BACKGROUND
porcine circovirus-like agent p <dig>  is a newly discovered virus.
p <dig> has a circular single-stranded genome of  <dig> nucleotides, and it is the smallest dna virus known to date.
the whole genome is highly homologous to the genome of porcine circovirus type  <dig>  with the exception of  <dig> nucleotides.
epidemiological investigation confirmed that the p <dig> virus has been epidemic in china’s pig farms  <cit> .
to further characterize the p <dig> virus, we constructed two clones, one with a single copy of p <dig> virus genome and one with a two copy tandem repeat.
it was confirmed that the tandem repeat clone can produce viral particles in transfected pk <dig> cells, demonstrating that this clone has in vitro viral activity.
virus particles are spherical in shape, non-enveloped with a diameter of approximately 25 nm.
in vivo studies have shown that the tandem repeat p <dig> clone can infect pigs and the infected animals show symptoms resembling postweaning multisystemic wasting syndrome .
clinical manifestations of p <dig> infection include pale skin, diarrhea and viremia.
generally, lesions include brain hyperemia, bladder mucosal bleeding, inguinal lymph node bleeding, and lung atrophy and bleeding.
histological examines identified diseases including subarachnoid vascular congestion, interstitial pneumonia, myocardial atrophy, tonsil tissue follicular cell hyperplasia, and necrosis of pancreatic cells.
p <dig> nucleic acids and antigens were detected in tissue samples of lung, brain, heart, liver, bladder, pancreas and gonads  <cit> .
the main symptoms of pmws include wasting and stunting, and sometimes pale skin, jaundice, diarrhea and breathing difficulties  <cit> .
pmws has become one of the major epidemic diseases that endanger the world’s pig industry  <cit> .
pcv <dig> is considered a primary pathogen of pmws  <cit> .
pcv <dig> belongs to the virus family circoviridae, and is a small, non- enveloped, single-stranded dna virus with a genomic size of  <dig> or  <dig> nucleotides .
there are three confirmed open reading frames encoded in the pcv <dig> genome.
orf <dig> encodes the viral replication enzyme  <cit> , orf <dig> encodes the viral capsid protein  <cit> , and orf <dig> encodes a protein which is not essential for viral replication but has apoptotic activity  <cit> .
an early study suggested that the virulence of different pcv <dig> strains correlates with the amino acid sequences of its capsid protein  <cit> .
several studies followed that focused on orf <dig> and based on these studies pcv <dig> can be divided into different genotypes  <cit> .
recent studies suggest that pcv <dig> genotype  <dig> is more virulent than genotype  <dig>  <cit> , however, there are discrepancies in the literature  <cit> .
the discrepancy is around the pathogenic characteristics of pcv <dig>  as it is difficult to reproduce the typical symptoms of pmws with pcv <dig> alone .
it is speculated that there may be another contributing factor in the occurrence of pmws.
the discovery of p <dig> provides a new etiological explanation for pmws since the nucleotide sequence of the p <dig> genome is highly homologous with orf <dig> of pcv <dig> and the encoded capsid protein is also homologous to that of pcv <dig>  the current conventional diagnostic techniques based on the orf <dig> of pcv <dig> may not be able to distinguish pcv <dig> from p <dig>  therefore, it is important to establish a rapid, highly sensitive diagnostic method to detect pcv <dig> and p <dig> which will aid the prevention and treatment of porcine circovirus-like virus infections.
real-time quantitative pcr has become the preferred method of quantitative detection of nucleic acids due to its’ high sensitivity, specificity and reproducibility as well as being rapid.
among them, the sybr green i dye method does not require probe labeling and is highly cost effective and therefore has been more widely used.
this study aims to establish an approach based on sybr green i dye real-time pcr to accurately and quantitatively detect p <dig> as well as distinguishing between p <dig> and pcv <dig> viruses in animals.
methods
ethics statement
the use of animal specimens in this study was approved by the jiangsu province animal regulations .
the protocol was approved by the committee on the ethics of animal experiments of the institute of veterinary medicine, jiangsu academy of agricultural sciences .
virus isolation
pseudo-rabies virus , porcine parvovirus , porcine reproductive and respiratory syndrome virus , porcine circovirus type  <dig>  and serum samples of p <dig> infected pigs were all isolated and stored in institute of veterinary medicine, jiangsu academy of agricultural sciences as previously described  <cit> .
primers
a pair of inverse primers was designed according to the p <dig> sequence  using the software oligo <dig> .
the sequence of the forward primer is 5′- gagaggcgggtgttgaagat- <dig> ′ and the reverse primer are 5′- aagaccccccacttaaaccc - <dig> ′.
the amplification product is l <dig> bp for p <dig> and 1238 bp or 1237 bp for pcv <dig> based on different genome size .fig.
<dig> genomic characterization of pcv <dig> and p <dig> and position of the primers in both genomes.
the high homologous sequences of pcv <dig> and p <dig> are shown in bold, red colour, the primers in both genomes are indicated by the arrowheads, and their nucleotide coordinates are marked in both genomes
preparation of viral dna template
prv, ppv, pcv <dig> infected cell culture samples and clinical samples  were used to prepare viral dna samples.
among them, the stool samples were diluted 1: <dig> with sterile phosphate buffer , subjected to three freeze-thaw cycles, centrifuged and then the supernatant was used for viral dna preparation.
