recently , a new coronavirus was identified as the suspected causative agent of an increased number of atypical pneumonia cases reported from hong kong , singapore , vietnam , and canada ( 14 ) .
subsequent publications demonstrated that this new coronavirus was detectable in patients with severe acute respiratory syndrome ( sars ) ( 5,6 ) , classified according to the world health organization 's case definition ( 7 ) . during the first 6 months of 2003
this fact , together with the reappearance of the sars - associated coronavirus ( sars - cov ) in china in late 2003 , makes it clear that rapid and reliable diagnostic tools are essential for accurate disease reporting and subsequent disease management . because a defined treatment program and vaccination strategy are lacking ,
the main strategy to counteract the spread of this emerging virus is timely identification and isolation of infected persons .
sars patients ' typical initial symptoms include fever , cough , and headache , similar to many acute viral respiratory infections .
therefore , molecular - based diagnostic methods are applied to rapidly identify sars - cov  infected persons .
recently , nested and real - time reverse transcription  polymerase chain reaction ( rt - pcr ) assays to detect sars - cov have been published ( 5,8 ) .
these assays  the first tools to detect sars - cov in patients with sars  were based on the short stretches of viral sequence identified as the rna - directed rna polymerase of a new microbe .
subsequently , sequences from several sars - cov isolates were determined , and all of these sequences were closely related , as would be expected during the clustered outbreaks in 2003 .
however , the genomes of rna viruses , including those of coronaviruses , tend to vary over time and with location ( 912 ) .
recently , the sequence variations of sars - cov during the first epidemic phases in china in 2003 were reported .
the neutral mutation rate for sars - cov was almost constant and similar to that of known rna viruses ; the s protein , responsible for virus - host receptor recognition , displayed the most extensive amino acid changes ( 13 ) .
in addition , the sequence analysis of isolates from recent sars patients in china in 2004 has shown that 98.8%99.4% of the 3,768 bases of s gene , 99% of 658 bases of m gene , and 99% of 1,068 bases of n gene are isogenous with those submitted to public databases , which date back to the first epidemic in spring 2003 ( 14 ) . however
, even these minimal changes could render existing pcr assays ineffective should sars - cov reemerge ( 15 ) . to improve the ability to detect sars - cov safely and reduce the risk of eliciting false - negative results caused by genome sequence variations , we established three individual real - time rt - pcr assays .
target sequences were chosen by using the following criteria : 1 ) the regions are distributed over the whole genome , including the nonstructural polyprotein 1a and 1ab genes and the spike glycoprotein gene ( table 1 ) ; 2 ) the regions are highly conserved among the 89 , 90 , and 100 respective sequences available in public sequence databases ; 3 ) the regions are suitable for the design of a real - time rt - pcr assay ; and 4 ) the designed primers , 5-nuclease probes , and amplicons displayed no considerable homology to other viruses , including human cov oc43 and 229e in blast searches ( available from http://www.ncbi.nlm.nih.gov/blast/ ) .
sars , severe acute respiratory syndrome ; s , sense ; a , antisense ; f , 6-carboxyfluorescein attached to 5-terminus ( fam ) ; t , 5-carboxytetramethylrhodamine ( 5-tamra ) attached to 5-ethylamino - dthymidin ; ns , nonstructural ; pp1ab , polyprotein 1ab gene ; tm , melting temperature ; tm , taqman . 
these assays were based on the fluorogenic oligoprobe chemistry , which uses the 5-exonuclease activity of the dna polymerase to generate a more specific signal than that produced by the use of sybr green i ( 8) . the real - time rt - pcr assays were successfully run on the applied biosystems real - time pcr systems ( sds7700 and sds7000 ; applied biosystems , foster city , ca ) as well as on the roche lightcycler ( roche diagnostics gmbh , mannheim , germany ) .
all assays were designed as one - step rt - pcr reactions to be run under identical conditions on the respective pcr platform .
this system allowed the simultaneous detection of different sars - cov regions in a single pcr run .
moreover , we could combine the three assays in a single tube , which might be important when clinical material is limited . finally , the assays were compared to the 5-nuclease assay published recently ( 5 ) and to a commercially available real - time pcr kit ( real - art hpa - coronavirus lc rt pcr reagents , artus gmbh , hamburg , germany ) .
after optimization of primer and 5-nuclease probe concentration and annealing temperature , reaction conditions for our 5-nuclease assay were as follows .
for the rt - pcr performed on the applied biosystems platforms , each 25-l reaction contained 12.5 l of 2xquantitect probe rt - pcr master mix ( qiagen , hilden , germany ) , 10 pmol of each primer , 3 pmol of 5-nuclease probe , and 0.25 l of quantitect probe rt mix .
rnase - free water was added up to 23 l , and 2 l of rna was used .
cycling conditions were 30 min at 50c for rt reaction , 15 min at 95c for inactivation of rt , activation of the taq dna polymerase , and cdna denaturation , followed by 45 cycles of 15 s at 95c and 30 s at 60c .
each 20-l reaction included 7.5 l of 2.7xlightcycler rna master hybridization probes mix ( roche diagnostics gmbh ) , 10 pmol of each primer , 3 pmol of the 5-nuclease probe , and 1.3 l mn ( oac)2 ( 50 mmol / l ) .
rnase - free water was added up to 18 l , and 2 l of rna was used .
cycling conditions were 20 min at 55c for the rt reaction , 30 s at 95c for initial denaturation , followed by 45 cycles of 1 s at 95c , 10 s at 55c , and 10 s at 72c .
the combined assays were set up by adding all primers and probes in the same concentration ; the amount of water was reduced accordingly .
protocols are also available from the robert koch - institut homepage ( available from www.rki.de/infekt/sars/pcrprotocol.pdf ) .
