yongonense. boldface bases denote the bases that differ from those in the consensus sequence. the strains included in this analysis were as follows: m. yongonense dsm 45126t; m. yongonense mott-36y; and m. yongonense mott-h4y specificity of the diagnostic assays the specificity of the real-time pcr assay developed for the identification of m. yongonenese was tested in reference strains of mycobacteria via cq and melting curve analyses. three m. yongonense strains were specifically identified through measurement of cq and tm, whereas none of the other reference strains showed any detectable cqs or melting temperatures. the real-time pcr assay was then applied to clinical isolates, including a number of mac species; again, only the 3 m. yongonense strains were identified, and not the other clinical mac isolates, based on the measurement of cqs and specific tms. agarose gel electrophoresis analysis of the real-time pcr products of 6 m. yongonense strains revealed a single electrophoretic band of the predicted size.fig. specificity test for the real-time pcr assay developed for the identification of m. yongonense with using reference strains of mycobacterium species. m. yongonense was specifically identified based on cq and melting temperature measurements. the tested strains are the same as those listed in additional file and were tested in duplicate via sybr green i real-time pcr. a, amplification curves; b, melting curve analysis sensitivity of the diagnostic assays to determine the detection limits of the real-time pcr assay for the detection of m. yongonense strains, serially diluted dna from all strains of the tested m. yongonense was subject to real-time pcr. the detection limit of the real-time pcr assay for m. yongonense species was 10 fg of genomic dna in one strain and  pg in all of the other tested strains.fig. analysis of the detection limits of the real-time pcr assay for the identification of m. yongonense species. all of the strains of m. yongonense species tested were detected, using as little as  pg of their genomic dna discussion among the members of the mac complex, for the diagnosis and epidemiology of 4 m. avium subspecies, at least four major is elements have been widely used, namely is is is and is. however, despite the increases in global clinical cases and in the species diversity of m. intracellulare-related strains, the description of is elements for their diagnosis and epidemiology has rarely been reported thus far. in this study, we first introduced a novel is element, ismyo specific to m. yongonense strains, which belong to the m. intracellulare-related species. this element can be used for the diagnosis and epidemiology of these strains, particularly to resolve the current taxonomic need of selectively discriminating m. yongonense from other m. intracellulare related strains. in this study, genome analysis using is finder revealed the presence of a total of is elements and is-like elements, consisting of at least types of is families, in the genome of m. yongonense dsm 45126t. of these is elements, a total of were shown to be present in only the genomes of 3 m. yongonense strains, and were absent in the genomes of 3 m. intracellulare strains. however, considering the copy numbers, blast search results, and sequence conservation between is alleles in a genome, we finally selected ismyo belonging to the is family, as a target is element for the diagnosis of m. yongonense strains. it is worth noting several characteristic features of ismyo that make it a useful diagnostic marker for m. yongonense. the first is the high specificity of ismyo for m. yongonense. to date, types of is family members have been described in the genus mycobacterium, three in m. tuberculosis and one in m. avium. despite belonging to the is family, the ismyo element identified in this study shows a very low level of sequence homology with other is members from mycobacteria, particularly in terms of dna sequences, suggesting the feasibility of using ismyo as a diagnostic marker for m. yongonense. furthermore, the application of a real-time pcr assay targeting ismyo in reference mycobacterial strains demonstrated that the assay was specific to only m. yongonense strains. furthermore, it did not produce any amplicons in any of the examined m. intracellulare clinical isolates, which included diverse genotypes such as int- int- and int- which are closely related to m. yongonense. second, the ismyo2- targeting assay was able to detect types of m. yongonense variants. our genome analysis revealed that ismyo is also present in the genomes of strains of m. yongonense, mott-36y and mott-h4y, which were previously designated as the m. intracellulare int- genotype, as well as in genome of m. yongonense dsm 45126t. although two int- strains exhibit rpob sequences