Sage Journals: Discover world-class research

Abstract

Porcine circovirus-2 (PCV-2) is considered the major etiological agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. The clinical manifestations of the disease are correlated with moderate to high amounts of PCV-2 DNA in biological samples of affected pigs. A threshold of 10⁷ DNA copies/ml is suggested as the trigger factor for symptoms. A comparative study was conducted to determine which quantitative method would be more suitable to estimate the PCV-2 DNA load. Two polymerase chain reaction (PCR) assays were developed: a competitive PCR (cPCR) and a SYBR Green–based real-time PCR. The assays were compared for their capacity to detect PCV-2 in DNA samples extracted from liver, lung, spleen, mesenteric lymph nodes, and kidney of PMWS-affected (n = 23) or non–PMWS-affected pigs (n = 9). Both assays could successfully quantify PCV-2 DNA in all tissue samples and were able to detect significant differences between the numbers of PCV-2 DNA copies found in tissues of PMWS-affected and non–PMWS-affected pigs (≥10^2.5). The highest mean viral loads were detected by the SYBR Green real-time PCR, up to 10^7.0±1.5 copies/100 ng of total DNA sample, while the cPCR detected up to 10^4.8±1.5. A mean difference of 10^1.8 was found between the amounts of PCV-2 DNA detected, using the SYBR Green real-time PCR and the cPCR, suggesting that the viral load threshold for PMWS should be determined for each particular assay.

Keywords

Competitive polymerase chain reaction DNA viral load Porcine circovirus-2 real-time polymerase chain reaction

Introduction

Porcine circovirus-2 (PCV-2; family Circoviridae, genus Circovirus) is distributed worldwide in swine herds and is an important cause of economic losses for the pig industry.⁶ The virus is regarded as the major infectious agent involved in the development of post-weaning multisystemic wasting syndrome (PMWS).^1,9,20 However, PMWS is a multifactorial disease where PCV-2 is necessary but not sufficient for its development.¹³ Typical PMWS signs include progressive weight loss, signs of respiratory impairment, and jaundice (Clark EG: 1997, Post-weaning wasting syndrome. In: Proceedings of the American Association of Swine Practitioners 28:499–501). Macroscopic lesions include granulomatous interstitial pneumonia, lymphadenopathy, and granulomatous hepatitis and nephritis.² Microscopically, PMWS is characterized by macrophage infiltration, syncytia formation, and inclusion bodies in cells of lymphoid tissues.²⁶ PCV-2 is also associated with other pathological conditions, including congenital tremors,³⁰ respiratory disease,¹⁴ porcine dermatitis and nephropathy syndrome (PDNS),³ abortions,³³ and other reproductive disorders.²¹ Diseases associated with PCV-2 are collectively termed porcine circovirus diseases (PCVD),²⁸ or porcine circovirus–associated disease (PCVAD).²² Healthy pigs can also be infected with PCV-2, but such pigs will bear lower viral loads than diseased animals. The sole presence of anti–PCV-2 antibodies and/or PCV-2 DNA is not sufficient to establish a definitive PMWS diagnosis, which is usually performed by analyzing data from clinical signs, histopathology, and detection of PCV-2 within tissue lesions.^6,28

Among the methods available to detect viral DNA, a substantial number of assays have been described based on variations of the polymerase chain reaction (PCR),^16,23,35 multiply primed rolling-circle amplification,⁸ loop-mediated isothermal amplification,⁷ and microarray analyses.¹² However, as PCV-2 is ubiquitously present in swine regardless of disease status, methods based on qualitative PCR are not appropriate for the establishment of a definitive PMWS diagnosis. In view of that, different types of quantitative PCR assays have been developed to measure the amount of PCV-2 DNA in animals, including a competitive PCR (cPCR)¹⁷ and several real-time PCR–based assays.^{5,19,29,32,34} In cPCR, the target DNA and an internal standard, the so-called competitor, are simultaneously amplified; the 2 fragments must be recognized by the same pair of primers.³⁶ The initial amount of target DNA is obtained by a comparison between the intensity of the bands of the target DNA and the competing DNA upon gel electrophoresis.³⁶ On the other hand, real-time PCR–based assays generate a fluorescent signal during the amplification cycle, which is directly correlated with the amount of target DNA molecules as well as inversely with the number of PCR cycles needed to reach a fluorescent signal threshold.¹⁰

In the present study, a cPCR and a SYBR Green–based real-time PCR were developed and evaluated to establish the most suitable method to determine the amount of PCV-2 DNA in swine tissue samples from PMWS-affected or healthy pigs, in an attempt to contribute to the diagnosis of PMWS.

