les infections  bordetella pertussis continuent dtre un important problme de sant publique au canada . les mthodes de raction en chane de la polymrase ( pcr ) pour dceler le b pertussis sont habituellement fondes sur la squence dinsertion multicopie is481 , do nt la sensibilit leve , mais do nt la spcificit despce est inexistante . une nouvelle mthode pcr en temps rel du b pertussis fonde sur le gne de porine a t mise  lessai paralllement  plusieurs mthodes dj publies qui ciblent des gnes comme lis481 , le promoteur de ptx , la pertactine et une thialase ventuelle . les mthodes ont t values  laide dun groupe de rfrence de bactries respiratoires communes , y compris diverses espces de bordetella et 107 chantillons nasopharyngs cliniques la mthode visant llment is481 avait la sensibilit analytique la plus leve , mais manquait de spcificit pour le b pertussis dans le groupe de rfrence et les chantillons cliniques . les mthodes ciblant les gnes du promoteur de ptx et de la perctatine ont galement donn des rsultats faux positifs . une mthode de pcr fonde sur le gne thialase tait hautement spcifique , mais ne dcelait pas toutes les souches de rfrence du b pertussis . cependant , une nouvelle mthode ciblant le gne de porine a dmontr une forte spcificit au b pertussis ,  la fois dans le groupe de rfrence et dans les chantillons cliniques et , daprs les rsultats confirms par squenage , prdit correctement tous les cas positifs au b pertussis dans les chantillons cliniques . daprs lanalyse de rgression probit , la limite de dtection de 95 % de la nouvelle mthode tait de quatre units formant colonies par raction . une nouvelle mthode faisant appel  la porine pour dceler le b pertussis donne un rendement suprieur et peut tre utile pour amliorer la dtection molculaire du b pertussis dans des chantillons cliniques . the reference panel was comprised of b pertussis ( american type culture collection [ atcc ] baa-589 , 9340 and 9797 , and a clinical strain isolated at bc children s hospital [ vancouver , british columbia ] from a patient nasopharyngeal aspirate ) , b parapertussis ( atcc 15237 and a clinical strain isolated at the bc centre for disease control [ vancouver , british columbia ] ) , b holmesii ( atcc 51541 ) , b bronchiseptica ( atcc baa-588 ) , haemophilus influenzae ( atcc 10211 ) , pseudomonas aeruginosa ( atcc 27853 ) , klebsiella pneumoniae ( atcc 13882 ) , staphylococcus aureus ( atcc 43300 ) , staphylococcus epidermidis ( atcc 12228 ) , streptococcus agalactiae ( atcc 13813 ) , streptococcus mitis ( atcc 6249 ) , streptococcus pneumoniae ( atcc 49619 ) , streptococcus pyogenes ( atcc 19615 ) , corynebacterium pseudodiphtheriticum ( atcc 10700 ) , mycoplasma pneumoniae ( atcc 29342 ) , chlamydophila pneumoniae ( atcc vr-1310 ) and a clinical strain of neisseria mucosa isolated at bc children s hospital . bordetella species were streaked on charcoal agar medium ( oxoid ) and grown at 37c for 72 h in a humidified environment . bacterial suspension was prepared in phosphate - buffered saline to a turbidity equivalent to a 0.5 mcfarland standard , titred by colony counting and cryopreserved at 80c . the titred bacterial stocks were diluted in te8 buffer ( 10 mm tris , 1 mm edta ; ph 8.0 ) and used directly in the pcr reactions . other bacteria were grown on blood or chocolate agar plates ( oxoid ) overnight at 37c in a 5% co2 atmosphere , and a bacterial suspension was prepared in te8 buffer to a turbidity equivalent to a 0.5 mcfarland standard . a total of 107 nasopharyngeal wash specimens were used in the present study , including selected pcr - positive ( by a sybr green pcr ) specimens since august 2009 , and specimens submitted between september 2011 to october 2012 to the microbiology and virology laboratory of bc children s hospital for b pertussis pcr . testing was performed exclusively on retrospective , residual samples that were stored at 80c . to maintain patient anonymity , each sample was coded and all patient identifiers were removed to ensure that personnel involved in the present study were unaware of any patient information . ethics approval was not considered to be necessary because studies that involve the secondary use of anonymous human biological materials are exempted from review by the local research ethics board of the university of british columbia . dna from 0.2 ml nasopharyngeal wash specimens was extracted using the qiasymphony virus / bacteria kit in an automated dna extraction platform qiasymphony sp ( qiagen , usa ) . the titred bacterial