300 μl of sample was mixed with 300 μl of cell lysis buffer  and 10 μl of proteinase k , and then the sample was transferred to 50 °c to digest for 2 h. after digestion the sample was mixed with an equal volume of tris-saturated phenol and centrifuged at 12000 r/min for 10 min.
the upper aqueous phase was transferred to a new  <dig>  ml eppendorf tube and mixed with an equal volume of phenol:chloroform:isoamyl alcohol  and centrifuged at 12000r/ min for 10 min.
the upper aqueous phase was extracted with phenol: chloroform: isoamyl alcohol  one more time and was transferred to a new  <dig>  ml eppendorf tube.
then the sample was mixed with  <dig> volumes of ice-cold ethanol and 1/ <dig> volume of 3 mol/l sodium acetate  and sit at room temperature  for 20 ~ 30 min to precipitate the genomic dna.
following centrifugation at 12000 r/min for 10 min, the supernatant was discarded and the precipitates were washed in 70 % ethanol and air dried.
dna was dissolved in te buffer containing 20 μg/ml rnasea and stored at -20 °c for later use.
preparation of viral rna template
total rna was extracted from prrsv infected cells using trizol ls reagent according to the manufacturer’s instructions .
reverse transcription was carried out immediately using the rna sample.
the reaction mix consisted of  <dig>  μl rna, 4 μl 5x buffer, 2 μl 10 mmol/l dntps mixture,  <dig>  μl  <dig> u/μl rnasin, 1 μl oligo  <dig>  and 1 μl  <dig> u/μl amv reverse transcriptase.
after incubation at 42 °c for 1- 2 h, the resulting cdna was stored at -20 °c for later use.
development of fluorescence quantitative standard curve
p <dig> genomic dna was extracted from serum specimens of infected pigs and used as template for a 50 μl pcr reaction.
the pcr conditions consisted of an initial denaturation at 95 °c for 5 min, followed by amplification for 35 cycles of 30 s at 95 °c, 30s at 60 °c, and 30 s at 72°cfollowed by one cycle of 72 °c for 10 min.
pcr product was separated on 2 % agarose gel by electrophoresis and recovered by agarose gel extraction.
the dna fragment was cloned into pmd18-t vector and transformed into e. coli top <dig>  the resulting recombinant plasmid was verified by pcr, restriction enzyme digestion and dna sequencing.
plasmids with an od 260 nm/od 280 nm ratio between  <dig> - <dig>  were used to establish a standard curve.
the concentration of the plasmids was calculated based on od260nm value and used to calculate copy numbers.
six 10-fold serial dilutions of plasmid samples with copy number from  <dig> to  <dig> copies/μl were used as template to run reaction on the abi  <dig> quantitative pcr machine to generate a standard curve.
the reaction mixture is 25 μl and contains  <dig>  μl ultrasybr mix  ,  <dig>  μm forward and reverse primers each, and 2 μl template.
the reaction cycles are as follows: 95 °c for 10 min followed by 40 cycles of 95 °c for 15 s, 60 °c for 30s.
after the final extension reaction added was the melting curve analysis.
after completion of the reaction, a standard curve is automatically generated by the abi software.
analysis of specificity, sensitivity and reproducibility of the sybr i qpcr
to test the specificity of the method, prv, ppv, pcv <dig>  and p <dig> virus genomic dna and prrsv cdna are used as templates in sybr i qpcr according to the aforementioned reaction conditions.
to test the sensitivity of the method, ten 10-fold serial dilutions of recombinant p <dig> dna with concentration from  <dig> to  <dig> copies/μl are used as templates to run real-time pcr.
to test the reproducibility of the method, sybr i qpcr reactions are repeated three times using 1: <dig> serial dilutions of p <dig> plasmids with copy numbers of  <dig> to  <dig> copies/μl and the obtained ct values are compared.
clinical samples
<dig> sera samples and  <dig> manure samples were tested in this study.
all samples were obtained from pig farms located in jiangsu province, anhui province, beijing city and tianjin city, respectively.
serum samples were collected in  <dig> or  <dig> from ~1 month old pigs with or without pmws.
manure samples were collected in  <dig> from piglets with severe diarrhea.
the extracted dnas from the aforementioned clinical samples were used as templates for sybr i qpcr and also used as template for whole genome amplification of pcv <dig> and p <dig> by conventional pcr .
RESULTS
pcr amplification of p <dig> gene fragment and construction of recombinant plasmid
p <dig> dna was used as the template for pcr amplification and an 119 bp dna fragment was obtained which is consistent with the size of the expected gene fragment.
the obtained fragment was used to construct a recombinant plasmid which was verified by both pcr and restriction enzyme digestion analysis.
then the plasmid was verified by dna sequencing.
generation of the standard curve and establishment of the linear regression equation
six serial dilutions of plasmid with copy number from  <dig> to  <dig> copies/μl were used as template to run sybr i qpcr.
following the reaction the system software automatically generated the amplification curves, melting curves, the standard curve and linear regression equation.
the amplification graph exhibited an s-shape fluorescence amplification curve.
the melting curve displayed a single peak and was consistent tm values of approximately  <dig>  °c.
standard curve analysis demonstrated that when copy numbers are in the range of  <dig> to  <dig> copies/μl, the copy numbers of p <dig> correlate linearly with ct values with a slope of - <dig> , the efficiency of the qpcr reaction as  <dig>  %, and the value of the correlation coefficient r <dig> of the linear regression equation as  <dig>   .fig.