Material and methods

Tissue samples

Tissue samples were collected from 23 PMWS-affected (PMWS group) and 9 healthy, non–PMWS-affected (non-PMWS group) pigs. The 5–8-week-old pigs of the PMWS group were obtained from pig farms in the state of Rio Grande do Sul, Brazil. At arrival, the pigs were showing clinical signs of PMWS, such as wasting, dyspnea, enlargement of superficial inguinal lymph nodes, pallor, jaundice, and diarrhea. These animals were subjected to necropsy following the general recommendations of COBEA (Brazilian Committee on Experimental Animal Care). Histopathological and immunohistochemical tests of tissues from animals in this group confirmed the PMWS diagnosis. The healthy, non–PMWS-affected group consisted of samples collected from finishing-age pigs (16–18 weeks old) randomly chosen in a slaughterhouse. Samples were collected from kidneys, livers, lungs, mesenteric lymph nodes, and spleens from all animals. The samples were stored at −70°C until used.

DNA extraction

The DNA extraction method was performed as described elsewhere,³¹ with minor modifications, as follows. Ten milligrams of tissue were minced and digested for 4 hr at 37°C in 1 ml of lysis buffer (10 mM Tris,^a 1 mM ethylenediamine tetra-acetic acid [EDTA],^b 100 mM NaCl^c) containing 0.5% sodium dodecyl sulfate^d and 0.1 mg proteinase K.^e The DNA was extracted twice with 1 ml phenol^e:chloroform^f:isoamyl alcohol^f (25:24:1). After centrifugation, 2 volumes of 100% ethanol^f were added to the water phase, and the mixture was kept at −20°C for 2 hr. After this, the mixture was centrifuged, and the obtained DNA pellet was washed with 70% ethanol,^f air-dried, and re-suspended in 100 µl of TE (10 mM Tris,^a 1 mM EDTA,^b pH 7.4).

Oligonucleotides used in the PCR assays

The nucleotide sequences of the primers used for the cPCR were based on a previously published sequence used in a qualitative PCV-2 PCR.¹⁵ The primers used were 1094F (5’-CGGATATTGTAGTCCTGG TCG-3’) and 1569R (5’-ACTGTCAAGGCTACCACAG TCA-3’). These oligonucleotides anneal within the open reading frame-2 region of the PCV-2 genome at nucleotides 1094–1114 (numbers based on PCV-2 genome 15/5P⁸ of GenBank accession no. DQ923523) and at nucleotides 1548–1569, respectively. Amplification with such primers should give rise to an amplicon with an expected size of 476 base pairs (bp).

The primers used for the SYBR Green real-time PCR were designed using PrimerSelect software^g and were chosen from within the region amplified by the cPCR (Fig. 1A). The designed primers were 1127F (5’-TCGAACGCAGT GCCGAG-3’) and 1190R (5’-AGCTGTGGCTGAGACTAC AAACT-3’), and were complementary to nucleotides 1027–1043 and 1190–1168 of the PCV-2 15/5P genome (GenBank accession no. DQ923523). The primers were expected to amplify a fragment of 64 bp and were different from the ones used for the cPCR because the maximum recommended size of an amplicon for a real-time PCR is 200 bp.²⁴

Figure 1.

Positions of the primers used in the current study in relation to nucleotide sequence of Porcine circovirus-2 (PCV-2) isolate 15/5P. A, the sequence of the 476-bp amplicon of PCV-2 DNA with the positions of primers 1094F and 1569R (underlined) used in competitive polymerase chain reaction (cPCR) and the positions of primers 1127F and 1190R (dashed underlined) used in SYBR Green real-time PCR. The position of the Stu I restriction enzyme site is indicated in bold. B, the sequence of the additional 96-bp fragment was inserted into the Stu I site of the 476-bp amplicon to create a competitive DNA template (p572) for cPCR. The positions of the primers (1672F and 1767R) that were used to obtain this 96-bp fragment are double underlined.