identical to that of m. intracellulare but not to that of m. parascrofulaceum, our phylogenetic analysis based on complete genome sequences showed that these strains and m. yongonense dsm 45126t are tightly clustered and are separated from other m. intracellulare strains, suggesting that they may be members of the same species, m. yongonense. our results also strongly supported the hypothesis previously put forth by tortoli et al. that there may be at least two variants in m. yongonense strains, one of which was subjected to the rpob lgt event from m. parascrofulaceum, including strains such as m. yongonense dsm 45126t, and another, phylogenetically older variant, that was not subject to the rpob lgt, including strains such as the int- strains mott-36y and mott-h4y. third, the ismyo targeting assay exhibited high sensitivity in detecting m. yongonense strains. our genome analysis showed that more than copies of ismyo are present in m. yongonense genomes. furthermore, the sequence conservation between the ismyo alleles found in the m. yongonense genomes facilitated the development of a common primer set capable of pcr amplification of all of these alleles. thus, we successfully developed a real-time pcr assay capable of pcr amplification of all of the alleles via performing multiple sequence alignments of ismyo alleles from the genomes of 3 m. yongonense strains. our real-time pcr assay can detect pcr amplicons at a dna level of 100 fg in all 6 m. yongonense strains, suggesting its feasibility as a diagnostic method for m. yongonense strains.  in conclusion, we identified a novel ismyo is element belonging to the is family that is specific to m. yongonense strains via genome analysis and developed a real-time pcr method based on its sequences. 
we identified a novel ismyo is element belonging to the is family specific to m. yongonense strains via genome analysis, and a real-time pcr method based on its sequences was developed. due to its close relatedness to other mac members, particularly m. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes. to identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence elements of 3 m. intracellulare and 3 m. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the m. yongonense-specific is element, ismyo, belonging to the is family. next, we developed a real-time pcr method using sybr green i for m. yongonense-specific detection targeting ismyo producing a 338-bp amplicon. using the mycobacterium yongonense genome information, we have identified a novel is-element, ismyo targeting the ismyo sequence, we developed a real-time pcr method and applied the technique to mycobacterial genomic dna. mycobacterium yongonense, as a novel member of the m. avium complex, was recently reported to be isolated from human specimens in south korea and italy. when this assay was applied to mycobacterium reference strains and mac clinical isolates, it produced amplicons in only the 6 m. yongonense strains, showing a sensitivity of 100 fg of genomic dna, suggesting its feasibility as a diagnostic method for m. yongonense strains.
 mycobacterium yongonense, as a novel member of the m. avium complex, was recently reported to be isolated from human specimens in south korea and italy. due to its close relatedness to other mac members, particularly m. intracellulare in taxonomic aspects, the development of a novel diagnostic method for its specific detection is necessary for clinical or epidemiologic purposes. methods using the mycobacterium yongonense genome information, we have identified a novel is-element, ismyo targeting the ismyo sequence, we developed a real-time pcr method and applied the technique to mycobacterial genomic dna.  to identify proper nucleic acid targets for the diagnosis, comparisons of all insertion sequence elements of 3 m. intracellulare and 3 m. yongonense strains, whose complete genome sequences we reported recently, led to the selection of a novel target gene, the m. yongonense-specific is element, ismyo, belonging to the is family. next, we developed a real-time pcr method using sybr green i for m. yongonense-specific detection targeting ismyo producing a 338-bp amplicon. when this assay was applied to mycobacterium reference strains and mac clinical isolates, it produced amplicons in only the 6 m. yongonense strains, showing a sensitivity of 100 fg of genomic dna, suggesting its feasibility as a diagnostic method for m. yongonense strains.  we identified a novel ismyo is element belonging to the is family specific to m. yongonense strains via genome analysis, and a real-time pcr method based on its sequences was developed. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mycobacterium yongonenseinsertion sequence elementismyo2real-time pcrdiagnostic markerissue-copyright-statement© the author 2015 