cultures , susceptibility testing and dna extraction - one hundred and 
 eight cultures had previously been tested for drug susceptibility using the ogawa - kudoh 
 standard proportion method ( canetti et al . 1963 ) 
 at the central laboratory of rio grande do sul ( ipb / lacen - rs ) , the regional reference 
 laboratory for mycobacterium - related disease in the city of porto 
 alegre , south region of brazil . this study was approved by the ethical committee of the 
 school of public health and the state foundation of health research and production of 
 brazil under protocols 465/09 and 03/2010 , respectively . nucleic acids were extracted from the m. tuberculosis culture using the 
 cetyl trimethylammonium bromide method , as described previously ( van soolingen et al . 1994 ) at the centre of scientific and 
 technological development of the state foundation in production and health research , 
 porto alegre . the genes encoding rpob , katg and the 
 inha promoter , which are related to rmp and inh , respectively , had 
 been sequenced as part of an earlier study ( verza et al . 
 2009 , maschmann et al . the h37rv 
 ( atcc27294 ) reference strain was used as a control for both dst and genotyping . amplification of part of rpob , katg , inha and is6110 - the multiplex 
 pcr was performed using the primers described in supplementary data . the reverse primers 
 were biotin labelled and the pcr - multiplex was standardised in a final volume of 50 l 
 containing 200 m of each dntp , 10 mm tris - hcl ( ph 8.3 ) , 50 mm kcl , 2 mm mgcl2 , 
 10 pmoles of primers rif1 and rif2 each , 25 pmoles of primers 
 katg1 and katg2 , inha1 and 
 inha2 and is1 and is2 each , 2.5 u of taq dna polymerase 
 ( invitrogen / usa ) and 1 l ( 100 ng/l ) of purified bacterial dna . the pcr reactions were 
 carried out as follows : 3 min at 95c , 30 cycles of 1 min at 95c , 1 min at 60c , 1.5 
 min at 72c and 4 min at 72c . each amplification experiment included a positive 
 ( m. tuberculosis h37rv dna , 100 ng ) and a negative control ( water ) . 
 for standardisation of the amplification reactions , the pcr products were analysed using 
 polyacrylamide gel electrophoresis , followed by detection in ultraviolet light after 
 staining with ethidium bromide and comparison of the amplicons to a molecular weight 
 marker . reverse - line blot hybridisation and colorimetric detection - reverse 
 hybridisation and colorimetric detection were performed based on the previously 
 described protocol ( maschmann et al . the 
 membranes were washed in 2x ssc/0.1% sodium dodecyl sulfate ( sds ) for 5 min at 50c and 
 were incubated in 2x ssc with 5% bovine serum albumin ( bsa ) at 50c for 15 min . twenty - five microlitres of each pcr reaction was added to 150 l of 2x ssc/0.1% sds and 
 the mixture was denatured at 100c for 10 min and then transferred to an ice bath . the 
 denatured pcr product was then added to a polyethylene tube containing a membrane in 1.4 
 ml of the hybridisation solution ( 2x ssc/0.1% sds ) . after hybridisation , the membranes were washed in 2x 
 ssc/0.5% sds at 57c for 10 min each . the membranes were then treated with tris - buffered 
 saline ( tbs ) ( 100 mm tris - hcl , 150 mm nacl , ph 7.5 ) containing 6% bsa for 30 min at 
 50c . the membranes were incubated in tbs containing 0.33 g / ml streptavidin - alkaline 
 phosphatase ( ap ) conjugate for 15 min at room temperature ( rt ) . the unbound conjugate 
 was removed by washing in tbs for 10 min followed by the addition of ap buffer ( 100 mm 
 tris - hcl , 150 mm nacl and 5 mm mgcl26h2o , ph 9.5 ) for 10 min at 
 rt . the hybridisation was visualised by adding 40 g / ml 5-bromo-4-chloro-3-indoyl 
 phosphatase and 82.5 g / ml nitro blue tetrazolium ( sigma ) in the ap buffer and 
 incubation for 10 min at rt . a purple precipitate was observed when there was a perfect 
 match between the probe and the biotinylated pcr product . the colorimetric reaction was 
 blocked with distilled water and the membranes were dried at rt . 