Competitive PCR

The cPCR reactions were performed using a fixed amount of DNA (100 ng) extracted from the tissue samples to be examined, plus a variable amount of competitor plasmid p572 (see below). The 25-µl reaction volume contained 0.2 mM of each deoxyribonucleotide triphosphate,^h 3 mM MgCl₂,^e 10 pmol of each primer (1094F and 1569R), 1 U of Taq polymerase,^e 100 ng of DNA sample, and 2 µl of competitor plasmid DNA p572. For each tested sample, 11 reactions were assembled using one of the p572 diluted DNA, which ranged from 10¹⁰ to 10⁰ DNA copies added per tube. A negative control sample was added between each 4 tissue samples under testing, where test DNA was substituted by 100 ng DNA extracted from PK-15 (porcine kidney epithelial) cells.ⁱ The reaction was performed as follows: initial incubation at 94°C for 4 min, 35 cycles at 94°C for 30 sec, 65°C for 1 min, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. Five microliters of the amplification products were analyzed on 1.5% agarose gel. The equivalence point, at which the bands of the target DNA (476 bp) and competitor (572 bp) had equal intensities, were determined on agarose gel by visual inspection and densitometric scanning using the KDS software.^j

Construction of a competing template

Using the cPCR protocol described above, an amplicon with the expected size (476 bp) was produced by amplification of DNA from the PCV-2 isolate 15/5P. The obtained amplicon was ligated into the cloning vector pCR 2.1-TOPO^eand used to transform competent Escherichia coli cells. The resulting plasmid was named p476. Plasmid DNA was isolated from an overnight culture of transformed E. coli cells using standard methods.²⁷ To construct the competitor template that would generate a slightly larger amplicon, p476 was cleaved with Stu I,^k and a 96-bp fragment was inserted into the site, resulting in plasmid p572. The 96-bp fragment was obtained through PCR using primers 1672F (5’-CTGGCCAAGATGGCTGCG-3’) and 1767R (5’-AATACTTACAGCGCACTT-3’), and the 15/5P PCV-2 isolate DNA as a template (Fig. 1B).

Quantitation of plasmid DNA copy numbers and co-amplification of PCV-2 DNA with competitor DNA

DNA from plasmids p476 and p572 was quantified in an ethidium bromide–stained agarose gel. The amount of plasmid DNA was determined by comparing the intensity of the obtained bands with those of the mass DNA ladder^k (0.75 µg/lane) performed with the aid of the KDS software.^j The number of plasmid DNA copies were calculated assuming an average molecular mass of 660 Da for each base pair of double-stranded DNA. The calculation was performed using the following equation: number of DNA copies per nanogram = (N_A × 10^-9)/(n × mw), where N_A is the Avogadro constant (6.02 × 10²³ molecules/mol), n is the size of the plasmid in base pairs (4407 bp for p476 or 4503 bp for p572), and mw is the molecular weight per bp (660 Da). Plasmid DNA was then diluted in DNAse-free water^e to a final concentration of 10¹⁰ molecules/2 µl, and 10-fold serially diluted. The dilutions were used with both the cPCR (p572) and the SYBR Green real-time PCR (p476).

To test whether the insertion of 96 bp within the PCV-2 target sequence changed the amplification performance of p572, 10-fold dilutions of equal amounts of the target DNA (p476) and competing DNA (p572) were co-amplified using the cPCR protocol mentioned above.

SYBR Green real-time PCR

The SYBR Green real-time PCR reactions were set up with 10 µl of SYBR Green PCR master mix,^l 5 pmol of each primer (1127F and 1190R), and 100 ng DNA sample in a total volume of 20 µl. As a negative control, 100 ng of DNA extracted from uninfected PK-15 cells were used. All real-time reactions (standards, test samples, and controls) were performed in duplicate. The reported results are an average of these duplicates. The SYBR Green real-time PCR were performed in a commercial system^l employing the following cycling conditions: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 sec at 94°C and 45 sec at 60°C. A dissociation curve was performed after amplification by a gradual rise in temperature from 60 to 95°C. Data analysis was performed with the aid of the SDS software 1.3.1.^l The number of copies of viral DNA was determined by comparison with a standard curve (see below).

Standard curve for the SYBR Green real-time PCR

To create a standard curve for the SYBR Green real-time PCR, 10-fold dilutions (10¹⁰–10⁰) of plasmid p476 were used as target DNA templates. The standard curve was constructed by plotting the plasmid copy number on a log₁₀ scale against the measured threshold cycle (Ct) values. One hundred nanograms of total DNA extracted from PK-15 cellsⁱ were used as negative control. The reactions were carried out as described above.