suspensions and dna extracts from 107 nasopharyngeal wash specimens were analyzed using taqman real - time pcr or by sybr green pcr with the primers and probes shown in table 1 . for taqman real - time pcr assays , 5 l of sample extract or diluted bacterial suspensions was mixed with 20 l of a master mix containing 12.5 l of taqman universal pcr master mix ( applied biosystems , usa ) and primers and probes to final concentrations shown in table 1 . thermal cycling was performed in a abi7500 fast instrument ( applied biosystems , usa ) with one cycle of 95c for 10 min , followed by 45 cycles consisting of 95c for 15 s and 60c for 60 s. the sybr green pcr was performed with the same parameters except that the reaction mix contained 12.5 l 2xitaq sybr green pcr master mix ( bio - rad , usa ) and the thermal cycling was performed in a smartcycler ( cepheid , usa ) with one cycle of 95c for 2 min , followed by 45 cycles consisting of 95c for 10 s and 64c for 60 s. the sybr green pcr reactions were subjected to meltcurve analysis after the final amplification cycle . samples with discrepant results from different assays were analyzed by sequencing pcr products using the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , usa ) in an abi 3130 genetic analyzer according to the manufacturer s instructions . for probit regression analysis , b pertussis strain atcc baa-589 stock suspension was serially diluted to 10 , 50 , 100 , 200 , 500 and 1000 colony forming units ( cfu)/ml , and eight replicates of each dilution were tested by real - time pcr ; 95% detection limit was determined as described previously ( 16 ) . the reference panel was comprised of b pertussis ( american type culture collection [ atcc ] baa-589 , 9340 and 9797 , and a clinical strain isolated at bc children s hospital [ vancouver , british columbia ] from a patient nasopharyngeal aspirate ) , b parapertussis ( atcc 15237 and a clinical strain isolated at the bc centre for disease control [ vancouver , british columbia ] ) , b holmesii ( atcc 51541 ) , b bronchiseptica ( atcc baa-588 ) , haemophilus influenzae ( atcc 10211 ) , pseudomonas aeruginosa ( atcc 27853 ) , klebsiella pneumoniae ( atcc 13882 ) , staphylococcus aureus ( atcc 43300 ) , staphylococcus epidermidis ( atcc 12228 ) , streptococcus agalactiae ( atcc 13813 ) , streptococcus mitis ( atcc 6249 ) , streptococcus pneumoniae ( atcc 49619 ) , streptococcus pyogenes ( atcc 19615 ) , corynebacterium pseudodiphtheriticum ( atcc 10700 ) , mycoplasma pneumoniae ( atcc 29342 ) , chlamydophila pneumoniae ( atcc vr-1310 ) and a clinical strain of neisseria mucosa isolated at bc children s hospital . bordetella species were streaked on charcoal agar medium ( oxoid ) and grown at 37c for 72 h in a humidified environment . bacterial suspension was prepared in phosphate - buffered saline to a turbidity equivalent to a 0.5 mcfarland standard , titred by colony counting and cryopreserved at 80c . the titred bacterial stocks were diluted in te8 buffer ( 10 mm tris , 1 mm edta ; ph 8.0 ) and used directly in the pcr reactions . other bacteria were grown on blood or chocolate agar plates ( oxoid ) overnight at 37c in a 5% co2 atmosphere , and a bacterial suspension was prepared in te8 buffer to a turbidity equivalent to a 0.5 mcfarland standard . a total of 107 nasopharyngeal wash specimens were used in the present study , including selected pcr - positive ( by a sybr green pcr ) specimens since august 2009 , and specimens submitted between september 2011 to october 2012 to the microbiology and virology laboratory of bc children s hospital for b pertussis pcr . testing was performed exclusively on retrospective , residual samples that were stored at 80c . to maintain patient anonymity , each sample was coded and all patient identifiers were removed to ensure that personnel involved in the present study were unaware of any patient information . ethics approval was not considered to be necessary because studies that involve the secondary use of anonymous human biological materials are exempted from review by the local research ethics board of the university of british columbia . dna from 0.2 ml nasopharyngeal wash specimens was extracted using the qiasymphony virus / bacteria kit in an automated dna extraction platform qiasymphony sp ( qiagen , usa ) . the titred bacterial suspensions and dna extracts from 107 nasopharyngeal wash specimens were analyzed using taqman real - time pcr or by sybr green pcr with the primers and probes shown in table 1 . for taqman real - time