 sensitivity and specificity of the assay - the analytical sensitivity 
 of the test was determined by performing the assay with a serial 10-fold dilution of 
 m. tuberculosis h37rv dna ( 100 ng to 1 pg ) . to determine the 
 specificity of line - tb / mdr , 100 ng of genomic dna from each of the following bacteria were submitted to the entire procedure : neisseria meningitidis , streptococcus 
 pneumoniae , salmonella enterica , haemophilus influenzae , escherichia coli 
 ( obtained from ipb / lacen - rs ) , mycobacterium marinum , mycobacterium 
 intracellulare , mycobacterium scrofulaceum , mycobacterium gordonae , mycobacterium 
 avium , mycobacterium smegmatis , mycobacterium kansasii , mycobacterium xenopi , 
 mycobacterium fortuitum - peregrinum and mycobacterium phlei 
 ( obtained from oswaldo cruz foundation , brazil ) ( verza et al . probe design and binding to membrane - the oligonucleotide probes were 
 designed using the software primer express v.2.0 ( applied biosystems ) . our final assay 
 setup was composed of ( i ) five probes for the wild type ( wt ) rpob 
 allele and five probes for each of the different rpob mutant alleles , 
 ( ii ) one probe each for the wt and mutant katg and ( iii ) one probe each 
 for the wt and mutant inha promoter alleles . additionally , we designed 
 a probe specific for hybridisation with the insertion sequence is6110 
 ( supplementary data ) . for the immobilisation of the dna probes , a negatively - charged nylon membrane ( biodyne 
 c , pall corporation ) was activated in a solution of 16% ( w / v ) 
 ( 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ) ( acros organics ) for 20 
 min at rt and was washed in distilled water for 2 min at rt . then , 10 l of each of the 
 oligonucleotide probes , containing a 5-terminal amino group , were diluted to the 
 appropriate concentration ( supplementary data ) in 0.5 m nahco3 ( ph 8.4 ) and 
 plotted into 15 circles ( 5 mm ) previously designed on the membrane , followed by 
 incubation for 1 min at rt . the membranes were then incubated in 0.1 m naoh for 10 min , 
 washed in distilled water for 2 min at rt , washed in 2x ssc/0.1% sds for 10 min at 50c 
 and finally incubated in 20 mm edta ( ph 8.0 ) for 15 min at rt . the membranes were 
 finally transferred into a polyethylene tube and stored at 4c for later use . assay interpretation - the panel f in figure demonstrates the positions of the probes for rpob , 
 katg , inha and is6110 . a 
 hybridisation result was considered positive when a purple colour was observed in the 
 circle where a particular probe had been plotted . the probes in rpob 
 were located within the 81 bp resistance - determining region ( rdr ) and hybridisation with 
 all five wt probes was considered to indicate an rmp - susceptible strain . when one of 
 these signals was lacking , independent of any hybridisation to probes 6 - 10 , the strain 
 was considered to be an rpob - mutated strain . a strain was considered 
 mutated for the inha gene when there was a lack of hybridisation to 
 probe 12 and/or hybridisation to probe 11 occurred ; the wt katg gene 
 was represented by hybridisation to probe 13 and a positive signal for probe 14 was 
 observed when the gene was mutated . the hybridisation to probe 15 
 ( is6110 ) was an additional confirmation of the m. 
