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for this study , all sybr green assays were modified from the previously described assays ( 5 ) , with the exception of the reverse primers for the newly designed pb1 and ns segments ( table 1 ) .
the subtype h1n1 swine influenza viruses isolated in hong kong during the past few years were mainly derived from the eurasian avian - like swine lineage ( 6,7 ) . to generate more precise genotyping data for our ongoing surveillance , the na segment 
specific assay was specifically designed to react with the pandemic ( h1n1 ) 2009 virus and a portion of eurasian avian - like swine viruses that are circulating in southeastern china ( 
 technical appendix figure 1 ) . to avoid overlapping the emission spectrum of sybr green
, we labeled all pandemic ( h1n1 ) 2009 virus  specific hydrolysis probes ( integrated dna technologies , inc . , coralville , ia , usa ) with cyanine 5 ( cy5 ) and black hole quencher-2 dyes at their 5 and 3 ends , respectively ( table 1 ) . to enable use of short oligonucleotide sequences without compromising the annealing temperature of these probes
rna extraction and complimentary dna synthesis were identical to the protocols described ( 5,8 ) .
one microliter of 10-fold diluted complimentary dna sample was amplified in a 20-l reaction containing 10 l of fast sybr green master mix ( applied biosystems , foster city , ca , usa ) and the corresponding primer probe set
all reactions were optimized and performed simultaneously in a 7500 sequence detection system ( applied biosystems ) with the following conditions : 20 s at 95c , followed by 30 cycles of 95c for 3 s and 62c for 30 s. sybr green and cy5 signals from the same reaction were captured simultaneously at the end of each amplification cycle .
the expected pcr results of virus segment derived from different swine viral lineages are shown in table 2 . *
pb , polymerase basic protein ; pa , polymerase acidic protein ; ha , hemagglutinin ; np , nucleocapsid protein ; na , neuraminidase ; m , matrix protein ; ns , nonstructural protein ; cy5 , cyanine 5 ; bhq2 , black hole quencher 2 . 
number represents nucleotide position of the first base in the target sequence ( crna sense ) . 
pb , polymerase basic protein ; pa , polymerase acidic protein ; ha , hemagglutinin ; np , nucleocapsid protein ; na , neuraminidase ; m , matrix protein ; ns , nonstructural protein .
specific probe results ; black symbols indicate sybr green results ; gray shading indicates sister clade of pandemic ( h1n1 ) 2009 virus for each virus segment . 
n2 and some of the n1 within swine euarasian avian - like lineage are expected to be double negative in the na test .
the dissociation kinetics of pcr amplicons were studied by a melting curve analysis at the end of the pcr ( 60c95c ; temperature increment 0.1c / s ) .
the condition described above gave the most robust and consistent dna amplification ( data not shown ) .
we tested 31 human pandemic ( h1n1 ) 2009 and 63 human seasonal influenza viruses ( 33 subtype h1n1 , 30 subtype h3n2 ) as controls .
as expected , all human pandemic influenza viruses were double positive ( i.e. , positive with sybr green and cy5 ) and all seasonal influenza samples were double negative in all 8 assays .
to evaluate the sensitivity of the assays , we tested serial diluted plasmid dna of the corresponding segments of influenza a / california/4/2009 virus as a standard .
the fluorescent signals generated from the sybr green reporter dye in all assays were highly similar to those previously reported ( 5 ) , and the modified assays had a linear dynamic detection range from 10 to 10 copies / reaction ( technical appendix figure 2 ) .
as expected , the threshold cycle values deduced from the cy5 reporter signal were generally higher than those from the sybr green reporter ( technical appendix figure 2 ) ( 9 ) .
this finding can be partly explained by the nature of these 2 kinds of real - time pcr chemistries : a single cy5 fluorophore of the hydrolysis probe was released from quenching for each amplicon synthesized while multiple sybr green dyes bound to a single amplicon ( 10 ) .
after 35 pcr amplification cycles , the linear dynamic detection range of cy5 signals generated from these reactions was 10 to 10 copies / reaction ( data not shown ) .
however , to avoid nonspecific sybr green signals , we purposely limited the number of amplification cycles to 30 . using these assays
in all 8 reactions , 10 pandemic ( h1n1 ) 2009 virus samples transmitted from humans to pigs ( 6 ) were double positive ( technical appendix figure 1 , pink ) . in these assays , gene segments of another 31 swine
isolates were either sybr green positive / cy5 negative ( technical appendix figure 1 , yellow ) or double negative ( technical appendix figure 1 , green ) , indicating that these virus segments were derived from the sister clade of pandemic ( h1n1 ) 2009 virus or other swine lineages ( except na ) , respectively .
for example , the reassortant of pandemic ( h1n1 ) 2009 virus ( a / swine / hong kong/201/2010 [ h1n1 ] ) was double positive for na , double negative for ha and matrix protein , and sybr green positive / cy5 negative for pb2 , pb1 , pa , np , and ns ( figure ; other data not shown ) .
all genotyping results of the studied viruses were consistent with results of previous phylogenetic analyses ( 5,6 ) , indicating that our modified probes and sybr green assays can provide more accurate genotyping results . with these genotyping data , viruses with atypical positive signal patterns
might suggest a novel viral reassortment event and can be highlighted for investigation with sequencing - based methods .
genotyping of a ) polymerase acidic protein , b ) hemagglutinin , and c ) neuraminidase segments of a / swine / hong kong/201/2010 influenza ( h1n1 ) virus .
black line , amplification signal of sybr green dye ; blue line , amplification signal of cyanine 5 dye ; green line , threshold level .
the x - axis denotes the cycle number of a quantitative pcr assay , and the y - axis denotes the fluorescence intensity over the background .
to demonstrate the potential use of these assays in studying swine viruses circulating in other geographic locations , we tested 7 recent swine isolates ( 1 pandemic influenza subtype h1n1 , 4 subtype h1n2 , and 2 subtype h3n2 ) collected in the united states .
genotyping results agreed 100% with data deduced from sequence analyses ( technical appendix table 1 ) .
we also analyzed all 436 contemporary ( 20082010 ) us swine virus segments available from the national center for biotechnology information influenza virus sequence database .
on the basis of the in silico analysis of sequences targeted by our primers and probes , 95% of the sequences ( n = 413 ) are predicted to yield the expected results ( technical appendix table 2 ) .
the emergence of pandemic ( h1n1 ) 2009 has highlighted the need for global systematic influenza surveillance in swine .
our results demonstrated that the addition of locked nucleic acid hydrolysis probes specific for pandemic ( h1n1 ) 2009 virus into previously established sybr green assays can help differentiate segments of pandemic ( h1n1 ) 2009 , eurasian avian - like , and triple reassortant virus lineages .
these assays might provide a rapid and simple genotyping method for identifying viruses that need to be fully genetically sequenced and characterized .
they may also help provide better understanding of the viral reassortment events and viral dynamics in pigs .
although at present , genes derived from human seasonal viruses can not be characterized with our modified assays , the performance of our assays warrants similar investigations for genotyping human influenza viruses .

 genotyping results , viral sequences , and combined sybr green / hydrolysis probe quantitative rt - pcr assays for swine influenza virus
the emergence of pandemic ( h1n1 ) 2009 virus highlighted the need for enhanced surveillance of swine influenza viruses . 
 we used real - time reverse  transcription pcr  based genotyping and found that this rapid and simple genotyping method may identify reassortants derived from viruses of eurasian avian - like , triple reassortant - like , and pandemic ( h1n1 ) 2009 virus lineages .