to address the need for sensitive multiplex assays in diagnostic molecular microbiology , we created a polymerase chain reaction ( pcr ) platform in which microbial gene targets are coded by a library of 64 distinct masscode tags ( qiagen masscode technology , qiagen , hilden , germany ) . microbial nucleic acids ( rna , dna , or both ) are amplified by multiplex reverse transcription ( rt)-pcr using primers labeled by a photocleavable link to molecular tags of different molecular weight . after removing unincorporated primers , the identity of the microbe in the clinical sample is determined by its cognate tags . schematic representation of mass tag polymerase chain reaction ( pcr ) . as a first test of this technology , we focused on respiratory disease because differential diagnosis is a common clinical challenge , with implications for outbreak control and individual case management . multiplex primer sets were designed to identify up to 22 respiratory pathogens in a single mass tag pcr reaction ; sensitivity was established by using synthetic dna and rna standards as well as titered viral stocks ; the utility of mass tag pcr was determined in blinded analysis of previously diagnosed clinical specimens . oligonucleotide primers were designed in conserved genomic regions to detect the broadest number of members for a given pathogen species by efficiently amplifying a 50- to 300-bp product . in some instances , we selected established primer sets ; in others , we used a software program designed to cull sequence information from genbank , perform multiple alignments , and maximize multiplex performance by selecting primers with uniform melting temperatures and minimal cross - hybridization potential ( table a1 ) . primers , synthesized with a 5  c6 spacer and aminohexyl modification , were covalently conjugated by a photocleavable link to masscode tags ( qiagen masscode technology ) ( 8,9 ) . masscode tags have a modular structure , including a tetrafluorophenyl ester for tag conjugation to primary amines ; an o - nitrobenzyl photolabile linker for photoredox cleavage of the tag from the analyte ; a mass spectrometry sensitivity enhancer , which improves the efficiency of atmospheric pressure chemical ionization of the cleaved tag ; and a variable mass unit for variation of the cleaved tag mass ( 8,1012 ) . thus , amplification of a microbial gene target produces a dual signal that allows assessment of specificity . gene target standards were cloned by pcr into pcr2.1-topo ( invitrogen , carlsbad , ca , usa ) by using dna template ( bacterial and dna viral targets ) or cdna template ( rna viral targets ) obtained by reverse transcription of extracts from infected cultured cells or by assembly of overlapping synthetic polynucleotides . assays were initially established by using plasmid standards diluted in 2.5-g / ml human placenta dna ( sigma , st . louis , mo , usa ) and subjected to pcr amplification with a multiplex pcr kit ( qiagen ) , primers at 0.5 mol / l each , and the following cycling protocol : an annealing step with a temperature reduction in 1c increments from 65c to 51c during the first 15 cycles and then continuing with a cycling profile of 94c for 20 s , 50c for 20 s , and 72c for 30 s in an mj ptc200 thermal cycler ( mj research , waltham , ma , usa ) . amplification products were separated from unused primers by using qiaquick 96 pcr purification cartridges ( qiagen , with modified binding and wash buffers ) . masscode tags were decoupled from amplified products through uv light - induced photolysis in a flow cell and analyzed in a single quadrapole mass spectrometer using positive - mode atmospheric pressure chemical ionization ( agilent technologies , palo alto , ca , usa ) . a detection threshold of 100 dna copies was determined for 19 of 22 cloned targets by using a 22-plex assay ( table 1 ) . * na , not assessed ; rsv , respiratory syncytial virus ; cov , coronavirus ; sars , severe acute respiratory syndrome ; hpiv , human parainfluenza virus . many respiratory pathogens have rna genomes ; thus , where indicated , assay sensitivity was determined by using synthetic rna standards or rna extracts of viral stocks . after quantitation by uv spectrometry , rna was serially diluted in 2.5-g / ml yeast trna ( sigma ) , reverse transcribed with random hexamers by using superscript ii ( invitrogen , carlsbad , ca , usa ) , and used as template for mass tag pcr . as anticipated , sensitivity was reduced by the use of rna instead of dna templates ( table 1 ) . the sensitivity of mass tag pcr to detect