 tuberculosis complex ( mtbc ) . a : example of the wild type ( wt ) result to the three genes 
 rpob , katg and inha ; b : 
 example of a strain mutated in the 531 region of the rpob gene 
 with no hybridisation with the rif5wt probe and hybridisation with the rif4 m ; 
 c : hybridisation with the 14 katgm probe ; d : hybridisation 
 with the probe number 11 ; e : hybridisation was not observed with the rif4 
 probe , what suggested that there are mutation in the rpob gene 
 codon 526 , although the method have not allowed to identified the mutation ; f : 
 schematic presentation of line - tb / mdr . probes position : rpob 
 wt probes , circles 1 - 5 ( rif 1 , rif 2 , rif 3 , rif 4 , rif 5 ) ; 
 rpob mutated probes , 6 - 10 ( rif1 m , rif2 m , rif3 m , rif4 m , 
 rif5 m ) ; 11 , inham ; 12 , 
 inhawt ; 13 , katgwt ; 14 , 
 katgm ; 15 , is6110 . 
 
 genotype 
  mtbdrplus assay - the genotype mtbdrplus assay was 
 performed according to the manufacturer s instructions ( crudu et al . 65 were tested 
 using the genotype mtbdrplus assay . in order to guarantee the highest 
 fidelity possible between the line - tb / mdr assay and the genotype 
 mtbdrplus assay , the same dna aliquot was used in both 
 experiments . a positive hybridisation signal was observed with the probe specific for 
 is6110 for all 108 isolates ; no non - mycobacterial dnas or 
 mycobacterium species other than those of the mtbc presented a 
 signal at this membrane region . the test was therefore 100% sensitive and specific for 
 strains belonging to the mtbc ( a in figure ) . among 
 the 108 cultures , 55 were considered sensitive to both of the drugs and presented the wt 
 alleles for rpob , katg and inha in 
 the dst . sequence analysis demonstrated the presence of at least one mutation in the 
 rpob gene in 53 of the mdr - tb samples and among these , 31 presented 
 an additional mutation in katg . there were also three samples that 
 presented mutations in the inha promoter region and 10 presented 
 mutations in both katg and inha . nine isolates ( 17% ) 
 had no mutation in either of the inh - r - associated genes ( table i ) . table icorrelation between line - tb / mdr and sequencingline - tb / mdr pattern   line - tb / mdr result - location of mutationsequencing data   positive  probesgene 
 ( probe )  nucleotide / amino acid changesstrains n ( % ) 1,2,3,4 * 
 rpob ( rif4 m ) 
 s531ltcg - ttg / ser - leu37 ( 69.8)1,2,3,4 * 
 rpob ( rif5m)s531wtcg - tgg / ser - trp1 ( 1.88)1,2,4,5 * 
 rpob ( rif3m)h526dcac - gac / his - asp4 ( 7.5)1,3,4,5 * 
 rpob ( rif2m)h526ycac - tac / his - tyr4 ( 7.5)1,2,3,5*lack 
 hybridisationh526lcac - gtc / his - leu1 ( 1.88)1,2,3,5*lack 
 hybridisation at ( rif2/3m)h526rcac - cgc / his - arg1 ( 1.88)1,2,3,5*lack 
 hybridisation at ( rif2/3m)h526pcac - ccc / his - pro1 ( 1.88)1,2,3,5*lack 
 hybridisation at ( rif2/3m)h526lcac - ctc / his - leu1 ( 1.88)1,3,4,5*lack 
 hybridisation at ( rif2/3m)d516ygac - tac / asp - tyr2 ( 3.77)1,3,4,5 * 
 rpob ( rif1m)d516vgac - gtc / asp - val1 ( 1.88)11 * 
 inha ( inham ) 
 -15c - t13 ( 24.6)12 * 
 inha ( inhawt)no mutationwt40 ( 75.4)14 * 
 katg ( katgm ) 
 s315tagc - acc / ser - thr41 ( 77.3)13 * 
 katg ( katgwt)no mutationwt12 ( 22.7)1,2,3,4,5,12,14*wtno mutationwt55 ( 100)wt : wild type . 