<dig> standard curve of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p <dig> plasmid standards.
x -axis is 10-fold serial dilutions of the plasmid standards , y axis represents the threshold cycle  values
analysis of specificity, sensitivity and reproducibility of reaction
sybr i qpcr reactions using the p <dig> plasmid as a standard gave rise to the corresponding product and produced a single melting peak, indicating no primer dimers or other non-specific products amplified.
the final product was further confirmed by dna sequencing.
reaction using pcv <dig> or p <dig> as template generated a melting curve with a sharp peak, while the melting curve of prv, ppv or prrsv was flat.
although pcv <dig> gene also could be amplified, the melting temperature is approximately 75 °c which is different from that of p <dig>  thus the method has enough specificity .
ten 10-fold serial dilutions of p <dig> with concentrations from  <dig> to  <dig> copies/μl were used as templates to run sybr i qpcr the results showed that even at the low concentration of  <dig> copies/μl, fluorescent signal could still be detected, indicating a high sensitivity and the concentration of templates correlates well with ct values .
a ct value of  <dig> was chosen as a cut-off value for standard positivity.
tests were repeated using the serial dilutions and further statistical analysis showed that the coefficient of variation within groups and between groups was less than 5 %, indicating good stability and reproducibility .fig.
<dig> specificity test of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p <dig>   p1;  pcv2;  prv;  prrsv; and  ppv
fig.
<dig> sensitivity test of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p <dig>  plasmid standard was used as template, x -axis is 10-fold serial dilutions of the plasmid standards , y axis represents the threshold cycle  values
107
106
105
104
results of clinical sample analysis
by fluorescence-based quantitative pcr analysis  <dig> out of  <dig> serum samples and all six stool samples were identified as p <dig> positive .
p <dig> virus titers of the majority of samples  were between 104- <dig> copies/ml with one manure sample up to  <dig> copies/ml.
the results are shown in table  <dig>  out of  <dig> serum samples,  <dig> and  <dig> serum samples from healthy and pmws pigs were postive for p <dig>  respectively.
the amount of p <dig> in healthy pigs were generally below p <dig> concentrations of  <dig> genome copies/ml of total dna, while the p <dig> concentrations in pmws pigs were consistently higher than  <dig> p <dig> dna copies/ml of total dna.table  <dig> p <dig> gene copy numbers of serum and fecal samples measured by quantitative pcr
103
the results of conventional pcr and quantitative pcr analysis of serum and manure samples were compared in table  <dig>  when applied to detection of pcv <dig> in the serum, conventional pcr identified  <dig> samples as pcv <dig> positive compared to the  <dig> samples by quantitative pcr.
neither conventional pcr nor quantitative pcr detected pcv <dig> in fecal samples.
conventional pcr can detect  <dig> serum samples as p <dig> positive compared to the  <dig> samples by quantitative pcr.
quantitative pcr detected  <dig> fecal samples as p <dig> positive compared to the  <dig> samples by conventional pcr.
the quantitative pcr failed to detect the sixteen double positives found by conventional pcr.
collectively the results demonstrate that when the p <dig> gene copy number is less than  <dig> copies/ml, it is difficult to use conventional pcr to detect.table  <dig> comparison of results obtained by quantitative pcr  and conventional pcr
discussion
circoviridae is a family of single-stranded dna viruses with a small genome size that can infect both poultry and pig.
two plant virus families, nanoviridae and geminiviridae, are considered to be closely related to circoviridae.
they all contain a stem-loop structure which encompasses a replication origin .
gibbs and weiller, based on bioinformatics results of the rep protein amino acid sequence of circovirus and nanoviridae, provided a hypothesis of the origin and evolution of circovirus.
in their study, they found that the n-terminal amino acid sequence of rep in circovirus is homologous to that of nanoviridae while the c-terminal amino acid sequence of the pcv rep is closely related to rna binding proteins 2c of vertebrate calicivirus.
therefore, they speculated that pcv is a combination of nanoviridae and calicivirus.
since calicivirus is a rna virus, the recombinant must involve a retrovirus or a retrotransposon  <cit> .
although the international committee on taxonomy of viruses  has not further classified viruses belonging to the same species, pcv <dig> could be divided into two major genotypes, namely pcv2a and pcv2b.
hesse et al.
reported a pcv <dig> strain which contains a recombination of pcv2a orf <dig> and pcv2b orf <dig>  <cit> .
thereafter, pcv2a/b recombinants have been frequently isolated .
we identified porcine circovirus agent p <dig> during a study of pcv <dig> and suggested it may be a result of recombination of pcv <dig> with other molecules, likely with pcv <dig>  because it also has circular single-stranded genome of  <dig> nucleotides.
