commercial porcine vaccines and viruses a total of 25 commercially available porcine vaccines were obtained from licensed manufacturers and pharmaceutical suppliers in taiwan ( table  1 ) .
viral dna was extracted from each vaccine using the qiaamp dna extraction kit ( qiagen , usa ) according to the manufacturer s instructions . for evaluating specificity of the lamp
, 21 virus isolates , comprising six pcv1 , five pcv2 , two porcine parvovirus ( ppv ) , three pseudorabies virus ( prv ) , three classic swine fever virus ( csfv ) , and two porcine reproductive and respiratory syndrome virus ( prrsv ) isolates , were used ; all of the isolates were previously identified by determining their partial nucleotide sequences ( wang et al .
the dna of pcv1 , pcv2 , ppv , prv , and cdna of csfv and prrsv isolates were produced as described previously ( huang et al .
2004 , 2008)table 1detection of pcv1 dna in commercial vaccines by the lamp and the nested pcrmanufactureragentlampnested pcracsfvaprvbcsfv++bprvccsfv++cprv , b. bronchiseptica , p. multocidadcsfvdfmdvecsfv++eprvfcsfvfprv , e. rhusiopathiae , p. multocida , m. hyopneumoniaeffmdvgfmdvgprrsvhfmdvhprv , m. hyopneumoniae , e. coliiprrsvjppvjfmdvkfmdvlppvmprv , ppv , e. rhusiopathiaencsfvnfmdvcsfv classic swine fever virus , prv pseudorabies virus , fmdv foot - and - mouth disease virus , prrsv porcine reproductive and respiratory syndrome virus , ppv porcine parvovirus detection of pcv1 dna in commercial vaccines by the lamp and the nested pcr csfv classic swine fever virus , prv pseudorabies virus , fmdv foot - and - mouth disease virus , prrsv porcine reproductive and respiratory syndrome virus , ppv porcine parvovirus primers specific primer sets of the lamp for pcv1 detection were designed by primerexplorer v4 software ( http://primerexplorer.jp/elamp4.0.0/index.html ) based on different reference pcv1 isolates ( genbank accession no .
af012107 , ay184287 , ay219836 , ay660574 , dq472013 , dq659154 , ef493843 , fj475129 , and gu722334 ) .
the primer sets consisted of two outer primers ( f3 and b3 ) and two inner primers ( fip and bip ) targeting a conserved region within the pcv1 orf2 gene . to further validate the lamp ,
the primers pcv - f1 , pcv - r1 , pcv - f2 , and pcv - r2 were used for nested polymerase chain reaction ( nested pcr ) targeting the conserved region of the pcv1 orf2 gene ( victoria et al .
nucleotide sequences and locations of the primers are shown in table  2.table 2oligonucleotide primer sets used for the lamp and nested pcr  primerpositiontypesequence ( 53)lampf31,2101,228forward outerttg ttt ggt cca gct cag gb31,4001,381backward outercag aga ccc cat cac ctc tafip1,2891,271 , 1,2301,248forward innerctc ctc ctc ccg cca cac cat af1c - tttt - f2-tttt- ttg ggg gtg aag tac ctg gbip1,3061,327 , 1,3721,354backward innercat agg cca agt tgg tgg acg gb1c - tttt - b2-tttt- ggt gtt ggg tcc act gtt gnested pcrpcv - f11,1461,167first forwardttg gtg tgg gta ttt aaa tgg apcv - r11,6771,659first reversegca gcc atc ttg gaa aca tpcv - f21,2971,318second forwardtat agg ggt cat agg cca agt tpcv - r21,4521,431second reverseccc tac ctt tcc aat act acc glocation of the primers based on the nucleotide sequence of the orf2 gene of pcv1 ( genbank accession no .
ay219836)both inner primers fip and bip contained two binding regions and were connected by a tttt bridge oligonucleotide primer sets used for the lamp and nested pcr location of the primers based on the nucleotide sequence of the orf2 gene of pcv1 ( genbank accession no .