  the line - tb / mdr method achieved 100% sensitivity ( 55/55 ) among the wt samples and , as 
 such , identified the inh - s and rmp - s isolates ; this was demonstrated by hybridisation to 
 the five wt rpob probes ( probes 1 - 5 ) and the wt inha 
 and katg probes ( probes 12 and 13 ) ( a in figure ) . among the 53 mdr strains , 38 had a mutation in codon 531 . the line - tb / mdr assay was able 
 to correctly identify all of the mutations occurring at codon 531 when the snp involved 
 resulted in an amino acid ( aa ) change of serleu ; these strains exhibited hybridisation 
 signals with probes 1 through 4 and 9 , but had a lack of hybridisation to probe five ( b 
 in figure ) . among the 12 additional isolates with 
 rpob mutations , the line - tb / mdr method correctly identified eight 
 codon 526 mutants , of which four presented a hisasp codon 516 in the line - tb / mdr assay correctly identified a single 
 strain that had the aspval aa change . four mutants at codon 526 and two mutants at 
 codon 516 did not have a complementary mutant probe in our assay , but they lacked 
 hybridisation signals with the wt 526 probe 4 , as expected , and probe 2 , which 
 represented the wt codon 516 ( e in figure ) . the 
 comparison of the results obtained for all of the isolates using the line - tb / mdr assay 
 ( n = 108 ) and the rpob rdr sequencing as a reference is summarised in 
 table ii . this assay reported a kappa value 
 ( k ) [ 95% confidence interval ( ci ) ] and agreement of 1 ( 99.5 - 100.0 ) . table iicomparison of the results obtained for the identification of mutation with 
 the sequencing and with drug susceptibility testing ( dst )  results  compared results  ( n = 
 108)(n = 
 108 )  line - tb / mdrline - tb / mdrline - tb / mdrline - tb / mdrkappa  sesp  line - tb / mdrseqwt / seq wtmut / seq wtwt / seq mutmut / seq mut(95% ci)p(%)(% ) 

 inh wt586458334488.9 
 ( 82.5 - 95.2 ) < 0.00177.690.6inh res5044rmp wt5555550053100 
 ( 99.5 - 100.0 ) < 0.001100100rmp res5353 

 line - tb / mdr  line - tb / mdrline - tb / mdrline - tb / mdrline - tb / mdrkappa  sesp  dstwt / dst - smut / dst - swt / dst - rmut / dst - r(95% ci)p(%)(% ) 

 inh wt5555   55  0124188.7 
 ( 82.2 - 95.1 ) < 0.00177100inh res4153  rmp wt5555550053100 
 ( 99.5 - 100.0 ) < 0.001100100rmp res5353  ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ; 
 sp : specificities . ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ; 
 sp : specificities . we also evaluated the performance of the line - tb / mdr method in 53 inh - resistant 
 isolates . according to the sequence data , 44 of these isolates carried mutations in 
 katg and/or inha and 41 ( 77.3% ) were 
 katg 315 ( agc - acc ) ( serthr ) . among these samples , the line - tb / mdr 
 method was able to correctly identify 36 ( 88% ) , with a positive signal at probe 14 
 ( katgm ) and a lack of hybridisation at probe 13 
 ( katgwt ) ( c in figure ) . three 
 isolates with a mutation in codon 315 gave a positive signal for probe 13 
 ( katgwt ) , while two of the isolates did not hybridise to any of the 
 katg probes ( e in figure ) . 