the 648 bp genome of p <dig> is the smallest dna viral genome identified to date.
its genome is comprised of a dna sequence highly homologous to pcv <dig> orf <dig> as well as exogenous sequences <dig>  the exogenous sequence of p <dig> is  <dig> nucleotides in size, and such a short fragment can be found in many species when a blast search was run to identify homologous sequence in genbank, thus making it difficult to trace its origin.
we can only speculate that p <dig> may be a product of a recombination between retrovirus and pcv <dig>
p <dig> infection of pigs can generate symptoms resembling pmws, but these symptoms are different from symptoms of pcv2-associated pmws in terms of anatomical and pathologic traits as well as viral distribution in tissues.
the investigation of virus replication and pathological mechanism requires quantitative determination of virus copy numbers.
the p <dig> genome has  <dig> nucleotides, and except ~ <dig> nucleotides, its sequence is highly homologous to pcv <dig> orf <dig> which contains  <dig> nucleotides.
this indicates that the p <dig> genome is almost completely overlapped with pcv <dig> orf <dig> and it is difficult for conventional pcr to distinguish between p <dig> and pcv <dig>  in this study we designed a pair of primers which gave rise to a p <dig> pcr product of 119 bp and a pcv <dig> pcr product of  <dig>  bp.
thus, pcv <dig> will inevitably be amplified when using optimized quantitative pcr to detect p <dig>  however its amplification efficiency and melting temperature are distinctively different from those of p <dig>  providing a base to distinguish between the two viruses.
the specificity of the sybr i qpcr method was further confirmed by the fact that the study identified less positive samples than conventional pcr when applied to pcv <dig> test, as well as that it preferred to amplify p <dig> genome when dna template contains both p <dig> and pcv <dig>
there are two commonly used quantitative real-time pcr techniques used, namely sybr green dye method and taqman probe method.
sybr green i has advantages over the taqman technique; it avoids the use of fluorescent-labeled probes and other expensive reagents, and it is applicable to any pcr amplification system and can recognize primer dimers and nonspecific amplifications by melting curve analysis.
in this study a method to detect p <dig> based on sybr i qpcr was successfully developed and the results show this approach is able to detect virus samples of a wide range of concentrations.
when virus concentration falls between  <dig> and  <dig> copies/μl, pcr results correlate linearly with virus concentrations.
with a high sensitivity the minimum virus concentration this method is able to detect is < <dig> copies/μl.
this test method has no cross-reactivity with ppv, prrsv, and prv viruses and can distinguish pcv <dig> by melting curve analysis despite the fact that a small amount of pcv <dig> amplifications can also be detected, thus manifesting a high specificity.
all results obtained by this method were very reproducible and the results of porcine serum samples showed that it can be easily applied to clinical analysis.
even some healthy pigs were found to be p <dig> positive.
and their serum p <dig> copy numbers were with the majority of them falling in between  <dig> and  <dig> copies/ml.
since p <dig> infection can cause pmws-like symptoms to some pigs, the relationship between p <dig> copy numbers and symptoms needs further investigation.
in 2010- <dig> a serious infectious diarrhea disease affected a large number of pig farms in china and caused high rates of morbidity and mortality.
no antibiotics or vaccines are effective to this disease and the economic loss was very significant.
a new strain of porcine epidemic diarrhea virus was found to be the major pathogen, but porcine bocavirus and crest virus were also detected in clinical specimens.
in this study high concentration of p <dig> virus was identified in all six stool samples obtained from diarrhea pigs, suggesting that p <dig> may play a role in this epidemic of diarrhea.
in previous studies using real-time quantitative pcr assay to detect pcv <dig>  either dye method  <cit>  or probe method  <cit> , orf <dig> gene was selected as the target.
this method cannot accurately measure pcv <dig> concentration under circumstances of p <dig> and pcv <dig> double infection.
p <dig> can only be distinguished when pcv <dig> orf <dig> is selected as the assay target  <cit> .
recent studies have shown that pcv <dig> can form intramolecular rearrangements of variable size which also have circular single-stranded genome consisting of nucleotide sequences derived entirely from the parent strains.
these molecules do not contain exogenous nucleotides and their cleavage sites are different from those of pcv <dig>  <cit> .
some rearrangements contain a part of orf <dig> fragment and rearrangements which contains longer fragment of orf <dig> also likely occur.
the emergence of pcv <dig> molecular rearrangements challenges the validity of quantitative pcr diagnostic methods based on orf <dig>
CONCLUSIONS
in summary, this study established a method with high specificity, sensitivity and reproducibility to detect p <dig> based on sybr green i real-time quantitative pcr.
this method can determine p <dig> copy number even in the presence of pcv <dig> and p <dig> double positive samples, rendering it possible to monitor in vivo p <dig> replication status.
thus our study paved a way for studies of p <dig> infection and proliferation, and for the study of the pathogenesis and prevention of p <dig>  the emerging pcv <dig> molecular rearrangements have brought trouble to p <dig> and pcv <dig> quantification.