live virus was tested by using rna extracted from serial dilutions of titered stocks of coronaviruses ( severe acute respiratory syndrome [ sars ] and oc43 ) and parainfluenzaviruses ( hpiv 2 and 3 ) . rna extracted from a 1-tcid50/ml dilution , representing 0.025 tcid50 per pcr reaction , was consistently positive in mass tag pcr . rna extracted from banked sputum , nasal swabs , and pulmonary washes of persons with respiratory infection was tested by using an assay panel comprising 30 gene targets that represented 22 respiratory pathogens . infection in each of these persons had been previously diagnosed through virus isolation , conventional nested rt - pcr , or both . reverse transcription was performed using random hexamers , and mass tag pcr results were consistent in all cases with the established diagnosis . infections with respiratory syncytial virus , human parainfluenza virus , sars coronavirus , adenovirus , enterovirus , metapneumovirus , and influenza virus were correctly identified ( table 2 and figure 2 ) . a panel comprising gene targets representing 17 pathogens related to central nervous system infectious disease ( influenza a virus matrix gene ; influenza b virus ; human coronaviruses 229e , oc43 , and sars ; enterovirus ; adenovirus ; human herpesvirus-1 and -3 ; west nile virus ; st . louis encephalitis virus ; measles virus ; hiv-1 and -2 ; and streptococcus pneumoniae , haemophilus influenzae , and nesseria meningitidis ) was applied to rna obtained from banked samples of cerebrospinal fluid and brain tissue that had been previously characterized by conventional diagnostic rt - pcr . eleven of 12 cases of enteroviral meningitis were detected representing serotypes cv - b2 , cv - b3 , cv - b5 , e-6 , e-11 , e-13 , e-18 , and e-30 ( data not shown ) . * rsv , respiratory syncytial virus ; hpiv , human parainfluenza virus ; cov , coronavirus ; sars , severe acute respiratory syndrome . positive and consistent with previous diagnosis / number tested ( with respective previous diagnosis ) . rna extracts from clinical specimens containing known pathogens were reverse transcribed into cdna ( superscript rt system , invitrogen , carlsbad , ca ; 20-l volume ) . five microliters of the reaction were subjected to mass tag pcr by using primers coupled to masscode tags ( qiagen masscode technology , qiagen , hilden , germany ) . detection of ( a ) influenza virus a ( h1n1 ) , ( b ) respiratory syncytial virus ( rsv ) group b , ( c ) human coronavirus sars ( hcov - sars ) , ( d ) human parainfluenza virus ( hpiv ) types 1 and ( e ) 3 , and ( f ) enterovirus ( ev ) by using a 30-plex assay , including 60 primers targeting influenza a virus matrix gene ( fluav - m ) , and for typing n1 , n2 , h1 , h2 , h3 , and h5 sequences , as well as influenza b virus ( flubv ) , rsv groups a and b , hcov-229e , -oc43 , and -sars , hpiv types 1 , 2 , 3 , and 4 ( groups a and b combined ; 4 primers ) , human metapneumovirus ( hmpv , 4 primers ) , measles virus ( mev ) , ev ( degenerate primer pair targeting all serogroups ) , human adenoviruses ( hadv , degenerate primer pair targeting all serogroups ) , human herpesvirus 1 ( hhv-1 , herpes simplex virus ) , human herpesvirus 3 ( hhv-3 ; varicella - zoster virus ) , mycoplasma pneumoniae , chlamydia pneumoniae , legionella pneumophila , streptococcus pneumoniae , haemophilus influenzae . the bar indicates an arbitrary cut - off threshold of 2.7 ( 4 times average background determined with random human dna ) . our results indicate that mass tag pcr is a sensitive and specific tool for molecular characterization of microflora . the advantage of mass tag pcr is its capacity for multiplex analysis . although the use of degenerate primers ( e.g. , enteroviruses and adenoviruses , table a1 and table 1 ) may reduce sensitivity , the limit of multiplexing to detect specific targets will likely be defined by the maximal primer concentration that can be accommodated in a pcr mix . although these steps are now performed manually , and mass spectrometers are not yet widely distributed in clinical laboratories , the increasing popularity of mass spectrometry in biomedical sciences and the advent of smaller , lower - cost instruments could facilitate wider use and integrated instrumentation . in addition to developing additional pathogen panels , our continuing work is focused on optimizing multiplexing , sensitivity , and throughput . potential applications include differential diagnosis of infectious diseases , blood product surveillance , forensic microbiology , and biodefense .