 among the 12 isolates , 11 ( 91.6% ) hybridised to probe 13 ( katgwt ) , as 
 expected , but one isolate again failed to hybridise to any of the katg 
 probes . in addition , 40 of the 53 mdr strains ( 75.4% ) presented a 
 wtinha genotype ; among these , 37 ( 92.5% ) showed hybridisation to 
 probe 12 ( inhawt ) and a lack of hybridisation to probe 11 
 ( inham ) ( d in figure ) . three 
 isolates presented discordant results when compared to the sequencing data ; one sample 
 hybridised to probe 11 , corresponding to a mutated region of the gene 
 ( inham ) , while two isolates did not hybridise to any of the inha 
 probes ( inham ) . the other strains ( n = 13 ; 25% ) contained the mutation 
 c-15 t in the promoter region of inha and 10 ( 77% ) strains correctly 
 demonstrated hybridisation to probe 11 ( inham ) , while three failed to 
 hybridise to either probe 11 or 12 . in conclusion , the overall results of the 
 line - tb / mdr assay ( n = 108 ) and the combined sequences of katg 
 and inha demonstrated a k ( 95% ci ) of 0.889 ( 82.5 - 95.2 ) for 
 katg and inha and an negative predictive value and 
 positive predictive value of 94.8% and 100% , respectively ( table ii ) . sixty - five of the 108 isolates were also analysed using the genotype 
 mtbdrplus assay and 25 susceptible strains with wt , inh and rmp 
 sequences were correctly identified . among the 40 rif - resistant strains that also had 
 rmp - r genotypes , 37 ( 92.5% ) the concordance rates between the line - tb / mdr 
 and mtbdrplus assays for drug resistance ( dr ) detection in these 
 samples were 93.6% ( 63/65 ) for the inh sequences and 87.4% ( 62/65 ) for the rmp sequences 
 ( table iii ) . the mtbdrplus 
 did not identify three rmp - r strains that were correctly identified by line - tb / mdr and 
 sequencing and three sequencing - confirmed inh - r isolates were not identified by 
 line - tb / mdr . table iiicomparison of the results obtained for the identification of genotype 
 mtbdrplus with the sequencing and with line - tb / mdr  resultscompared results  ( n = 
 65)(n = 
 65 )  hain 
 strip  hain 
 striphain 
 striphain 
 striphain 
 stripkappa  sesp  seqwt / seq wtmut / seq wtwt / seq mutmut / seq mut(95% ci)p(%)(% ) 

 inh wt272525324096.9 < 0.00187.0100.0inh res3840(89.9 - 98.6)rmp wt282525033790.5 < 0.00192.5100.0rmp res3740(84.3 - 86.4 )  hain striphain striphain striphain stripkappa  hain stripline - tb / mdrwt / line - tb / mdr wtmut / line - tb / mdr wtwt / line - tb / mdr mutmut / line - tb / mdr mut(95% ci)p 

 inh wt252525023893.6 < 0.001  inh res4040(86.7 - 97.5 )  rmp wt282525033787.4 < 0.001  rmp res3740(84.3 - 96.2 )  ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ; 
 sp : specificities . ci : confidence interval ; inh : isoniazid ; rmp : rifampicin ; se : sensitivities ; 
 sp : specificities . mdr - tb is defined by simultaneous resistance to inh and rmp , the main drugs used for tb 
 treatment ( laurenzo & mousa 2011 ) . because 
 m. tuberculosis is a clonal organism , most of the acquisition of dr 
 in m. tuberculosis is due to point mutations in the chromosome ( sreevatsan et al . 1997 , ramaswamy & musser 1998 ) that accumulate and are preserved in 
 the different lineages ( brosch et al . these traits of 
 mycobacterium render it suitable for the development of assays for 
 the detection and eventual characterisation of the presence of well - described inh - r and 
 rmp - r snps ( cardoso et al . 2011 ) . in brazil , as recommended by the ministry of health , 
 tb diagnosis and dst can be performed using solid media ( lj or ogawa - kudoh ) and liquid 
 media ( middlebrook 7h10 ) , but these methods have several limitations , mainly related to 
 the delay of diagnosis , which can be as long as 12 weeks ( ms 2010 ) . for dr detection , tests based on reverse blot 
 hybridisation probes , such as the line probe assay ( inno - lipa assay rif tb , 
 innogenetics ) and the genotype mtbdrplus assay , are currently widely 
 used in many countries . these tests have sensitivities ranging from 71 - 100% and 
 specificities of 96 - 100% ( bwanga et al . while inno - lipa is 
 restricted to the identification of rmp resistance , genotype is able to identify both 
 rmp and inh resistance . however , both of these methods are expensive and not widely 
 available in developing countries ( mkinen et al . 