pcr assays , 5 l of sample extract or diluted bacterial suspensions was mixed with 20 l of a master mix containing 12.5 l of taqman universal pcr master mix ( applied biosystems , usa ) and primers and probes to final concentrations shown in table 1 . thermal cycling was performed in a abi7500 fast instrument ( applied biosystems , usa ) with one cycle of 95c for 10 min , followed by 45 cycles consisting of 95c for 15 s and 60c for 60 s. the sybr green pcr was performed with the same parameters except that the reaction mix contained 12.5 l 2xitaq sybr green pcr master mix ( bio - rad , usa ) and the thermal cycling was performed in a smartcycler ( cepheid , usa ) with one cycle of 95c for 2 min , followed by 45 cycles consisting of 95c for 10 s and 64c for 60 s. the sybr green pcr reactions were subjected to meltcurve analysis after the final amplification cycle . samples with discrepant results from different assays were analyzed by sequencing pcr products using the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , usa ) in an abi 3130 genetic analyzer according to the manufacturer s instructions . for probit regression analysis , b pertussis strain atcc baa-589 stock suspension was serially diluted to 10 , 50 , 100 , 200 , 500 and 1000 colony forming units ( cfu)/ml , and eight replicates of each dilution were tested by real - time pcr ; 95% detection limit was determined as described previously ( 16 ) . real - time pcr assays targeting the is481 element ( is481 ) , b pertussis/ b parapertussis gene for an outer membrane porin protein ( omp ) , pertussis toxin ( ptx ) promoter ( bptp ) , pertactin gene ( prn ) , and a putative thialase gene ( bp283 ) ( table 1 ) ( 1115 ) were selected from the literature . in addition , two new sets of primers and taqman probes were designed based on ptx promoter ( pt - p ) and b pertussis gene for a porin protein ( bptd_0837 ) ( por ) . the new ptx promoter assay was designed through modification of the primer and probe sequences described by grogan et al ( 12 ) in an attempt to further improve the specificity of the assay . by aligning the genomes of several different bordetella species , it was noted that the published probe sequence has only a single nucleotide mismatch with the corresponding sequences of b bronchiseptica and b parapertussis genome . in contrast , the newly designed probe has > 3 nucleotide differences with the corresponding sequences of b bronchiseptica and b parapertussis genome . the new por assay was designed by exploiting the nucleotide sequence variation between the b pertussis gene for a porin protein and the corresponding sequences in the genomes of b parapertussis , b bronchiseptica and b holmesii ( figure 1 ) . all pcr assays were first tested using a panel comprised of atcc strains and patient isolates of various bordetella species and other bacteria commonly found in the respiratory tract ( table 2 ) . the omp assay ( 15 ) is a sybr green - based , shared primer assay designed to amplify both b pertussis and b parapertussis - specific dna , but with different size pcr products that can subsequently be differentiated by their dissociation curves . when this pcr assay was applied to the panel of different bordetella species and other respiratory bacteria , false - positive amplification was observed with b holmesii , b bronchiseptica , s mitis and s pyogenes , but the dissociation curves were distinct from that of b pertussis and b parapertussis strains ( table 2 ) . apart from this assay , none of the other assays showed any cross - reactivity with other bacteria tested in the present study that do not belong to the genus bordetella . the analytical sensitivity of is481 assay for b pertussis was much higher ( by cycle threshold > 7 ) than other assays . the assay detected all b pertussis strains in the test panel , but also resulted in the false - positive detection of b parapertussis , b holmesii and b bronchiseptica strains , confirming the nonspecific nature of this target . among the single - target taqman assays , the bptp assay nonspecifically detected b bronchiseptica along with b pertussis strains , while the ptx - promoter assay , pt - p , designed in the present study lacked sensitivity and failed to detect all b pertussis strains . the bp283 assay was specific to b pertussis but failed to detect two of the four b pertussis strains tested . the prn assay detected all b pertussis strains but it weakly detected b bronchiseptica at a relatively high concentration ( 5.410 cfu / ml ) . in contrast , the newly designed por assay did not exhibit any cross - reactivity with dna from any of the non - b pertussis strains . based on these results , the is481 , bptp , bp283 , prn and por assays were selected for further analysis of clinical samples . among the 107 nasopharyngeal wash specimens , 21 ( 19.6% ) samples were positive according to the is481 assay , 20 ( 18.7% ) samples were positive according to the bptp and por assays , and 19 ( 17.7% ) samples were positive according to the bp283 and prn assays ( table 3 ) . there were three discrepant samples : a sample that was positive according to all assays except the bp283 assay ; a sample that was missed by the prn and bptp assays ; and a sample that was only positive using the bptp and is481 assays . sequencing of pcr products confirmed that the first two samples were , in fact , b pertussis , while the third sample was b bronchiseptica ( figure 2 ) . taking into account the definitive sequence identification , the performances of all five b pertussis - specific assays were comparable , but the newly designed por assay correctly detected all samples with a clinical sensitivity equal to that of is481 assay . based on a probit regression analysis , the diagnosis of whooping cough caused by b pertussis could be difficult , particularly at the early stages because the signs and symptoms of the disease closely resemble those of other common respiratory illnesses , such as those caused by mycoplasma pneumoniae , corynebacterium pneumoniae and adenoviruses . moreover , other related bordetella species , such as b parapertussis , b bronchiseptica and b holmesii , are also known to cause respiratory illness in humans , although generally with a milder clinical presentation ( 3,8 ) . specificity of diagnostic and surveillance methodologies for b pertussis is , therefore , critical for the effective management and control of pertussis disease . however , pcr testing for pertussis , which is now in widespread use , has variable specificity and needs further standardization and optimization ( 1,9 ) . in the present study , we aimed to identify or develop a highly specific and sensitive , real - time pcr assay for b pertussis that can replace the pcr assays based on is481 , which is a repeat element present in the genomes of different bordetella species at variable copy numbers ( 5 ) . we tested and compared the performance characteristics of both previously published and in - house developed pcr assays that target different genes and genomic regions specific to b pertussis , in addition to that of is481-based pcr assays . we observed remarkable variation in the specificity of different pcr assays while assessing a sample panel , comprised of reference strains of various bordetella species . however , the sensitivity and specificity of these assays remained approximately 95% in the clinical samples . the discrepant clinical sample results obtained by the different b pertussis specific real - time pcr assays are consistent with those obtained using the atcc panel of different bordetella species . the bp283 assay , which failed to detect two known atcc strains of b pertussis , also failed to detect one of the b pertussis - positive clinical samples . the is481 and bptp assay , which detected a known atcc strain of b bronchiseptica , also produced one false - positive result among the clinical samples , which was eventually confirmed to be positive for b bronchiseptica by sequencing . the results of using the prn assay on the atcc panel suggests that this assay could also falsely detect b bronchiseptica , particularly at bacterial concentrations > 10 cfu / ml in clinical samples . the novel real - time pcr assay that targets the porin gene of b pertussis demonstrated superior performance over all other b pertussis pcr assays tested in the present study . the higher analytical sensitivity , but poor specificity of the is481 pcr , is well described in the literature and is a generally accepted phenomenon ( 57,11 ) . however , an independent comparison of the most common single - target pcr assays for b pertussis has not been reported to date . therefore , the results of the present study could be useful for guiding clinical microbiologists in the selection of an appropriate real - time pcr assay for b pertussis . the new por gene - based pcr assay described may serve as a valuable new tool for diagnostic laboratories seeking to improve the specificity of b pertussis detection , with sensitivity comparable with that of is481 pcr assays .