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

Brunborg, Inger Marit; Moldal, Torfinn; Jonassen, Christine Monceyron

doi:10.1016/j.jviromet.2004.08.014

Cited by 179 publications

(182 citation statements)

References 28 publications

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“…The animals with high PCV2 DNA load in liver and spleen were not bled before death, and there is a possibility that the observed PCR results were caused by PCV2 in the retained blood. In cases of post-weaning multisystemic wasting syndrome, the PCV2 load in plasma can be very high, 6 and this may contribute to a background level of PCV2 DNA in tissues with a high retention of blood or plasma. In a recent study in which pregnant sows were inoculated intranasally with PCV2, Park et al were unable to detect any virus in the myocardium from fetuses either by immunohistochemistry or in situ hybridization.…”

Section: Discussionmentioning

confidence: 99%

“…DNA was isolated from 5-30-mg tissue using a commercial kit according to manufacturer's instructions b ; tissue weights and DNA concentrations were recorded. Quantitative real-time PCR was performed on the DNA samples as described previously 6 using primers and a probe designed to perfectly match the target sequence (PCV-C-1256U21 59-ATA GCG GGA GTG GTA AGA GAA-39, PCV-F-1319L21 59-GCA ACA GCC CTA ACC TAT GAC-39, TaqMan3-PCV2 59-6-Fam-ATG TAA ACT ACT CCT CCC GCC ATA CCA TT-Tamra-39). Results were recorded as the number of PCV2 copies per 500-ng DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Statistical analyses of PCV2 load in different tissues were performed using JMP 5.0.1a. e Samples below the detection limit of 1 copy per 500-ng dsDNA were set to 0.5 copies per 500 ng dsDNA, representing the mean of the values, and likewise, the samples calculated by the LightCycler software to be between the detection limit and the quantification limit of 1.0 3 10 2 copies per 500 ng dsDNA, 6 were set to 5.0 3 10 1 copies per 500 ng dsDNA. As the distribution was not normal even after log-transformation, Wilcoxon rank sum test was performed for comparison between groups.…”

Section: Methodsmentioning

confidence: 99%

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Association of Myocarditis with High Viral Load of Porcine Circovirus Type 2 in Several Tissues in Cases of Fetal Death and High Mortality in Piglets. A Case Study

et al. 2007

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Abstract. During a period of 1.5 months, a newly established pig herd experienced a high number of mummifications and stillbirths, a high neonatal mortality rate, and many piglets with congenital tremors or hind leg ataxia. After clinical and histological investigations, the submitted animals were divided into 4 groups: mummified or stillborn (N 5 6), live born with myocarditis (N 5 5) (average age 22.8 days), live born without myocarditis (N 5 14) (average age 20.0 days), and control animals from a different herd (N 5 5) (newborn). Statistically significant differences were observed in the mean porcine circovirus 2 (PCV2) load among the 4 groups in the liver (P , 0.0001). The presence of PCV2 antigen within the myocardial lesions was confirmed by immunohistochemistry. A high load of PCV2 DNA was observed in myocardium, liver, and spleen from mummified or stillborn piglets (.1 3 10 7 copies per 500 ng DNA), lower in piglets with myocarditis (.1 3 10 5 copies per 500 ng DNA), and even further lower in pigs without myocarditis (,1 3 10 5 copies per 500 ng DNA), whereas no PCV2 DNA was detected in the control animals. Myocardium, liver, and spleen were well suited for routine testing of fetuses and young piglets by quantitative real-time polymerase chain reaction. Neither porcine parvovirus nor encepaholomyocarditis virus was detected. These results indicate that the PCV2 infection might have been of etiological importance for the fetal deaths and piglet mortality observed in this herd.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Association of Myocarditis with High Viral Load of Porcine Circovirus Type 2 in Several Tissues in Cases of Fetal Death and High Mortality in Piglets. A Case Study

et al. 2007

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“…Real-time PCR methods described so far are based on TaqMan, FRET or Molecular Beacon principles (Rovira et al, 2002;Brunborg et al, 2004;Olvera et al, 2004;Chung et al, 2005;McKillen et al, 2006). In general, these systems require perfect hybridisation between the probe and the viral target region for comparative quantification of viral load, since the results might be influenced by point mutations in the PCV2 genomes.…”

Section: Discussionmentioning

confidence: 99%

“…Since PCV2 commonly occurs in swine populations, it is advisable to determine not only the qualitative, but also the quantitative aspects of PCV2. Several quantitative realtime PCR assays have been developed to improve the detection of PCV2 and to determine the viral quantity based on TaqMan (Rovira et al, 2002;Brunborg et al, 2004;Olvera et al, 2004), FRET (Chung et al, 2005), PriProET (LadekjaerMikkelsen et al, 2002) and molecular beacon (McKillen et al, 2006) chemistries. The main disadvantage of TaqMan systems is the need for a perfect match between the probe and the viral target region.…”

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confidence: 99%

Development of Primer-Probe Energy Transfer real-time PCR for the detection and quantification of porcine circovirus type 2

Bálint

Tenk

Deim

et al. 2009

Acta Veterinaria Hungarica

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A real-time PCR assay, based on Primer-Probe Energy Transfer (PriProET), was developed to improve the detection and quantification of porcine circovirus type 2 (PVC2). PCV2 is recognised as the essential infectious agent in postweaning multisystemic wasting syndrome (PMWS) and has been associated with other disease syndromes such as porcine dermatitis and nephropathy syndrome (PDNS) and porcine respiratory disease complex (PRDC). Since circoviruses commonly occur in the pig populations and there is a correlation between the severity of the disease and the viral load in the organs and blood, it is important not only to detect PCV2 but also to determine the quantitative aspects of viral load. The PriProET real-time PCR assay described in this study was tested on various virus strains and clinical forms of PMWS in order to investigate any correlation between the clinical signs and viral loads in different organs. The data obtained in this study correlate with those described earlier; namely, the viral load in 1 ml plasma and in 500 ng tissue DNA exceeds 10 7 copies in the case of PMWS. The results indicate that the new assay provides a specific, sensitive and robust tool for the improved detection and quantification of PCV2.

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Circoviruses

Segalés¹,

Allan²,

Domingo³

2019

Diseases of Swine

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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

Cited by 179 publications

References 28 publications

Association of Myocarditis with High Viral Load of Porcine Circovirus Type 2 in Several Tissues in Cases of Fetal Death and High Mortality in Piglets. A Case Study

Association of Myocarditis with High Viral Load of Porcine Circovirus Type 2 in Several Tissues in Cases of Fetal Death and High Mortality in Piglets. A Case Study

Development of Primer-Probe Energy Transfer real-time PCR for the detection and quantification of porcine circovirus type 2

Circoviruses

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