 2006 , maschmann et al . 2013 ) . recently , 
 a molecular test for the detection of m. tuberculosis and mutations 
 conferring rmp resistance directly from sputum ( genexpert , cepheid sunnyvale , ca , usa ) 
 was developed , but the performance characteristics of this procedure might be different 
 when implemented in environments with low or high tb burdens ( dorman et al . 2013 ) . for the evaluation of our reverse hybridisation - based assay for the 
 detection of rmp - r and inh - r , we chose a panel of strains with mutations similar to 
 those reported in other publications . the mutations were predominantly localised in 
 codons 531 , 526 and 516 of the rpob gene ( sekiguchi et al . 1996 , arago et al . 2007 ) and at position -15 of the inha 
 regulatory region ( palomino 2009 ) . all of the 108 
 isolates analysed by line - tb / mdr showed specific hybridisation to the is6110 
 probe , which was used for m. tuberculosis identification 
 ( amongst other members of the mtbc ) , indicating that this method can be a reliable tool 
 for tb diagnosis . this result is similar to that found previously by our research group 
 when testing a method to detect tb ( detec - tb ) ( michelon 
 et al . in addition , the 55 dna samples that did not contain mutations 
 in the genes associated with resistance and were drug susceptible according to the dst , 
 were correctly identified by the line - tb / mdr method . of the 53 samples with mutations in 
 the rpob gene , 100% ( 53/53 ) hybridised to at least one 
 rpob - mutated probe or showed no hybridisation to the wt probe , 
 confirming that the isolate was not susceptible to rmp ; there was complete agreement ( k 
 = 1 ) between the current test and the sequencing results ( table ii ) . these data suggest that the line - tb / mdr method can be 
 used to reliably detect resistance to this drug , as observed by aslan et al . ( 2008 ) , when using a commercial kit and samples , both 
 mutated or not , in the rpob gene . when snps for which a mutant probe 
 was developed were present , we observed a corresponding signal for that mutant and a 
 lack of hybridisation to the wt probe . four isolates carried snps that had no mutant 
 probe represented and , as expected , demonstrated a lack of signal in the wt probe only 
 ( e in figure ) , which indicated the presence of an 
 snp associated with rmp - r . the sensitivity for detecting rmp resistance was 100% , with 
 sequencing used as the reference method . this is promising for the rapid detection of 
 mdr / tb isolates because rmp mono - resistant isolates are rarely observed ( malhotra et al . previous studies reported 
 that approximately 95 - 100% of rmp - resistant clinical isolates carry mutations in an 81 
 bp region of the rpob gene ( palomino 
 2009 ) . therefore , in cases with a lack of snps in this region , we do not 
 expect our assay to be useful for detecting rmp - r . although most of the methods used to 
 detect mutations related to inh resistance use only the katg gene as a 
 target , maschmann et al . ( 2011 ) demonstrated that 
 the concomitant evaluation of katg and the inha 
 regulatory region improves sensitivity for identifying inh resistance ( aslan et al . 2008 ) . in this study , only three 
 inh - resistant isolates had mutations in the inha gene , but the 
 frequency of inh - resistant isolates containing the snp ( cg ) at position 315 of 
 katg ( serthr ) ranged from 50 - 100% in the literature , including 
 literature from brazil ( hillemann et al . 2005 , 
 costa et al . this aa change seems to 
 lead to a decrease in catalase peroxidase enzyme activity , which has been described as 
 essential for the activation of the drug ( zhang et al . when the 
 line - tb / mdr method was compared to dst , which is a reference for mdr / tb identification , 
 the overall agreement was 88.7% ( 82.2 - 95.1 ) . among the 12 dna samples that were not 
 correctly identified as inh - resistant , nine were found to have an inha 
 and katg wt genotype by sequencing even though they were 
 identified as mdr by dst ; only three showed no hybridisation to the probes used in our 
 assay . some studies report that approximately 10 - 30% of inh - r m. 