Specificity and sensitivity of cPCR and SYBR Green real-time PCR

The specificity of cPCR and SYBR Green real-time PCR were studied with respect to Porcine circovirus-1 (PCV-1). Duplicates of 100 ng total DNA of PK-15 cells persistently infected with PCV-1 were analyzed under the optimal conditions of the assays as determined above. SYBR Green real-time PCR and cPCR sensitivity was tested in triplicates using 10-fold dilutions (10¹⁰–10⁰ copies/reaction) of p476 plasmid DNA as standard and 100 ng of DNA extracted from PCV-2–free tissue samples.

Statistical analysis

In order to normalize the acquired data for statistical analysis, the number of copies of PCV-2 DNA obtained by the 2 PCR assays under comparison was transformed in log₁₀. Descriptive statistics, Pearson correlation coefficient, and analysis of variance were performed using the SPSS 16.0 software^m; differences were considered significant when P ≤ 0.05. In order to determine the agreement between the 2 assays, the Bland–Altman plot was constructed based on differences of PCV-2 copy numbers (SYBR Green real-time minus cPCR) against their means.⁴ The MedCalc softwareⁿ (version 11.5.0.0) was used for this analysis, and the significance was accepted at P ≤ 0.05.

Results

Co-amplification of PCV-2 DNA with competitor DNA

The amplification of the competitor template produced a band of predicted size (572 bp), which was clearly distinguishable from the 476-bp viral amplicon (Fig. 2). The efficiency of amplification was equivalent for both target and competitor DNA, because over the range of 10¹⁰–10¹ DNA copies, the intensities of the amplified bands were the same for the p476 and p572 templates (data not shown).

Figure 2.

Electrophoretic analysis of the products of the competitive polymerase chain reaction (cPCR) on 1.5% agarose gel stained with ethidium bromide. Lane M: a 100-bp ladder,^o the left arrow points to the 500-bp fragment; lanes 1–5: cPCR products from reactions performed on 100 ng of tissue DNA plus variable amounts (10⁶–10² molecules) of competitor plasmid (p572) DNA; lane 3: equivalence point—10⁴ molecules of PCV-2 DNA in the tissue sample and 10⁴ molecules of competitor plasmid p572; lane 6: negative control (100 ng of PK-15 DNA).

Specificity and sensitivity of the cPCR and SYBR Green real-time PCR

Neither a specific band with the cPCR (Fig. 2, lane 6) nor any significant fluorescence signal could be detected by SYBR Green real-time PCR when PCV-1 DNA sample was used. Negative controls did not give rise to any amplification product; therefore, no peaks were observed on the dissociation curve. The observed temperature of dissociation of the 64-bp amplicon occurred between 79.3°C and 80.3°C (Fig. 3B). Nevertheless, the PK-15 cells used as PCV-2–negative control were indeed positive for PCV-1, as demonstrated with a primer pair specifically designed for PCV-1¹⁵ amplification in a qualitative PCR (data not shown). On the sensitivity assays, SYBR Green real-time PCR and cPCR showed similar sensitivity, detecting from 10¹⁰ to 10¹ copies of plasmid DNA per reaction.

Figure 3.

A, standard curve constructed based on the amount of Porcine circovirus-2 (PCV-2) DNA copies versus the threshold cycle (Ct) obtained on SYBR Green real-time polymerase chain reaction (PCR). A 10-fold dilution series of plasmid p476 was used as PCV-2 DNA template. Each point in the graph represents the mean Ct value of a duplicate measurement. The amounts of p476 molecules are given on a log₁₀ scale. B, dissociation curves of the SYBR Green real-time PCR products of PCV-2–positive samples.

Standard curve for the SYBR Green real-time PCR

The generated standard curve covered a linear range of 10¹–10¹⁰ p472 DNA copies (Fig. 3A), and the value of the linear regression (R²) calculated between DNA copy number and Ct value was 0.993. The detection limit found on this assay was 10 copies of PCV-2 DNA per sample.

Determination of viral load

The average PCV-2 DNA load detected by cPCR in the PMWS-affected group ranged from 10^3.9 to 10^4.8 per 100 ng of total DNA (Fig. 4A). This was significantly higher than the viral load measured on tissues samples of the non–PMWS-affected group (10^0.22–10^0.57 copies/100 ng of sample DNA; P ≤ 0.05). No statistically significant differences were found on amounts of PCV-2 DNA detected in different types of tissues. None of the tissue samples from the non–PMWS-affected pigs had PCV-2 DNA loads greater than 10² copies per 100 ng DNA sample, as determined by cPCR.