in future studies, amplification of full-length genome by conventional pcr followed by dna sequencing to exclude rearrangements should be applied to accurately quantify p <dig> and pcv <dig>
abbreviations
pcv2porcine circovirus type 2
p1porcine circovirus-like virus p1
prvpseudo-rabies virus
ppvporcine parvovirus
prrsvporcine reproductive and respiratory syndrome virus
pmwspostweaning multisystemic wasting syndrome
qpcrreal-time quantitative pcr detecting system
ctthreshold cycle
competing interests
the authors declare that they have no competing interests.
author’ contributions
conceived and designed the experiments: cpl.
performed the experiments: kwh lbw ysw.
wrote the paper: kwh.
all authors approved the final version of the manuscript to be published.
the study is funded by the national natural science foundation of china  and the fund for independent innovation of agricultural sciences in jiangsu province cx  <dig>

 porcine circovirus-like agent p is a newly discovered virus. p has a circular single-stranded genome of nucleotides, and it is the smallest dna virus known to date. the whole genome is highly homologous to the genome of porcine circovirus type with the exception of nucleotides. epidemiological investigation confirmed that the p virus has been epidemic in china’s pig farms. to further characterize the p virus, we constructed two clones, one with a single copy of p virus genome and one with a two copy tandem repeat. it was confirmed that the tandem repeat clone can produce viral particles in transfected pk cells, demonstrating that this clone has in vitro viral activity. virus particles are spherical in shape, non-enveloped with a diameter of approximately 25 nm. in vivo studies have shown that the tandem repeat p clone can infect pigs and the infected animals show symptoms resembling postweaning multisystemic wasting syndrome. clinical manifestations of p infection include pale skin, diarrhea and viremia. generally, lesions include brain hyperemia, bladder mucosal bleeding, inguinal lymph node bleeding, and lung atrophy and bleeding. histological examines identified diseases including subarachnoid vascular congestion, interstitial pneumonia, myocardial atrophy, tonsil tissue follicular cell hyperplasia, and necrosis of pancreatic cells. p nucleic acids and antigens were detected in tissue samples of lung, brain, heart, liver, bladder, pancreas and gonads. the main symptoms of pmws include wasting and stunting, and sometimes pale skin, jaundice, diarrhea and breathing difficulties. pmws has become one of the major epidemic diseases that endanger the world’s pig industry. pcv is considered a primary pathogen of pmws. pcv belongs to the virus family circoviridae, and is a small, non- enveloped, single-stranded dna virus with a genomic size of or nucleotides. there are three confirmed open reading frames encoded in the pcv genome. orf encodes the viral replication enzyme, orf encodes the viral capsid protein, and orf encodes a protein which is not essential for viral replication but has apoptotic activity. an early study suggested that the virulence of different pcv strains correlates with the amino acid sequences of its capsid protein. several studies followed that focused on orf and based on these studies pcv can be divided into different genotypes. recent studies suggest that pcv genotype is more virulent than genotype, however, there are discrepancies in the literature. the discrepancy is around the pathogenic characteristics of pcv as it is difficult to reproduce the typical symptoms of pmws with pcv alone. it is speculated that there may be another contributing factor in the occurrence of pmws. the discovery of p provides a new etiological explanation for pmws since the nucleotide sequence of the p genome is highly homologous with orf of pcv and the encoded capsid protein is also homologous to that of pcv the current conventional diagnostic techniques based on the orf of pcv may not be able to distinguish pcv from p therefore, it is important to establish a rapid, highly sensitive diagnostic method to detect pcv and p which will aid the prevention and treatment of porcine circovirus-like virus infections. real-time quantitative pcr has become the preferred method of quantitative detection of nucleic acids due to its’ high sensitivity, specificity and reproducibility as well as being rapid. among them, the sybr green i dye method does not require probe labeling and is highly cost effective and therefore has been more widely used. this study aims to establish an approach based on sybr green i dye real-time pcr to accurately and quantitatively detect p as well as distinguishing between p and pcv viruses in animals. methods ethics statement the use of animal specimens in this study was approved by the jiangsu province animal regulations. the protocol was approved by the committee on the ethics of animal experiments of the institute of veterinary medicine, jiangsu academy of agricultural sciences. virus isolation pseudo-rabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, porcine circovirus type and serum samples of p infected pigs were all isolated and stored in institute of veterinary medicine, jiangsu academy of agricultural sciences as previously described. primers a pair of inverse primers was designed according to the p sequence using the software oligo. the sequence of the forward primer is 5′- gagaggcgggtgttgaagat- ′ and the reverse primer are 5′- aagaccccccacttaaaccc - ′. the amplification product is l bp for p and 1238 bp or 1237 bp for pcv based on different genome size.fig. genomic characterization of pcv and p and position of the primers in both genomes. the high homologous sequences of pcv and p are shown in bold, red colour, the primers in both genomes are indicated by the arrowheads, and their nucleotide coordinates are marked in both genomes preparation of viral dna template prv, ppv, pcv infected cell culture samples and clinical samples were used to prepare viral dna samples. among them, the stool samples were diluted 1: with sterile phosphate buffer, subjected to three freeze-thaw cycles, centrifuged and then the supernatant was used for viral dna preparation. 