BACKGROUND
porcine reproductive and respiratory syndrome  causes significant economic losses in most pig-producing countries  <cit> .
the causative agent, the prrs virus , was identified in the early 1990s  <cit> .
prrsv is an enveloped, positive-strand rna virus with a genome of approximately 15 kb, and it belongs to family arteriviridae and order nidovirales <cit> .
a remarkable amount of genetic variation has been observed among the prrsvs isolates worldwide, particularly in nsp <dig>  <cit> , orf <dig>  <cit>  and the nucleocapsid   <cit> .
the genetic characteristics of the prrsv strains clearly indicate the existence of two major genotypes, the european type  and the north american type   <cit> .
during prrsv infection, clinical disease is detectable in all of ages of pigs but is usually observed in nursery-grown pigs  <cit> .
the clinical presentation of prrsv can range from asymptomatic to devastating, with symptoms such as listlessness, emaciation, hyperpnea, dyspnea, chemosis, abortion, stillbirth and a reduction in semen quality  <cit> .
however, infection with prrsv predominantly exists at a subclinical level, participating as a cofactor in porcine respiratory disease complex  and porcine circovirus associated disease   <cit> .
prrsv can be detected using molecular methods from nasal fluid, salivary, serum and tonsil specimens from naturally infected pigs  <cit> .
however, because prrsv is common within the swine population, no quantitative real-time pcr assays have been described using the serum samples of both symptomatic and asymptomatic prrsv-infected pigs.
the diagnosis of prrsv infection has relied on probe-based real-time pcr  <cit> , sybr green-based pcr  <cit> , rt-pcr  <cit> , virus isolation  <cit> , immunohistochemistry  <cit>  and serological methods  <cit> .
zip nucleic acids  are oligonucleotide-oligocation conjugates with multiple cationic spermine moieties attached to the nucleic acid oligomer  <cit> .
the melting temperature of a hybridized zna is easily predictable and increases linearly with the length of the oligocation  <cit> .
zna were shown to enable specific and sensitive reactions when used as a primer for pcr and reverse transcription  <cit> .
the present study describes a sensitive method for detecting type  <dig> prrsv using real-time fluorescent quantitative pcr with zna probes.
RESULTS
zna probe-based real-time pcr amplification and the limit of detection
tenfold serial plasmid dilutions  were tested and used to construct the standard curve by plotting the logarithm of the plasmid copy number against the measured quantification cycles  values.
the generated standard curve covered a linear range of six orders of magnitude of the standard plasmid dna.
the linear correlation  between the cq and the logarithm of the plasmid copy number was  <dig>   .
to assess the limit of detection of the assay,  <dig> to  <dig> copies/μl of standard plasmid dna per reaction were tested in  <dig> replicates.
at more than  <dig> copies, 100% of the replicates were positive, and at  <dig> copies,  <dig>  of the replicates were positive .
anumber positive/number tested.
reproducibility and specificity of the zna probe-based real-time pcr
the coefficient of variation of the mean cq values in the within-run and between-run for standard plasmid dna precision experiments ranged from  <dig>  to  <dig> % and from  <dig>  to  <dig> %, respectively .
the cv values in the within-run and between-run for clinical specimens that spanned the whole ranged from  <dig>  to  <dig> % and from  <dig>  to  <dig> %, respectively .
we analyzed other swine viruses to test the specificity of the zna probe-based real-time pcr.
no specific amplifications were detected for any of these samples .
alog <dig> copy number per microliter of prrsv.
detection of the viral load in serum samples from in the prrsv naturally infected pigs
the assay was tested on serum from both healthy and prdc pigs.
as determined by zna probe-based real-time pcr,  <dig>  of the  <dig> prdc pigs and  <dig>  of the  <dig> asymptomatic pigs were positive .
using a chi-square test, the correlation between prrsv and prdc was calculated.
among the  <dig> pigs that were analyzed, a positive result for prrsv appeared to be highly significantly correlated with the presence of prdc .
in addition, the prrsv load was significantly higher  in the prdc pigs  compared to that in the asymptomatic pigs   .
these data indicated that the levels of viral load correlated with the severity of the clinical presentation of the prrsv-infected pigs.
alog  <dig> copies number per microliter in serum.
discussion
the molecular diagnosis of prrsv infection has relied on probe-based real-time pcr  <cit>  and sybr green-based pcr  <cit> .
this study first describes a new zna probe-based real-time pcr for prrsv and evaluates this method as a diagnostic tool for prrsv infection.
this assay was sensitive, specific and reliable for the amplification of prrsv cdna, with a reproducible limit of detection of approximately  <dig> copies of target dna per reaction.
both the intra- and inter-assay cvs for standard plasmid dna and clinical specimens were satisfactorily low.
a novel type of modified oligonucleotides known as zna was recently developed as a method for the clinical diagnosis of pathogens, such as the hepatitis b virus  <cit> .