Figure 4.

Mean values and standard deviations of the number of Porcine circovirus-2 (PCV-2) DNA molecules detected by competitive polymerase chain reaction (cPCR; A) and the SYBR Green real-time PCR (B) in 5 different tissues from either postweaning multisystemic wasting syndrome (PMWS) pigs (n = 23) or non-PMWS pigs (n = 9). The data are given in log₁₀ scales.

Results obtained with the SYBR Green real-time PCR showed that, in the PMWS-affected group, the average PCV-2 DNA loads were significantly higher (10^6.96 PCV-2 DNA copies/100 ng DNA sample) than in the non-PMWS group (10^0.92 PCV-2 DNA copies/100 ng DNA sample; Fig. 4B). Within the groups, no statistical differences were detected among the values of PCV-2 DNA copies found in the different types of tissues. An exception was the mean PCV-2 DNA load detected in the spleens of the non-PMWS group, where PCV-2 DNA concentration was significantly higher than in the mesenteric lymph nodes (P ≤ 0.05). None of the sample from the non–PMWS-affected group had a PCV-2 DNA load higher than 10^4.4 copies per 100 ng of DNA sample.

Comparison of the PCR-based assays

A strongly positive correlation (0.77) was found between the assays, as determined by Pearson correlation coefficient. On the Bland–Altman plot, the results showed that the viral loads detected from SYBR Green real-time PCR were significantly higher than those from cPCR. The mean difference between the assays was 10^1.8 PCV-2 DNA copies per 100 ng of DNA sample, with limits of agreement being 10^-2.0–10^5.6 (Fig. 5). The prevalence of PCV-2 DNA on the sample was analyzed, and approximately 90% of the samples were positive or negative in both assays (Table 1). Eleven out of the 160 samples analyzed were positive for PCV-2 in SYBR Green real-time PCR but negative in cPCR, whereas the opposite was observed on 4 samples.

Figure 5.

Bland–Altman plot of the differences between Porcine circovirus-2 DNA loads detected by and competitive polymerase chain reaction (cPCR) and SYBR Green real-time PCR. The solid line indicates the mean difference among the paired measurements, and the dashed lines indicate a 95% limit of agreement.

Table 1.

Prevalence and viral loads of Porcine circovirus-2 DNA detected on swine tissue samples using SYBR Green real-time PCR or competitive polymerase chain reaction (cPCR).*

	No. of samples	cPCR (mean ± SD.log₁₀)	SYBR Green real-time (mean ± SD.log₁₀)
Positive results in both assays	128 (79.4)	4.1 ± 1.63	6.35 ± 2.07
Positive results only in cPCR	4 (2.5)	1.0 ± 0.0	Negative
Negative results only in cPCR	11 (6.9)	Negative	2.13 ± 0.93
Negative results in both assays	18 (11.2)	Negative	Negative

Numbers in parentheses are percentages.

Discussion

In the current study, a cPCR and SYBR Green real-time PCR were developed and compared to establish which of the 2 tests would be more appropriate to quantify PCV-2 DNA in PMWS-suspected pigs. Both methods examined were able to detect significant differences between the viral loads in tissues of PMWS-affected and non-affected pigs. However, the amounts of PCV-2 DNA detected by the cPCR were 10^1.8 lower than those detected by the SYBR Green real-time PCR. A possible explanation for the difference might be the degree of similarity of the putative binding sites of the primers and the actual nucleotide sequences of the isolates. In comparing 587 PCV-2 genome sequences deposited in GenBank with the primers used with the cPCR (1569R and 1094F)¹⁵ assay, most of the PCV-2 genome sequences display up to 3 mismatches in the binding site for primer 1569R and up to 1 mismatch for primer 1094F. In contrast, the binding sites for the primers developed for the SYBR Green real-time PCR, 1190R and 1127F, were much more conserved; most of the PCV-2 genome sequences displayed up to 1 mismatch for 1190R and no mismatch for 1127F.

Mismatches might alter the amplification performance, resulting in the detection of lower amounts of DNA.²⁵ It has been shown that 5 mismatches in primers and probe resulted in an underestimation of copy numbers by 1 log₁₀ unit, while a total of 6 mismatches resulted in 2 log₁₀ units underestimation of the copy numbers.⁵ A 3–5 log₁₀ difference between perfectly matched primers and 3 mismatches in one primer has been reported in quantitative PCR.¹⁸ Another possible explanation for the different sensitivity detected between the assays is that clinical samples contain contaminants that affect cPCR more than SYBR Green real-time PCR. However, this explanation is unlikely, as the amplification of the competitor DNA was not affected.