 tuberculosis strains have no mutations in dr - associated genes ( hillemann et al . 2005 ) and may have other resistance 
 mechanisms , such as efflux pumps or cell envelope permeability ( mkinen et al . the prevalence of katg and 
 inha mutations , as well the frequency and type of gene mutations , 
 varies greatly among different geographic regions , with ranges of 35 - 91% and 20 - 35% , 
 respectively ( piersimoni & scarparo 2003 , 
 silva et al . the inclusion of other genes , such as the 
 oxyr - ahpc intergenic region , may increase the sensitivity of the 
 test , although the relationship between inh resistance and mutations in this region are 
 not yet fully understood . in previous work , mutations in this gene have frequently been 
 observed to be associated with mutations in other genes and have been found alone in 
 very few isolates ( costa et al . 2009 ) . the line - tb / mdr method achieved satisfactory results for the identification of snps 
 involved in inh resistance , with a sensitivity of 91.6% ( 99/108 ) , which is similar to 
 the performance of genotype mtbdrplus and other commercial kits ( bonington et al . our aim was to evaluate the potential 
 contribution of a new  in - house test for accelerating the laboratory detection of rif 
 and inh resistant strains , particularly when mdr - tb is suspected . the discrepancies between the line - tb / mdr method and dna sequencing might be due to ( i ) 
 the specificity of the probes , ( ii ) the analysis of hetero - resistant strains or ( iii ) 
 the lack of sensitivity of the hybridisation . it should be noted that the line - tb / mdr 
 test identified three isolates as inh - resistant using inham probe 
 hybridisation , while the sequencing data identified them as wtinha . 
 this could be due to the higher sensitivity of the hybridisation - based assays compared 
 to sequencing - based assays for detecting mixed bacterial populations ( van deun et al . the line - tb / mdr method described in this study offers advantages over 
 alternative techniques and provides results 6 h after amplification of the target , 
 considerably decreasing the time needed for dst . we are now in a second - phase trial for 
 the evaluation of the reproducibility of this test in different laboratories . the hope 
 is that this test will be an additional tool for the diagnosis of mdr - tb and will be 
 routinely applied in laboratories in regions with high tb and tb / hiv burdens and limited 
 financial resources . probe set for identification of the wild type ( wt ) and mutant 
 genotypes  sequencelength  probes concentrationprobes(5'-3')(bp )  ( pmoll)rif1wtcagccagctgagccaattcat21wt5.0rif2wtttcatggaccagaacaaccc20wt10.0rif3wtcgctgtcggggttgacc17wt10.0rif4wtttgacccacaagcgccgact20wt5.0rif5wtctgtcggcgctggggc16wt10.0rif1mccaattcatggtccagaa21mutant5.0rif2mgttgacctacaagcgccg18mutant10.0rif3mggttgaccgacaagcgcc18mutant10.0rif4mctgttggcgctggggc16mutant10.0rif5mcgactgtgggcgctgg16mutant10.0 
 katg wttcaccagcggcatcgag17wt0.4katgmtcaccaccggcatcgag17mutant0.4inhawtcggcgagacgataggttgtc20wt0.6is6110tgcccgtcccgccgatctc18wt1.0 
 
 primers used for amplification and standardisation of multiplex pcr for 
 genes rpob , katg , inha and is6110  sizegeneprimer sequence ( 5'-3')(bp)is6110is1 
 cgtgagggcatcgaggtggc245  is2 
 bio - gcgtaggcgtcggtgacaaa  rpobrif1 
 ggtcgccgcgatcaaggagt157  rif2 
 bio - tgcacgtcgcggacctcca  katgkatg1 
 catgaacgacgtcgaaacag232  katg2 
 bio - cgaggaaactgttgtcccat  inhainha1 
 cctcgctgcccagaaaggga248  inha2 
 bio - atcccccggtttcctccggt