ay219836 ) both inner primers fip and bip contained two binding regions and were connected by a tttt bridge lamp reaction a 25-l reaction mixture consisted of 12.5  l of 2 lamp reaction buffer ( eiken chemical , japan ) , 8  u bst dna polymerase ( new england biolabs , usa ) , 2  l of the extracted target dna sample , 40  pmol each of fip and bip primers , and 5  pmol each of f3 and b3 primers .
the lamp reaction was conducted by incubating the reaction mixture at 60c for 60  min and then inactivating the bst dna polymerase at 80c for 2  min .
the reaction was monitored in real - time at 6-s intervals by measuring the turbidity at a650 using a loopamp real - time turbidimeter ( la-320 ; eiken chemical , japan ) .
the cutoff turbidity was set at 0.1  turbidity units and was determined by analyzing negative controls in replicates . a sample with turbidity remaining below 0.1  turbidity units throughout the 60-min incubation period was considered to be negative for pcv1 dna .
if the turbidity of a sample increased above 0.1  turbidity units within the 60-min incubation period , it was considered to be positive , indicating contamination with pcv1 dna .
the lamp amplicons were further analyzed by electrophoresis through a 2% agarose gel containing 0.5  mg / ml sybr safe dna gel stain ( invitrogen , usa ) in tris  acetate 
nested pcr reaction a total of 25 pcr reaction mixtures of the first round pcr in the nested pcr consisted of 2.5  l of 10 buffer ( 100  mmol / l tris 
hcl at ph  8.8 , 500  mmol / l kcl , 15  mmol / l mgcl2 , 1% triton x-100 ) , 1.25  mol / l each of four nucleoside triphosphates , 20  mol / l of each primer , 0.5  l of dna polymerase power taq ( 2  u/l ; bertec , taipei , taiwan ) , and 2  l of the dna sample .
the first round pcr was performed at 95c for 5  min , followed by 30 cycles of denaturation at 95c for 20  s , annealing at 55c for 30  s , and extension at 72c for 30  s. finally , the samples were subjected to a terminal extension step at 72c for 10  min . from this reaction , 2  l of the first round pcr product was used as a template for the nested pcr . in the nested pcr , 25  l of reaction mixture consisted of 2.5  l of 10 buffer ( 100  mmol / l tris 
hcl at ph  8.8 , 500  mmol / l kcl , 15  mmol / l mgcl2 , 1% triton x-100 ) , 1.25  mol / l each of four nucleoside triphosphates , 20  mol / l of each primer , and 0.5  l of dna polymerase power taq ( 2  u/l ; bertec , taiwan ) .
the nested pcr was performed at 95c for 5  min , followed by 30 cycles each of denaturation at 95c for 20  s , annealing at 55c for 20  s , and extension at 72c for 20  s. finally , the samples were subjected to a terminal extension step at 72c for 10  min .
amplicon of the nested pcr were analyzed by electrophoresis through a 2% agarose gel as described above .
construction of recombinant plasmids full - length orf2 gene of pcv1 ( genbank accession no .
the amplicon was ligated into the pcrii - topo plasmid that was supplied with the dual promoter topo ta cloning kit ( invitrogen , usa ) .
top10fescherichia coli competent cells ( invitrogen , usa ) were transformed with the recombinant plasmid according to the manufacturer s instructions .
the inserted target gene carried by the recombinant plasmid was directly sequenced as described previously ( wang et al .
the recombinant plasmid was quantified by measuring its absorbance a260 on a u2100 pro uv / vis spectrophotometer ( general electronic healthcare , singapore ) and diluted to 10  copies/l to develop a standard curve for evaluating the analytical sensitivity of the lamp .
analytical specificity and sensitivity of the lamp to determine the analytical specificity of the lamp , dna samples extracted from the 21 virus isolates were tested by the lamp .
dna extracted from specific - pathogen - free ( spf ) swine tissue samples was used as the negative control . to evaluate the analytical sensitivity of the assay , 10-fold serial dilutions of the recombinant plasmid
were performed to prepare the dilutions at concentration from 10  copies/l to 1  copy/l , and each dilution was tested by both the lamp and the nested pcr ( victoria et al .