In the cPCR developed in the current study, according to the tissue sample analyzed, the detected average varied from 10^>3.9 to 10^4.8 PCV-2 DNA copies per 100 ng DNA sample in the PMWS group. A similar cPCR-based test detected an average of 10^6.6 copies of PCV-2 DNA in 1 ml of serum from PMWS-affected pigs,¹⁷ which is equivalent to 10^5.3 copies per100 ng DNA sample. Therefore, the detected viral load of the cPCR employed in the present study was approximately 3–25-fold lower than that achieved in a prior study.¹⁷ This difference between the 2 cPCR assays may be due, inter alia, to different sensitivities of the methods, the stage of the disease, or the type of sample used in the assays.

SYBR Green real-time PCR detected higher mean amounts of PCV-2 DNA in PMWS-affected (≤10⁷) than in non–PMWS-affected pigs (≤10^2.3). The differences between groups varied from 2.5 to 4 log₁₀ units, depending on the tissue samples examined (see Fig. 4B). Using a SYBR Green–based real-time PCR^19,34 or a TaqMan-based real-time PCR,⁵ other authors detected similar viral loads to those reported in the current study with the real-time PCR. A statistically significant difference was found among spleen and mesenteric lymph node from the non-PMWS group, with higher viral loads being detected in the spleen samples. This result suggests that mesenteric lymph node may not be the appropriate sample when monitoring PCV-2 levels in swine herds.

It has been proposed that a threshold of 10⁷ PCV-2 DNA copies per ml serum or 2 × 10⁶ copies per 100 ng DNA sample would be suggestive of PMWS.⁵ However, a study comparing 2 routinely used real-time PCR assays found a bias of 10^1.4 on the PCV-2 DNA copies detected between the assays.¹¹ These findings revealed that the results of viral load among assays could vary, suggesting that the establishment of the viral load threshold to characterize PMWS should be determined by each particular assay. Variation in the detected PCV load was also observed in the quantitative PCR assays used in the current study. Based on the highest viral load detected in the non-PMWS group in the current study, the suggested viral load thresholds for clinically detectable PMWS are 10² and 10^4.4 copies of PCV-2 DNA per 100 ng DNA (or 10^3.3 and 10^5.7 copies of PCV-2 DNA/500 ng DNA), for cPCR and SYBR Green real-time PCR, respectively.

Other authors have observed that the mean PCV-2 concentration (per gram or ml) was 10³ higher in naturally PWMS- affected versus non–PWMS-affected pigs.¹⁹ Therefore, another approach in support of a PMWS diagnosis is to compare DNA viral loads found on samples of PMWS-affected and non–PMWS-affected pigs. Using such criterion, both described assays would allow proper diagnosis, since both methods detected differences of at least 10^2.5 per 100 ng DNA.

With respect to the decision about which of the 2 assays would be more suitable to support a PMWS diagnosis, SYBR Green real-time PCR has a number of practical advantages.³⁶ This type of assay is less prone to false-positive results in comparison to cPCR because the risk of contamination is less, as post-PCR manipulation is unnecessary. As well, real-time PCR is less time consuming than cPCR, since no competitive DNA template is necessary, and multiple reaction dilutions as well as electrophoresis analyses are not needed. In addition, SYBR Green real-time PCR was more sensitive than cPCR, indicating that this method amplified the target DNA more efficiently, probably because more conserved primers were used.

In summary, both cPCR and SYBR Green real-time PCR can be applied to discriminate between PMWS-affected and non–PMWS-affected pigs. Either a threshold value for the PCV-2 DNA load or the difference in PCV-2 DNA load between PMWS-affected and non–PMWS-affected pigs could be used for diagnostic purposes. The difference between the assays may not be inherent to the cPCR method and could probably be solved by the use of more conserved primers. Although both methods may thus be used to support a PMWS diagnosis, SYBR Green real-time PCR is preferable because of a number of practical advantages and because it was able to detect higher amounts of PCV-2 DNA copies.

Footnotes

Acknowledgements

The authors thank Dr. David Driemeier, from the Veterinary Faculty, Federal University of Rio Grande do Sul (UFRGS), for providing tissue samples of swine with PMWS. To FAMR, in memoriam.

a.