300 μl of sample was mixed with 300 μl of cell lysis buffer and 10 μl of proteinase k, and then the sample was transferred to 50 °c to digest for 2 h. after digestion the sample was mixed with an equal volume of tris-saturated phenol and centrifuged at 12000 r/min for 10 min. the upper aqueous phase was transferred to a new  ml eppendorf tube and mixed with an equal volume of phenol:chloroform:isoamyl alcohol and centrifuged at 12000r/ min for 10 min. the upper aqueous phase was extracted with phenol: chloroform: isoamyl alcohol one more time and was transferred to a new  ml eppendorf tube. then the sample was mixed with volumes of ice-cold ethanol and 1/ volume of 3 mol/l sodium acetate and sit at room temperature for 20 ~ 30 min to precipitate the genomic dna. following centrifugation at 12000 r/min for 10 min, the supernatant was discarded and the precipitates were washed in 70 % ethanol and air dried. dna was dissolved in te buffer containing 20 μg/ml rnasea and stored at -20 °c for later use. preparation of viral rna template total rna was extracted from prrsv infected cells using trizol ls reagent according to the manufacturer’s instructions. reverse transcription was carried out immediately using the rna sample. the reaction mix consisted of  μl rna, 4 μl 5x buffer, 2 μl 10 mmol/l dntps mixture,  μl u/μl rnasin, 1 μl oligo and 1 μl u/μl amv reverse transcriptase. after incubation at 42 °c for 1- 2 h, the resulting cdna was stored at -20 °c for later use. development of fluorescence quantitative standard curve p genomic dna was extracted from serum specimens of infected pigs and used as template for a 50 μl pcr reaction. the pcr conditions consisted of an initial denaturation at 95 °c for 5 min, followed by amplification for 35 cycles of 30 s at 95 °c, 30s at 60 °c, and 30 s at 72°cfollowed by one cycle of 72 °c for 10 min. pcr product was separated on 2 % agarose gel by electrophoresis and recovered by agarose gel extraction. the dna fragment was cloned into pmd18-t vector and transformed into e. coli top the resulting recombinant plasmid was verified by pcr, restriction enzyme digestion and dna sequencing. plasmids with an od 260 nm/od 280 nm ratio between - were used to establish a standard curve. the concentration of the plasmids was calculated based on od260nm value and used to calculate copy numbers. six 10-fold serial dilutions of plasmid samples with copy number from to copies/μl were used as template to run reaction on the abi quantitative pcr machine to generate a standard curve. the reaction mixture is 25 μl and contains  μl ultrasybr mix,  μm forward and reverse primers each, and 2 μl template. the reaction cycles are as follows: 95 °c for 10 min followed by 40 cycles of 95 °c for 15 s, 60 °c for 30s. after the final extension reaction added was the melting curve analysis. after completion of the reaction, a standard curve is automatically generated by the abi software. analysis of specificity, sensitivity and reproducibility of the sybr i qpcr to test the specificity of the method, prv, ppv, pcv and p virus genomic dna and prrsv cdna are used as templates in sybr i qpcr according to the aforementioned reaction conditions. to test the sensitivity of the method, ten 10-fold serial dilutions of recombinant p dna with concentration from to copies/μl are used as templates to run real-time pcr. to test the reproducibility of the method, sybr i qpcr reactions are repeated three times using 1: serial dilutions of p plasmids with copy numbers of to copies/μl and the obtained ct values are compared. clinical samples sera samples and manure samples were tested in this study. all samples were obtained from pig farms located in jiangsu province, anhui province, beijing city and tianjin city, respectively. serum samples were collected in or from ~1 month old pigs with or without pmws. manure samples were collected in from piglets with severe diarrhea. the extracted dnas from the aforementioned clinical samples were used as templates for sybr i qpcr and also used as template for whole genome amplification of pcv and p by conventional pcr.  pcr amplification of p gene fragment and construction of recombinant plasmid p dna was used as the template for pcr amplification and an 119 bp dna fragment was obtained which is consistent with the size of the expected gene fragment. the obtained fragment was used to construct a recombinant plasmid which was verified by both pcr and restriction enzyme digestion analysis. then the plasmid was verified by dna sequencing. generation of the standard curve and establishment of the linear regression equation six serial dilutions of plasmid with copy number from to copies/μl were used as template to run sybr i qpcr. following the reaction the system software automatically generated the amplification curves, melting curves, the standard curve and linear regression equation. the amplification graph exhibited an s-shape fluorescence amplification curve. the melting curve displayed a single peak and was consistent tm values of approximately  °c. standard curve analysis demonstrated that when copy numbers are in the range of to copies/μl, the copy numbers of p correlate linearly with ct values with a slope of -, the efficiency of the qpcr reaction as  %, and the value of the correlation coefficient r of the linear regression equation as.fig. standard curve of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p plasmid standards. x -axis is 10-fold serial dilutions of the plasmid standards, y axis represents the threshold cycle values analysis of specificity, sensitivity and reproducibility of reaction sybr i qpcr reactions using the p plasmid as a standard gave rise to the corresponding product and produced a single melting peak, indicating no primer dimers or other non-specific products amplified. the final product was further confirmed by dna sequencing. reaction using pcv or p as template generated a melting curve with a sharp peak, while the melting curve of prv, ppv or prrsv was flat. although pcv gene also could be amplified, the melting temperature is approximately 75 °c which is different from that of p thus the method has enough specificity. ten 10-fold serial dilutions of p with