Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green

Zheng, Lan-Lan; Wang, Yabin; Li, Mingfeng; Chen, Hongying; XianPo, Guo; Geng, Junyou; Wang, Zhenya; Wei, Zhanyong; Bao-an, Cui

doi:10.1016/j.jviromet.2012.06.024

Cited by 31 publications

(17 citation statements)

References 23 publications

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“…The diagnosis of CPV 2, FPV and PPV infection has relied on probe-based real-time PCR, 18-23 SYBR Greenbased real-time PCR, [24][25][26] conventional PCR, 23,27 electron microscopy, 28 and methods provided as commercial kits. 29 In the present study, we established a SYBR Green-based PCR assay to detect new virus types of CPV 2, FPV and PPV.…”

Section: Discussionmentioning

confidence: 99%

Development of Sybr Green-Based Real-Time PCR for the Detection of Canine, Feline and Porcine Parvoviruses

et al. 2014

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Parvovirus is now considered to be one of the most important infectious agents a®ecting canine, feline and porcine domestic animals. Canine parvovirus 2 (CPV 2) and feline parvovirus (FPV) are major infectious causes of mortality in puppies and kittens. Both CPV 2 and FPV have recently been shown to infect both dogs and cats. The characteristic symptoms of canine, feline and porcine parvovirus disease are intestinal hemorrhage with severe bloody diarrhea, leukopenia and reproductive failure, respectively. In this work, we describe the development of a novel real-time PCR system that is based on the use of SYBR Green and that allows the simultaneous detection of VP2 gene of CPV, FPV and porcine parvovirus (PPV). This system yielded low coe±cients of variation for intra-assay and inter-assay variabilities. This novel SYBR Green-based real-time PCR assay was sensitive, speci¯c and reliable for the ampli¯cation of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/L of target DNA per reaction. The methods described in this study have been used successfully in our veterinary diagnostic laboratory and have been shown to be helpful tools for the diagnosis and quanti¯cation of parvovirus infection in canines, felines and swine.

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Section: Discussionmentioning

confidence: 99%

Development of Sybr Green-Based Real-Time PCR for the Detection of Canine, Feline and Porcine Parvoviruses

et al. 2014

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“…Real-time qPCR assay for detection of PCV2 DNA and PCV1 DNA were both performed on Agilent Mx3005P thermocycler machine (Life Technologies, USA) as previously described [20, 21]. The primers PCV2 F (5′-ATGGCGGGAGGAGTAGTTT-3′) and PCV2 R (5′-CCCTTTGAATACTACAGCG-3′) were used to amplify a 171 bp fragment within ORF2 region of the PCV2 genome.…”

Section: Methodsmentioning

confidence: 99%

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

Yang

Qin

Sun

et al. 2017

BioMed Research International

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Porcine circovirus virus type II (PCV2) is the etiology of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis, nephropathy syndrome (PDNS), and necrotizing pneumonia. Rapid diagnosis tool for detection of PCV2 plays an important role in the disease control and eradication program. Recombinase polymerase amplification (RPA) assays using a real-time fluorescent detection (PCV2 real-time RPA assay) and RPA combined with lateral flow dipstick (PCV2 RPA LFD assay) were developed targeting the PCV2 ORF2 gene. The results showed that the sensitivity of the PCV2 real-time RPA assay was 102 copies per reaction within 20 min at 37°C and the PCV2 RPA LFD assay had a detection limit of 102 copies per reaction in less than 20 min at 37°C. Both assays were highly specific for PCV2, with no cross-reactions with porcine circovirus virus type 1, foot-and-mouth disease virus, pseudorabies virus, porcine parvovirus, porcine reproductive and respiratory syndrome virus, and classical swine fever virus. Therefore, the RPA assays provide a novel alternative for simple, sensitive, and specific identification of PCV2.

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“…The second ORF encodes capsid proteins VP1, VP2, and VP3 with masses of 84, 64, and 60 kDa, respectively. Capsid protein VP2 harbors major antigenic domains of PPV which can induce PPV-neutralizing antibodies to neutralize PPV infection and plays a key role in PPV diagnosis and immune prophylaxis [9,10]. Virus isolation in cell culture is laborious and time consuming and it cannot be achieved for all PPV strains.…”

Section: Introductionmentioning

confidence: 99%

Molecular Detection of Pseudorabies Virus (PrV), Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV2) in Swine in Republic of Montenegro

et al. 2016

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The presence of pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus 2 (PCV2) was examined in sixty samples (spleen and lymph nodes) and thirty samples of sacral ganglia collected from non-vaccinated swine by virus isolation and polymerase chain reaction (PCR). Using PCR method PrV was detected in three samples, PPV in seven samples and six samples were found positive for PCV2. The phylogenetic analysis of the nucleotide sequences of three PrV isolates identifi ed in this study showed high similarity and signifi cant clustering within the PrV genotype I strains such as Kaplan and Bartha isolated from pigs in Hungary, strain Becker isolated in USA and strain Kolchis isolated in Greece. The nucleotide sequences of two PPV isolates showed high level of similarity with the strain Challenge isolated from pigs in UK, strain Kresse isolated in USA and strains 77 and LZ isolated in China. The phylogenetic analysis of the nucleotide sequences of two PCV2 isolates showed high level of similarity and signifi cant clustering within genotype PCV2b strains such as NIVS-3, NIVS-5 and NIVS-6 isolated in Serbia, strain 3959 isolated in Austria, strain PM165 isolated from pigs in Brasil, and strain XT2008 isolated in China. The results of our study present the molecular characterization of PrV, PPV and PCV2 identifi ed in swine in Republic of Montenegro. Besides that, these results confi rmed that PCR is a very useful method for rapid detection of these viruses in subclinically infected swine.

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Simultaneous detection of porcine parvovirus and porcine circovirus type 2 by duplex real-time PCR and amplicon melting curve analysis using SYBR Green

Cited by 31 publications

References 23 publications

Development of Sybr Green-Based Real-Time PCR for the Detection of Canine, Feline and Porcine Parvoviruses

Development of Sybr Green-Based Real-Time PCR for the Detection of Canine, Feline and Porcine Parvoviruses

Development of Isothermal Recombinase Polymerase Amplification Assay for Rapid Detection of Porcine Circovirus Type 2

Molecular Detection of Pseudorabies Virus (PrV), Porcine Parvovirus (PPV) and Porcine Circovirus 2 (PCV2) in Swine in Republic of Montenegro

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