Gibco BRL, Grand Island, NY.

b.

Sigma-Aldrich, St. Louis, MO.

c.

Vetec, Duque de Caxias, Rio de Janeiro, Brazil.

d.

Promega Corp., Madison, WI.

e.

Invitrogen Corp., Carlsbad, CA.

f.

Synth, Diadema, São Paulo, Brazil.

g.

DNASTAR Inc., Madison, WI.

h.

GE Life Sciences, Piscataway, NJ.

i.

CCL-33 American Type Culture Collection, Manassas, VA.

j.

Eastman Kodak Company, Rochester, NY.

k.

New England Biolabs, Ipswich, MA.

l.

7500 Real-Time PCR System, Applied Biosystems, Foster City, CA.

m.

SPSS Inc., Chicago, IL.

n.

MedCalc, Mariakerke, Belgium.

o.

Fermentas Life Science, Glen Burnie, MD.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Financial support was provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by the Financiadora de Estudos e Projetos (FINEP). D. Dezen was in receipt of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). P.M. Roehe is a CNPq 1B research fellow.

References

Allan

Meehan

Todd

.: 1998, Novel porcine circoviruses from pigs with wasting disease syndromes. Vet Rec 142:467–468.

Allan

McNeilly

Kennedy

.: 1998, Isolation of porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet Diagn Invest 10:3–10.

Allan

McNeilly

Kennedy

.: 2000, PCV-2-associated PDNS in Northern Ireland in 1990. Porcine dermatitis and nephropathy syndrome. Vet Rec 146:711–712.

Bland

Altman

: 1986, Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307–310.

Brunborg

Moldal

Jonassen

: 2004, Quantitation of porcine circovirus type 2 isolated from serum/plasma and tissue samples of healthy pigs and pigs with postweaning multisystemic wasting syndrome using a TaqMan-based real-time PCR. J Virol Methods 122:171–178.

Chae

: 2004, Postweaning multisystemic wasting syndrome: a review of aetiology, diagnosis and pathology. Vet J 168:41–49.

Chen

Zhang

Sun

.: 2008, Rapid detection of porcine circovirus type 2 by loop-mediated isothermal amplification. J Virol Methods 149:264–268.

Dezen

Rijsewijk

Teixeira

.: 2010, Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: applications on detection, sequencing and virus isolation. Res Vet Sci 88:436–440.

Ellis

Hassard

Clark

.: 1998, Isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome. Can Vet J 39:44–51.

10.

Higuchi

Fockler

Dollinger

Watson

: 1993, Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 11:1026–1030.

11.

Hjulsager

Grau-Roma

Sibila

.: 2009, Inter-laboratory and inter-assay comparison on two real-time PCR techniques for quantification of PCV2 nucleic acid extracted from field samples. Vet Microbiol 133:172–178.

12.

Jiang

Shang

.: 2010, Detection and genotyping of porcine circovirus in naturally infected pigs by oligo-microarray. Res Vet Sci 89:133–139.

13.

Kennedy

Moffett

McNeilly

.: 2000, Reproduction of lesions of postweaning multisystemic wasting syndrome by infection of conventional pigs with porcine circovirus type 2 alone or in combination with porcine parvovirus. J Comp Pathol 122:9–24.

14.

Kim

Chung

Chae

: 2003, Association of porcine circovirus 2 with porcine respiratory disease complex. Vet J 166: 251–256.

15.

Kim

Han

Choi

Chae

: 2001, Differentiation of porcine circovirus (PCV)-1 and PCV-2 in boar semen using a multiplex nested polymerase chain reaction. J Virol Methods 98:25–31.

16.

Larochelle

Morin

Antaya

Magar

: 1999, Identification and incidence of porcine circovirus in routine field cases in Québec as determined by PCR. Vet Rec 145:140–142.

17.

Liu

Wang

Willson

Babiuk

: 2000, Quantitative, competitive PCR analysis of porcine circovirus DNA in serum from pigs with postweaning multisystemic wasting syndrome. J Clin Microbiol 38:3474–3477.

18.

Markowski-Grimsrud

Miller

Schat

: 2002, Development of strain-specific real-time PCR and RT-PCR assays for quantitation of chicken anemia virus. J Virol Methods 101:135–147.

19.

McIntosh

Tumber

Harding

.: 2009, Development and validation of a SYBR green real-time PCR for the quantification of porcine circovirus type 2 in serum, buffy coat, feces, and multiple tissues. Vet Microbiol 133:23–33.