znas are able to discriminate between complementary sequence that are a perfect match and those that are mismatched by a single base pair  <cit> .
znas are particularly efficient at low magnesium concentrations, low primer concentrations and high annealing temperatures  <cit> .
zna probes provide broad flexibility with respect to experimental design and represent an effective alternative to minor groove binder-dna and locked nucleic acid probes  <cit> .
the viral load is an indicator of active infection, virus-host interaction and disease progression  <cit> .
the asymptomatic pigs are believed to serve as important reservoirs for the transmission of prrsv to uninfected pigs  <cit> .
seventeen point eight percent  of the asymptomatic pigs were positive in this study.
similar findings  were reported in a study that employed traditional rt-pcr in china  <cit> .
however, this is first study to report the viral load using serum samples in asymptomatic prrsv-infected pigs using zna probe-based real-time pcr.
four asymptomatic prrsv-infected pigs were detected at levels as high as  <dig> log <dig> viral copies/μl .
this result may be explained by differences in the susceptibility to prrsv infection.
the present study also reinforces a the previous suggestion that prrsv is one of the major viral agent for prdc  <cit> .
prrsv viraemia was detected in  <dig> %  of the prdc pigs, ranging from  <dig>  to  <dig>  log <dig> prrsv genome/μl .
in contrast to the symptomatic pigs, the viraemia of the asymptomatic pigs ranged from  <dig>  to  <dig>  log <dig> prrsv genome/μl .
moreover, based on the present results, it can be concluded that when pigs are infected with prrsv, the amount of prrsv in serum samples is significantly higher in prdc pigs.
similar reports had been made for another major viral agent of prdc, pcv <dig>  <cit> .
however, there were no previous studies performed for this disease.
the results obtained in the present study indicated that the prrsv load in serum might be used to assess the importance of prrs infection in a symptomatic case of prdc.
CONCLUSIONS
zna probe-based real-time pcr can be a useful tool to diagnose asymptomatic prrsv-infected pigs.
the presence of high prrsv loads in a sample of animals  is correlated to the presence of prdc in pigs.

 porcine reproductive and respiratory syndrome causes significant economic losses in most pig-producing countries. the causative agent, the prrs virus, was identified in the early 1990s. prrsv is an enveloped, positive-strand rna virus with a genome of approximately 15 kb, and it belongs to family arteriviridae and order nidovirales. a remarkable amount of genetic variation has been observed among the prrsvs isolates worldwide, particularly in nsp, orf and the nucleocapsid. the genetic characteristics of the prrsv strains clearly indicate the existence of two major genotypes, the european type and the north american type. during prrsv infection, clinical disease is detectable in all of ages of pigs but is usually observed in nursery-grown pigs. the clinical presentation of prrsv can range from asymptomatic to devastating, with symptoms such as listlessness, emaciation, hyperpnea, dyspnea, chemosis, abortion, stillbirth and a reduction in semen quality. however, infection with prrsv predominantly exists at a subclinical level, participating as a cofactor in porcine respiratory disease complex and porcine circovirus associated disease. prrsv can be detected using molecular methods from nasal fluid, salivary, serum and tonsil specimens from naturally infected pigs. however, because prrsv is common within the swine population, no quantitative real-time pcr assays have been described using the serum samples of both symptomatic and asymptomatic prrsv-infected pigs. the diagnosis of prrsv infection has relied on probe-based real-time pcr, sybr green-based pcr, rt-pcr, virus isolation, immunohistochemistry and serological methods. zip nucleic acids are oligonucleotide-oligocation conjugates with multiple cationic spermine moieties attached to the nucleic acid oligomer. the melting temperature of a hybridized zna is easily predictable and increases linearly with the length of the oligocation. zna were shown to enable specific and sensitive reactions when used as a primer for pcr and reverse transcription. the present study describes a sensitive method for detecting type prrsv using real-time fluorescent quantitative pcr with zna probes.  zna probe-based real-time pcr amplification and the limit of detection tenfold serial plasmid dilutions were tested and used to construct the standard curve by plotting the logarithm of the plasmid copy number against the measured quantification cycles values. the generated standard curve covered a linear range of six orders of magnitude of the standard plasmid dna. the linear correlation between the cq and the logarithm of the plasmid copy number was. to assess the limit of detection of the assay, to copies/μl of standard plasmid dna per reaction were tested in replicates. at more than copies, 100% of the replicates were positive, and at copies, of the replicates were positive. anumber positive/number tested. reproducibility and specificity of the zna probe-based real-time pcr the coefficient of variation of the mean cq values in the within-run and between-run for standard plasmid dna precision experiments ranged from to % and from to %, respectively. the cv values in the within-run and between-run for clinical specimens that spanned the whole ranged from to % and from to %, respectively. we analyzed other swine viruses to test the specificity of the zna probe-based real-time pcr. no specific amplifications were detected for any of these samples. alog copy number per microliter of prrsv. detection of the viral load in serum samples from