we conducted a preliminary comparison of the relative sensitivity of a cross - section of published hrv - specific pcr primer pairs ( most of which were first published before hrv - c was reported ) , independent of most variables described above , by testing a panel of 57 clinical specimen nucleic acid extracts from combined nose and throat swabs from preschool children with colds and influenza - like illnesses in melbourne , australia .
the panel included representatives of the 3 hrv species ( figure ) , human enteroviruses ( hevs ) , and extracts negative for picornaviruses .
the hrvs had been previously detected by using a nested primer pair ( table a1 ) ( 5 ) .
we used 10 different hrv primer pairs and also retested specimens by using the original primer pair with our standard reagents and equipment ( 5 ) .
the original descriptions of primer pairs 7 and 10 ( table a1 ) lacked tm information , and after in - house calculations , we used tms of 50c and 58c , respectively .
we also deliberately standardized the reagents ( onestep rt - pcr kit , qiagen , doncaster , victoria , australia ) and thermal cyclers used ( veriti , applied biosystems , foster city , ca , usa ) for conventional pcr and the rotorgene 3000 real - time cycler ( qiagen ) .
because primer pair 1 had a published history of detecting types from all hrv species , we chose it to genotype hrv - positive samples by sequencing the amplified products .
distribution of human rhinovirus ( hrv ) and human enterovirus ( hev ) sequences used for primer pair studies .
the hrv and hev genotypes from the testing panel ( indicated by filled circles ) were aligned with the central 154 nt of the 5 untranslated region ( utr ) region of all complete hrv genomes and poliovirus-1 .
hrv - ca and hrv - cc refer to hrv - cs with 5 utr sequences that have phylogenetic origins from either hrv - as or hrv - cs , respectively .
the tree was constructed by neighbor joining of maximum composite likelihood distance implemented in mega ( www.megasoftware.net ) .
we found that no primer pair detected the same hrvs and hevs typed when the original pair ( 5 ) or pair 1 ( table a1 ) was used .
five primer pairs , including real - time pcr ( rtpcr ) pair 5 , did not amplify the hevs , a positive feature for hrv - specific studies . only 2 primer pairs amplified anything from a specimen that was positive for both hrv and hev , a problem for accurate estimation of the frequency of co - detections .
the original primer pair screen detected 3 untypeable picornaviruses , which were not detected by any other pair or by repeat testing using the same pair .
only the second - round amplicon of the 3 nested sets of nested primer pairs ( 2,3 , and,9 ) was considered because the second round increased the total number of positive specimens over the first round .
the longest amplicon , produced by primer pair 7 , was also a valuable genotyping target , but it detected only 14 of the original 27 hrv - positive specimens in this population .
we next selected 4 frequently published primer pairs ( 1,5,7 , and,8 ) to examine 44 picornavirus - positive specimens ( 39 hrvs , 3 hevs , and 2 untypeable picornaviruses ) from nonhospitalized children with acute asthma exacerbation ( 6 ) .
as before , primer pair 1 detected the greatest number of hrv- and hev- positive specimens and all positive specimens detected by other primer sets ( n = 41 ) , followed by pair 7 ( n = 40 ) , pair 5 ( n = 36 ) , and pair 8 ( n = 31 ) .
most notably , primer pair 7 performed better than it had in the previous population , detecting only 1 fewer hrv than primer pair 1 and 9 more hrvs than pair 8 .
no species - specific bias was apparent , but generally , a specimen with a lower rna concentration , as indicated by the cycle threshold from primer pair 5 , was less likely to be detected or typed by using other primer pairs .
we noted in both populations that primer pair 1 sometimes amplified a region of human genomic dna from chromosome 6 ( gq497714 ) , for which amplicon size was indistinguishable from that expected due to hrv .
it was not possible to use the precise conditions reported for the 10 compared assays ; 1 was published > 2 decades ago and used phenol chloroform extraction .
some of the original enzyme formulations or reagents are no longer available , and production processes have changed in the interim .
in addition , the previously published primers were used in assays divided between those using 1-step rt - pcr and those using a separate rt cdna synthesis step .
a review of studies that detected hrvs with adequately described conditions during 20092010 found that fewer used a single - tube rt - pcr approach than a 2-step system .
we conducted single - tube rt - pcr to maintain the benefits of the so - called closed amplification system of rtpcr .
thus , we chose to use a single common set of reagents as the fairest way to compare the primer pairs examined in this study .
we believe the nature of this relative comparison best reflects performance for the likely end users : clinical microbiology laboratories or researchers .
we compared primers rather than assay function using clinical material instead of cultured virus , plasmid or synthetic rna standards , or screening contemporary or archived extracts , which are sometimes of low viral load . when picornavirus epidemiology is the primary research focus , we recommend using > 2 primer pairs to maximize the detection of hrvs . under our conditions ,
pairs 14 returned the highest number of positive results , and the rtpcrs behaved similarly but with reduced sensitivity .
many possible reasons could cause discrepant virus testing results between different sites , including changes to specimen integrity resulting from transport and variable amplification resulting from low viral loads .
the effects of viral load can be seen in this study : specimens in population 1 that were positive with multiple ( > 6 separate pairs ) primer pairs had a mean cycle threshold of 33.3 ( combining results from both rtpcrs ) , whereas those with < 6 positive results had means of 39.3 cycles .
amplification variability can also be attributed to the substantial nucleotide sequence diversity between hrvs and the different temporal and clinical characteristics of the 2 specimen populations we used .
our selection of published primer pairs includes those from studies that have informed our current understanding of hrv epidemiology . finding such a high degree of variability in performance was thus noteworthy .
comparison of data between different hrv studies is confounded as are data from studies seeking to determine the effects of other respiratory viruses .
the prevalence , seasonality , transmission , and clinical effects of hrv types and species require reexamination with tools that have been comparatively validated to ensure their sensitivity .
 we conducted a preliminary comparison of the relative sensitivity of a cross - section of published human rhinovirus ( hrv)specific pcr primer pairs , varying the oligonucleotides and annealing temperature . 
 none of the pairs could detect all hrvs in 2 panels of genotyped clinical specimens ; > 1 pcr is required for accurate description of hrv epidemiology . 
The Study
Conclusions