20.

Nayar

Hamel

Lin

: 1997, Detection and characterization of porcine circovirus associated with postweaning multisystemic wasting syndrome in pigs. Can Vet J 38:385–386.

21.

O’Connor

Gauvreau

West

.: 2001, Multiple porcine circovirus 2-associated abortions and reproductive failure in a multisite swine production unit. Can Vet J 42:551–553.

22.

Opriessnig

Meng

Halbur

: 2007, Porcine circovirus type 2 associated disease: update on current terminology, clinical manifestations, pathogenesis, diagnosis, and intervention strategies. J Vet Diagn Invest 19:591–615.

23.

Park

Kwon

.: 2009, Detection of porcine circovirus 2 in mammary and other tissues from experimentally infected sows. J Comp Pathol 140:208–211.

24.

Qvarnstrom

James

Xayavong

.: 2005, Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis. J Clin Microbiol 43:5491–5497.

25.

Ratcliff

Chang

Kok

Sloots

: 2007, Molecular diagnosis of medical viruses. Curr Issues Mol Biol 9: 87–102.

26.

Rosell

Segalés

Plana-Durán

.: 1999, Pathological, immunohistochemical, and in-situ hybridization studies of natural cases of postweaning multisystemic wasting syndrome (PMWS) in pigs. J Comp Pathol 120:59–78.

27.

Sambrook

Russell

: 2001, Preparation and transformation of competent E. coli using calcium chloride. In: Molecular cloning: a laboratory manual, 3rd ed., vol. 1, pp. 116-118. Spring Harbour Laboratory Press, New York.

28.

Segalés

Allan

Domingo

: 2005, Porcine circovirus diseases. Anim Health Res Rev 6:119–142.

29.

Segalés

Calsamiglia

Olvera

.: 2005, Quantification of porcine circovirus type 2 (PCV2) DNA in serum and tonsillar, nasal, tracheo-bronchial, urinary and faecal swabs of pigs with and without postweaning multisystemic wasting syndrome (PMWS). Vet Microbiol 111:223–229.

30.

Stevenson

Kiupel

Mittal

.: 2001, Tissue distribution and genetic typing of porcine circoviruses in pigs with naturally occurring congenital tremors. J Vet Diagn Invest 13:57–62.

31.

Van Engelenburg

FAC

Maes

van Oirschot

Rijsewijk

: 1993, Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen. J Clin Microbiol 31:3129–3135.

32.

Vilcek

Vlasakova

Jackova

: 2010, LUX real-time PCR assay for the detection of porcine circovirus type 2. J Virol Methods 165:216–221.

33.

West

Bystrom

Wojnarowicz

.: 1999, Myocarditis and abortion associated with intrauterine infection of sows with porcine circovirus 2. J Vet Diagn Invest 11:530–532.

34.

Yang

Habib

Shuai

Fang

: 2007, Detection of PCV2 DNA by SYBR Green I-based quantitative PCR. J Zhejiang Univ Sci B 8:162–169.

35.

Yue

Cui

Zhang

Yoon

: 2009, A multiplex PCR for rapid and simultaneous detection of porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, and porcine reproductive and respiratory syndrome virus in clinical specimens. Virus Genes 38:392–397.

36.

Zentilin

Giacca

: 2007, Competitive PCR for precise nucleic acid quantification. Nat Protoc 2:2092–2104.

Comparative evaluation of a competitive polymerase chain reaction (PCR) and a SYBR Green–based real-time PCR to quantify Porcine circovirus-2 DNA in swine tissue samples

Abstract

Keywords

Introduction

Material and methods

Tissue samples

DNA extraction

Oligonucleotides used in the PCR assays

Competitive PCR

Construction of a competing template

Quantitation of plasmid DNA copy numbers and co-amplification of PCV-2 DNA with competitor DNA

SYBR Green real-time PCR

Standard curve for the SYBR Green real-time PCR

Specificity and sensitivity of cPCR and SYBR Green real-time PCR

Statistical analysis

Results

Co-amplification of PCV-2 DNA with competitor DNA

Specificity and sensitivity of the cPCR and SYBR Green real-time PCR

Standard curve for the SYBR Green real-time PCR

Determination of viral load

Comparison of the PCR-based assays

Discussion

Footnotes

Acknowledgements

a.

b.

c.

d.

e.

f.

g.

h.

i.

j.

k.

l.

m.

n.

o.

References