concentrations from to copies/μl were used as templates to run sybr i qpcr the results showed that even at the low concentration of copies/μl, fluorescent signal could still be detected, indicating a high sensitivity and the concentration of templates correlates well with ct values. a ct value of was chosen as a cut-off value for standard positivity. tests were repeated using the serial dilutions and further statistical analysis showed that the coefficient of variation within groups and between groups was less than 5 %, indicating good stability and reproducibility.fig. specificity test of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p p1; pcv2; prv; prrsv; and ppv fig. sensitivity test of sybr green i fluorescence quantitative pcr detection of porcine circovirus-like agent p plasmid standard was used as template, x -axis is 10-fold serial dilutions of the plasmid standards, y axis represents the threshold cycle values 107 106 105 104 results of clinical sample analysis by fluorescence-based quantitative pcr analysis out of serum samples and all six stool samples were identified as p positive. p virus titers of the majority of samples were between 104- copies/ml with one manure sample up to copies/ml. the results are shown in table  out of serum samples, and serum samples from healthy and pmws pigs were postive for p respectively. the amount of p in healthy pigs were generally below p concentrations of genome copies/ml of total dna, while the p concentrations in pmws pigs were consistently higher than p dna copies/ml of total dna.table p gene copy numbers of serum and fecal samples measured by quantitative pcr 103 the results of conventional pcr and quantitative pcr analysis of serum and manure samples were compared in table  when applied to detection of pcv in the serum, conventional pcr identified samples as pcv positive compared to the samples by quantitative pcr. neither conventional pcr nor quantitative pcr detected pcv in fecal samples. conventional pcr can detect serum samples as p positive compared to the samples by quantitative pcr. quantitative pcr detected fecal samples as p positive compared to the samples by conventional pcr. the quantitative pcr failed to detect the sixteen double positives found by conventional pcr. collectively the results demonstrate that when the p gene copy number is less than copies/ml, it is difficult to use conventional pcr to detect.table comparison of results obtained by quantitative pcr and conventional pcr discussion circoviridae is a family of single-stranded dna viruses with a small genome size that can infect both poultry and pig. two plant virus families, nanoviridae and geminiviridae, are considered to be closely related to circoviridae. they all contain a stem-loop structure which encompasses a replication origin. gibbs and weiller, based on bioinformatics results of the rep protein amino acid sequence of circovirus and nanoviridae, provided a hypothesis of the origin and evolution of circovirus. in their study, they found that the n-terminal amino acid sequence of rep in circovirus is homologous to that of nanoviridae while the c-terminal amino acid sequence of the pcv rep is closely related to rna binding proteins 2c of vertebrate calicivirus. therefore, they speculated that pcv is a combination of nanoviridae and calicivirus. since calicivirus is a rna virus, the recombinant must involve a retrovirus or a retrotransposon. although the international committee on taxonomy of viruses has not further classified viruses belonging to the same species, pcv could be divided into two major genotypes, namely pcv2a and pcv2b. hesse et al. reported a pcv strain which contains a recombination of pcv2a orf and pcv2b orf. thereafter, pcv2a/b recombinants have been frequently isolated. we identified porcine circovirus agent p during a study of pcv and suggested it may be a result of recombination of pcv with other molecules, likely with pcv because it also has circular single-stranded genome of nucleotides. the 648 bp genome of p is the smallest dna viral genome identified to date. its genome is comprised of a dna sequence highly homologous to pcv orf as well as exogenous sequences the exogenous sequence of p is nucleotides in size, and such a short fragment can be found in many species when a blast search was run to identify homologous sequence in genbank, thus making it difficult to trace its origin. we can only speculate that p may be a product of a recombination between retrovirus and pcv p infection of pigs can generate symptoms resembling pmws, but these symptoms are different from symptoms of pcv2-associated pmws in terms of anatomical and pathologic traits as well as viral distribution in tissues. the investigation of virus replication and pathological mechanism requires quantitative determination of virus copy numbers. the p genome has nucleotides, and except ~ nucleotides, its sequence is highly homologous to pcv orf which contains nucleotides. this indicates that the p genome is almost completely overlapped with pcv orf and it is difficult for conventional pcr to distinguish between p and pcv in this study we designed a pair of primers which gave rise to a p pcr product of 119 bp and a pcv pcr product of bp. thus, pcv will inevitably be amplified when using optimized quantitative pcr to detect p however its amplification efficiency and melting temperature are distinctively different from those of p providing a base to distinguish between the two viruses. the specificity of the sybr i qpcr method was further confirmed by the fact that the study identified less positive samples than conventional pcr when applied to pcv test, as well as that it preferred to amplify p genome when dna template contains both p and pcv there are two commonly used quantitative real-time pcr techniques used, namely sybr green dye method and taqman probe method. sybr green i has advantages over the taqman technique; it avoids the use of fluorescent-labeled probes and other expensive reagents, and it is applicable to any pcr amplification system and can recognize primer dimers and nonspecific amplifications by melting curve analysis. in this study a method to detect p based on sybr i qpcr was