for quantification of the diluted samples , a standard curve was generated for the lamp , with serial 10-fold dilutions of the recombinant plasmid that were positive for pcv1 dna on the x - axis and the time of positivity ( min ) on the y - axis .
evaluation of lamp with commercial swine vaccines two batches of each vaccine were tested for detection of pcv1 dna by the lamp and the nested pcr , respectively . to evaluate the vaccine matrices for interference or inhibition of the lamp reaction ,
all extracted dna of tested vaccine samples were spiked with plasma containing the recombinant plasmid .
the final concentration of the recombinant plasmid for each spiked vaccine sample was 10  copies / ml .
detection of pcv1 dna for each spiked vaccine sample was performed by the lamp as an internal control .
the analytical specificity of lamp was evaluated using dna extracted from pcv1 , pcv2 , ppv , and prv , and cdna derived from csfv and prrsv .
pcv1 dna was successfully detected , and a ladder - like amplicon was generated ( fig .  1 ) .
the lamp amplified the pcv1 target gene , but not the nucleic acids extracted from pcv2 , ppv , prv , csfv , and prrsv ( fig . 
lane m 2,000100  bp ladder marker ( 50  u/l ; protech , taiwan ) ; lanes 17 extracted dna of pcv1 , pcv2 , ppv , prv , cdna of csfv , prrsv , and negative control , respectively analytical specificity of the lamp was observed using agarose gel electrophoresis .
lane m 2,000100  bp ladder marker ( 50  u/l ; protech , taiwan ) ; lanes 17 extracted dna of pcv1 , pcv2 , ppv , prv , cdna of csfv , prrsv , and negative control , respectively by testing 10-fold serial dilutions ( 10  copies/l to 1  copy/l ) of the recombinant plasmid , the lamp demonstrated a detection limit of 10 copies ( fig .  2 , lane 7 ) ,
lane 15 ) . the standard curve for lamp was generated from the amplification plots of the 10-fold serial dilutions of the recombinant plasmids ( 10  copies/l to 10  copies/l ) vs. time ( min ) ( fig . 
3a ) . a linear regression line with a coefficient of r  =  0.97 was plotted for the lamp ( fig . 
2analytical sensitivities of the lamp ( lanes 18 ) and the nested pcr ( lanes 916 ) were compared by detecting 10-fold serial dilutions of the recombinant plasmid ( 10  copies/l to 1  copy/l ) by agarose gel electrophoresis .
lane m 2,000100  bp ladder marker ( 50  u/l ; protech , taiwan ) .
the pcr products of lanes 915 were 155 base pairs ( bp ) in lengthfig .
3analytical sensitivity of the lamp ( a ) was performed by real - time monitoring with 10-fold serial dilutions of the recombinant plasmid : 10  copies/l to 1  copy/l and negative control ( nt ) . the linear function fitted to the plot of the mean threshold time against the log of the input pcv1 the recombinant plasmid
is also shown ( b ) analytical sensitivities of the lamp ( lanes 18 ) and the nested pcr ( lanes 916 ) were compared by detecting 10-fold serial dilutions of the recombinant plasmid ( 10  copies/l to 1  copy/l ) by agarose gel electrophoresis .
lane m 2,000100  bp ladder marker ( 50  u/l ; protech , taiwan ) .
the pcr products of lanes 915 were 155 base pairs ( bp ) in length analytical sensitivity of the lamp ( a ) was performed by real - time monitoring with 10-fold serial dilutions of the recombinant plasmid : 10  copies/l to 1  copy/l and negative control ( nt ) .
the linear function fitted to the plot of the mean threshold time against the log of the input pcv1 the recombinant plasmid is also shown ( b ) pcv1 dna was detected in three commercial csfv vaccines by the lamp and the nested pcr ( table  2 ) .
these vaccines were manufactured by three different commercial companies ( b , c , and e ) . all extracted dna of tested vaccine samples were spiked with pcv1 recombinant plasmids and yielded positive results for detection of pcv1 dna by the lamp ( data not shown ) .