the human l13 gene and the human cyclophilin gene ( 16 ) were amplified under identical reaction conditions as the sars - cov  specific assays on the abi platforms and the lightcycler , respectively , to act as amplification controls . to evaluate the sensitivity of the sars - cov  specific assays ,
rt - pcrs were performed repeatedly on serial dilutions of rna extracted with the viral rna kit ( qiagen ) from cultured sars - cov with defined amounts of genome equivalents ( ge ) by using the international standard of the european network for the diagnostics of imported viral diseases ( enivd ) , distributed through the robert koch - institut ( available from : http://www.rki.de/infekt/sars/datasheet.pdf ) .
detection limits of the three new assays were < 10 ge . comparison of the threshold cycle ( ct ) values showed that the new assays were at least as sensitive as the previously described assays ( 5,8 ) and the commercially available kit .
when we combined the assays in a single tube targeting three different regions on the same rna template , the ct was reduced by 1 to 2 cycles ( sds7700 ) , which suggests either that sensitivity was unchanged or , when there was an increase , that it was attributable to the combination of all three signals ( lightcycler ) .
subsequent agarose gel analysis during optimization steps of the pcr confirmed the simultaneous amplification of the three rt - pcr products ( figure ) .
ct , threshold cycle ; sars - cov , severe acute respiratory syndrome  associated coronavirus ; rt - pcr , reverse transcription  polymerase chain reaction ; ge , genome equivalents / assay ; pp1ab , nonstructural polyprotein 1ab gene ; n.d . , not determined . 
lightcycler amplification curves of the three single sars - coronavirus ( cov)-specific assays and the combined assay .
five hundred genome equivalents ( ge ) of sars - cov were amplified with the three single assays - ns pp1a , ns pp1ab , and ss gp - as well as with the combination of the three assays .
, the respective agarose gel analysis of the amplification products in a 3% gel is shown .
lane 1 : 50-bp marker , lane 2 : 500 ge sars - cov amplified with the assay combination ( products of assays ns pp1ab and ss gp are not clearly distinguishable because of sizes of 88 bp and 79 bp , respectively ) , lane 3 : human cov 229e , lane 4 : respiratory syncytial virus , lane 5 : rhinovirus , lane 6 : human metapneumovirus , lane 7 : mycoplasma pneumoniae , or lane 8 : chlamydia pneumoniae .
using the single or combined assays , we analyzed 27 bronchoalveolar - lavage fluid samples from 19 suspected sars case - patients and 8 probable sars patients ( according to the robert koch - institut case definition , available from http://www.rki.de/infekt/sars/aolg-falldef-arsuu.pdf ) .
in agreement with the previously published assay results ( 5 ) , sars - cov was detectable in three samples from eight probable sars patients , without explicit differences in the ct value of individual assays when the single or combined assays were used .
respiratory samples and stool samples taken 8 days later from the remaining 5 probable patients as well as the 19 persons with suspected disease were negative by rt - pcr
in addition , 35 serum samples from patients with sars - cov infection obtained 152 days after disease onset were analyzed . between 20 and 1,000 ge / ml of sars - cov - specific rna was detected in 21 of 35 serum samples , even when serum was obtained from patients 1 day after disease onset .
none of the assays displayed cross - reactivity to clinical samples containing human cdna from blood ; human cov 229e ; influenza viruses a and b ; parainfluenzaviruses 1 , 2 , and 3 ; respiratory syncytial virus ; rhinoviruses ; enteroviruses ; adenoviruses 110 ; human metapneumovirus ; mycoplasma pneumoniae ; or chlamydia pneumoniae . for these pathogens
, we obtained neither a fluorescent signal nor an amplification product in subsequent agarose gel analysis ( selection shown in figure ) .
although we focused on a one - step rt - pcr to decrease handling and total assay time , the three real - time rt - pcr assays can also be performed as two - step rt - pcr , including a separate cdna synthesis step followed by pcr , and then finally using appropriate ready - to - use master mixes and the same cycling conditions , omitting the rt step .
the single assays and the combined assay were also used in an external quality assessment to detect sars - cov organized by the enivd .
all assays could detect sars - cov in 7 of 11 samples with virus loads ranging from 5x10 to 2x10 ge of two isolates of sars - cov per milliliter sample without false - positive or false - negative results . while the application of three single assays to detect sars - cov leads to a higher reliability of negative results , reflecting the negative outcome of three independent amplification reactions , it is a more expensive approach than combining the assays . in conclusion ,
the real - time rt - pcr assays we describe provide a fast and reliable tool that can complement and improve recently introduced techniques for sars diagnostics .
parallel amplification of two human reference genes , l13 and cyclophilin , confirmed negative results in clinical samples by demonstrating amplifiable rna .
the separation of the control reaction was chosen to guarantee the high sensitivity of the sars - cov detection of < 10 ge of sars - cov per reaction .
an rt - pcr run is completed in < 1 h , depending on the real - time pcr platform . in cases of small amounts of material or in an emergency situation with a high throughput of samples , the three sars - cov  specific assays can be combined into one rt - pcr reaction without loss of sensitivity . furthermore , as the ambiguous diagnostic results in a hospital in canada have recently shown ( 17 ) , targeting three different regions distributed over the whole genome considerably reduces the risk for false - negative results caused by virus sequence modifications .
 we developed a set of three real - time reverse transcription  polymerase chain reaction ( pcr ) assays that amplify three different regions of the sars - associated coronavirus ( sars - cov ) , can be run in parallel or in a single tube , and can detect < 10 genome equivalents of sars - cov . 
 the assays consider all currently available sars - cov sequences and are optimized for two prominent real - time pcr platforms . 
The Study
Conclusions