successfully developed and the results show this approach is able to detect virus samples of a wide range of concentrations. when virus concentration falls between and copies/μl, pcr results correlate linearly with virus concentrations. with a high sensitivity the minimum virus concentration this method is able to detect is < copies/μl. this test method has no cross-reactivity with ppv, prrsv, and prv viruses and can distinguish pcv by melting curve analysis despite the fact that a small amount of pcv amplifications can also be detected, thus manifesting a high specificity. all results obtained by this method were very reproducible and the results of porcine serum samples showed that it can be easily applied to clinical analysis. even some healthy pigs were found to be p positive. and their serum p copy numbers were with the majority of them falling in between and copies/ml. since p infection can cause pmws-like symptoms to some pigs, the relationship between p copy numbers and symptoms needs further investigation. in 2010- a serious infectious diarrhea disease affected a large number of pig farms in china and caused high rates of morbidity and mortality. no antibiotics or vaccines are effective to this disease and the economic loss was very significant. a new strain of porcine epidemic diarrhea virus was found to be the major pathogen, but porcine bocavirus and crest virus were also detected in clinical specimens. in this study high concentration of p virus was identified in all six stool samples obtained from diarrhea pigs, suggesting that p may play a role in this epidemic of diarrhea. in previous studies using real-time quantitative pcr assay to detect pcv either dye method or probe method, orf gene was selected as the target. this method cannot accurately measure pcv concentration under circumstances of p and pcv double infection. p can only be distinguished when pcv orf is selected as the assay target. recent studies have shown that pcv can form intramolecular rearrangements of variable size which also have circular single-stranded genome consisting of nucleotide sequences derived entirely from the parent strains. these molecules do not contain exogenous nucleotides and their cleavage sites are different from those of pcv. some rearrangements contain a part of orf fragment and rearrangements which contains longer fragment of orf also likely occur. the emergence of pcv molecular rearrangements challenges the validity of quantitative pcr diagnostic methods based on orf  in summary, this study established a method with high specificity, sensitivity and reproducibility to detect p based on sybr green i real-time quantitative pcr. this method can determine p copy number even in the presence of pcv and p double positive samples, rendering it possible to monitor in vivo p replication status. thus our study paved a way for studies of p infection and proliferation, and for the study of the pathogenesis and prevention of p the emerging pcv molecular rearrangements have brought trouble to p and pcv quantification. in future studies, amplification of full-length genome by conventional pcr followed by dna sequencing to exclude rearrangements should be applied to accurately quantify p and pcv abbreviations pcv2porcine circovirus type 2 p1porcine circovirus-like virus p1 prvpseudo-rabies virus ppvporcine parvovirus prrsvporcine reproductive and respiratory syndrome virus pmwspostweaning multisystemic wasting syndrome qpcrreal-time quantitative pcr detecting system ctthreshold cycle competing interests the authors declare that they have no competing interests. author’ contributions conceived and designed the experiments: cpl. performed the experiments: kwh lbw ysw. wrote the paper: kwh. all authors approved the final version of the manuscript to be published. the study is funded by the national natural science foundation of china and the fund for independent innovation of agricultural sciences in jiangsu province cx 
this method doesn’t detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type from p by melting temperature analysis. a pair of primers was designed and used to amplify a 119 bp dna fragment to generate a recombinant plasmid which was served as the standard. a sybr i qpcr protocol was established using the p recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. the results demonstrate a strong correlation with p recombinant plasmid titers when virus dna copy numbers fall in between 100 ~  copies/μl. this study aims to develop a rapid, sensitive and specific method to detect p the porcine circovirus-like agent p is a newly discovered dna virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type p infection can cause symptoms resembling postweaning multisystemic wasting syndrome.
 the porcine circovirus-like agent p is a newly discovered dna virus with a single-stranded circular genome that is highly homologous to that of porcine circovirus type p infection can cause symptoms resembling postweaning multisystemic wasting syndrome. this study aims to develop a rapid, sensitive and specific method to detect p  a pair of primers was designed and used to amplify a 119 bp dna fragment to generate a recombinant plasmid which was served as the standard. a sybr i qpcr protocol was established using the p recombinant plasmid standard and the sensitivity, specificity and stability of this method was analyzed. the results demonstrate a strong correlation with p recombinant plasmid titers when virus dna copy numbers fall in between 100 ~  copies/μl. this method doesn’t detect pseudo rabies, porcine parvovirus or porcine reproductive and respiratory syndrome virus; moreover it can distinguish porcine circovirus type from p by melting temperature analysis. coefficient of variation for each batch of reaction is less than 5 %. the serum virus titers of p positive in this study were measured by this protocol to be to copies/ml.  the established qpcr is sensitive, specific, and reliable, which could be a useful tool when applied to quantification of p in a variety of samples from infected pigs. keywords porcine circovirus-like virus p1pcv2fluorescence quantitative pcrmelting curveissue-copyright-statement© the author 2015 