these results also indicated that inhibition was not observed in the extracted dna of 25 commercial swine vaccine samples .
the products generated by this method can be analyzed not only by conventional agarose gel electrophoresis but also spectrophotometry , which enables real - time analysis and quantification of the amplicons in commercial swine vaccines .
amplification of a specific genomic region by conventional or nested pcr alone , or by pcr in combination with gene sequencing , has long been used to confirm the accuracy of amplicons generated by pcr ( muhling et al .
however , with or without gene sequencing , conventional detection procedures for pcv1 require several hours to complete .
in contrast , dna detection with the lamp was much faster than with conventional pcr or nested pcr .
after preparation of the sample dna , the lamp could be completed in approximately 1  h , whereas other methods required several hours to a few days .
furthermore , pcv1 dna could be specifically and sensitively amplified using the two lamp primer sets ; no amplification was observed when the method was used to detect nucleic acids of pcv2 , ppv , prv , csfv , fmdv , and prrsv .
these results indicate the specificity and wide applicability of our lamp method in the detection of viral contaminants in commercial swine vaccines .
our results showed that the lamp is highly sensitive and can detect small amounts of pcv1 dna present in samples .
this method enables the detection and quantification of pcv1 dna in commercial swine vaccines . in this study , using the lamp , we found that three commercially available csfv vaccines were contaminated with pcv1 dna , although the source of pcv1 dna contamination in some commercial porcine vaccines has remained unknown .
recently , pcv1 dna was also found in two commercial human rotavirus vaccines ( baylis et al .
although pcv1 is not regarded as a potential pathogen in humans ( victoria et al .
2010 ) , avoiding virus contamination during the process of vaccine production is absolutely essential .
the detected pcv1 dna may originate from the cell lines used for vaccine production ( quintana et al .
presence of pcv1 dna in the vaccines does not necessarily imply that infectious pcv1 particles are present in the vaccines .
the contaminating pcv1 may have been inactivated during the manufacturing process or may have been present in the live vaccine products in too small amounts to cause an infection ( quintana et al .
the presence of viral dna other than the vaccine dna itself is absolutely unacceptable . to fulfill this requirement ,
a rapid , specific , and sensitive method such as lamp can be easily incorporated in the production process to screen for pcv1 contamination in vaccines and in cell lines used for cultivation of the vaccine viruses .
the spiking study was performed by deliberately spiking pcv1 virus into the vaccine samples to account for inhibition ; this avoids a potential problem that may result in the under - detection of pcv1 ( i.e. , false negatives ) .
a previous study indicated that some human medicines contain heparin , adjuvant , phenol , or formaldehyde , which are common pcr inhibitors and always remain with the extracted nuclear acid ( yokota et al .
, no evidence of inhibition has been observed in any extracted dna of swine vaccine sample for spiked recombinant plasmids .
however , this was necessary to perform spiking studies as an internal control for the lamp reaction ; this assessed individual vaccine matrices that may be the cause of amplification product inhibition . in conclusion ,
this method can be used to rapidly detect cell lines and commercial swine vaccines for pcv1 contamination with high sensitivity and specificity .
 a loop - mediated isothermal amplification ( lamp ) method with a real - time monitoring system was developed for the detection of porcine circovirus type 1 ( pcv1 ) in commercial swine vaccines . 
 this method was highly specific for pcv1 . 
 no cross - reaction to porcine circovirus type 2 , porcine parvovirus , pseudorabies virus , classical swine fever virus , and porcine reproductive and respiratory syndrome virus was observed . 
 the analytical sensitivity of the lamp for pcv1 dna was 10  copies/l in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction ( nested pcr ) . 
 furthermore , 25 commercial swine vaccines were tested by both the lamp and the nested pcr , and three of them were tested positive for pcv1 dna . 
 these results indicate that pcv1 dna can be real - time detected by the lamp ; the method was highly specific , sensitive , and rapid for the detection of pcv1 dna , particularly in commercial swine vaccines . 
Materials and methods
Results
Discussion