in the prrsv naturally infected pigs the assay was tested on serum from both healthy and prdc pigs. as determined by zna probe-based real-time pcr, of the prdc pigs and of the asymptomatic pigs were positive. using a chi-square test, the correlation between prrsv and prdc was calculated. among the pigs that were analyzed, a positive result for prrsv appeared to be highly significantly correlated with the presence of prdc. in addition, the prrsv load was significantly higher in the prdc pigs compared to that in the asymptomatic pigs. these data indicated that the levels of viral load correlated with the severity of the clinical presentation of the prrsv-infected pigs. alog copies number per microliter in serum. discussion the molecular diagnosis of prrsv infection has relied on probe-based real-time pcr and sybr green-based pcr. this study first describes a new zna probe-based real-time pcr for prrsv and evaluates this method as a diagnostic tool for prrsv infection. this assay was sensitive, specific and reliable for the amplification of prrsv cdna, with a reproducible limit of detection of approximately copies of target dna per reaction. both the intra- and inter-assay cvs for standard plasmid dna and clinical specimens were satisfactorily low. a novel type of modified oligonucleotides known as zna was recently developed as a method for the clinical diagnosis of pathogens, such as the hepatitis b virus. znas are able to discriminate between complementary sequence that are a perfect match and those that are mismatched by a single base pair. znas are particularly efficient at low magnesium concentrations, low primer concentrations and high annealing temperatures. zna probes provide broad flexibility with respect to experimental design and represent an effective alternative to minor groove binder-dna and locked nucleic acid probes. the viral load is an indicator of active infection, virus-host interaction and disease progression. the asymptomatic pigs are believed to serve as important reservoirs for the transmission of prrsv to uninfected pigs. seventeen point eight percent of the asymptomatic pigs were positive in this study. similar findings were reported in a study that employed traditional rt-pcr in china. however, this is first study to report the viral load using serum samples in asymptomatic prrsv-infected pigs using zna probe-based real-time pcr. four asymptomatic prrsv-infected pigs were detected at levels as high as log viral copies/μl. this result may be explained by differences in the susceptibility to prrsv infection. the present study also reinforces a the previous suggestion that prrsv is one of the major viral agent for prdc. prrsv viraemia was detected in % of the prdc pigs, ranging from to log prrsv genome/μl. in contrast to the symptomatic pigs, the viraemia of the asymptomatic pigs ranged from to log prrsv genome/μl. moreover, based on the present results, it can be concluded that when pigs are infected with prrsv, the amount of prrsv in serum samples is significantly higher in prdc pigs. similar reports had been made for another major viral agent of prdc, pcv. however, there were no previous studies performed for this disease. the results obtained in the present study indicated that the prrsv load in serum might be used to assess the importance of prrs infection in a symptomatic case of prdc.  zna probe-based real-time pcr can be a useful tool to diagnose asymptomatic prrsv-infected pigs. the presence of high prrsv loads in a sample of animals is correlated to the presence of prdc in pigs. 
the clinical presentation of prrsv ranges from asymptomatic to devastating. these results suggest that a high viral load is a major feature of prrsv-affected pigs. porcine reproductive and respiratory syndrome virus is a rna virus with high genetic variation. porcine reproductive and respiratory syndrome virusviral loadzip nucleic acidreal-time pcr zna probe-based real-time pcr can be a useful tool to diagnose symptomatic and asymptomatic prrsv-infected pigs. the presence of this marker in a sample of animals with high prrsv loads seems to indicate that it correlates with the presence of prdc in pigs. in this study, we developed a sensitive and specific zip nucleic acid probe-based real-time pcr assay to evaluate the viremia of natural prrsv-infected pigs in taiwan. these include clinically healthy pigs and symptomatic pigs were confirmed to have porcine respiratory disease complex. serum samples were collected from pigs aged 5–12 weeks. this virus causes significant economic losses in most pig-producing countries.
 porcine reproductive and respiratory syndrome virus is a rna virus with high genetic variation. this virus causes significant economic losses in most pig-producing countries. the clinical presentation of prrsv ranges from asymptomatic to devastating. in this study, we developed a sensitive and specific zip nucleic acid probe-based real-time pcr assay to evaluate the viremia of natural prrsv-infected pigs in taiwan. serum samples were collected from pigs aged 5–12 weeks. these include clinically healthy pigs and symptomatic pigs were confirmed to have porcine respiratory disease complex.  viremia was quantified in of the clinically healthy pigs and in of the prdc cases. viremias were significantly more common in pigs with prdc compared with the clinically healthy pigs. these results suggest that a high viral load is a major feature of prrsv-affected pigs.  zna probe-based real-time pcr can be a useful tool to diagnose symptomatic and asymptomatic prrsv-infected pigs. the presence of this marker in a sample of animals with high prrsv loads seems to indicate that it correlates with the presence of prdc in pigs. porcine reproductive and respiratory syndrome virusviral loadzip nucleic acidreal-time pcr 