Simultaneous detection of porcine reproductive and respiratory syndrome virus and porcine circovirus 3 by SYBR Green І-based duplex real-time PCR

Porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 can cause reproductive failure in pigs, and swine are often simultaneously infected by combinations of the three viruses. We here report the development of a SYBR Green I-based multiplex real time PCR assay for simultaneous detection of porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2. Three pairs of specific primers were designed for the porcine parvovirus-VP2, porcine pseudorabies virus-gH and porcine circovirus type 2-ORF2 genes. Viral genomes were identified based on their distinctive melting temperatures in singleplex PCR reactions. The melting temperature was 74.5 °C for the 313 bp amplicon of porcine parvovirus-VP2 gene, 87.5 °C for the 355 bp amplicon of porcine pseudorabies virus-gH gene and 80.5 °C for the 171 bp amplicon of the porcine circovirus type 2-ORF2 gene, respectively. The detection limit of the method ranged from 0.01–0.03 TCID<sub>50</sub>/ml for the three viruses. In addition, porcine parvovirus, porcine pseudorabies virus and porcine circovirus type 2 viral loads were measured in 100 field samples, and the result showed that the concordance between real-time PCR and conventional PCR was 60.42%. The sensitivity and specificity of real-time PCR were 100% and 100%, while those of conventional PCR were 40.83% and 72.22%, respectively.

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Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green

Research in Veterinary Science ◽

10.1016/j.rvsc.2007.10.003 ◽

2008 ◽

Vol 85 (1) ◽

pp. 184-193 ◽

Cited By ~ 24

Author(s):

E. Martínez ◽

P. Riera ◽

M. Sitjà ◽

Y. Fang ◽

S. Oliveira ◽

...

Keyword(s):

Real Time ◽

Simultaneous Detection ◽

Melting Curve ◽

Curve Analysis ◽

Sybr Green ◽

Respiratory Syndrome Virus ◽

Melting Curve Analysis ◽

Rt Pcr

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Detection and species identification of microsporidial infections using SYBR Green real-time PCR

Journal of Medical Microbiology ◽

10.1099/jmm.0.026781-0 ◽

2011 ◽

Vol 60 (4) ◽

pp. 459-466 ◽

Cited By ~ 29

Author(s):

Spencer D. Polley ◽

Samuel Boadi ◽

Julie Watson ◽

Alan Curry ◽

Peter L. Chiodini

Keyword(s):

Real Time ◽

Species Identification ◽

Real Time Pcr ◽

Simultaneous Detection ◽

Enterocytozoon Bieneusi ◽

Sybr Green ◽

Pcr Assay ◽

Real Time Analysis ◽

Highly Sensitive ◽

Stool Samples

Diagnosis of microsporidial infections is routinely performed by light microscopy, with unequivocal non-molecular species identification achievable only through electron microscopy. This study describes a single SYBR Green real-time PCR assay for the simultaneous detection and species identification of such infections. This assay was highly sensitive, routinely detecting infections containing 400 parasites (g stool sample)−1, whilst species identification was achieved by differential melt curves on a Corbett Life Science Rotor-Gene 3000. A modification of the QIAamp DNA tissue extraction protocol allowed the semi-automated extraction of DNA from stools for the routine diagnosis of microsporidial infection by real-time PCR. Of 168 stool samples routinely analysed for microsporidian spores, only five were positive by microscopy. By comparison, 17 were positive for microsporidial DNA by real-time analysis, comprising 14 Enterocytozoon bieneusi, one Encephalitozoon cuniculi and two separate Pleistophora species infections.

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Development of a TaqMan-based real-time PCR assay for the specific detection of porcine circovirus 3

Journal of Virological Methods ◽

10.1016/j.jviromet.2017.07.007 ◽

2017 ◽

Vol 248 ◽

pp. 177-180 ◽

Cited By ~ 27

Author(s):

Jianchang Wang ◽

Yongning Zhang ◽

Jinfeng Wang ◽

Libing Liu ◽

Xiaoyu Pang ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Porcine Circovirus ◽

Specific Detection ◽

Pcr Assay ◽

Porcine Circovirus 3

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DEVELOPMENT OF SYBR GREEN-BASED REAL-TIME PCR FOR THE DETECTION OF CANINE, FELINE AND PORCINE PARVOVIRUSES

Taiwan Veterinary Journal ◽

10.1142/s1682648514500012 ◽

2014 ◽

Vol 40 (01) ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Chao-Nan Lin ◽

Chi-Hsien Chien ◽

Ming-Tang Chiou ◽

Jou-Wei Wang ◽

Ya-Ling Lin ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

Simultaneous Detection ◽

Sybr Green ◽

Porcine Parvovirus ◽

Diagnostic Laboratory ◽

Vp2 Gene ◽

Coefficients Of Variation ◽

Causes Of Mortality

Parvovirus is now considered to be one of the most important infectious agents affecting 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 coefficients of variation for intra-assay and inter-assay variabilities. This novel SYBR Green-based real-time PCR assay was sensitive, specific and reliable for the amplification of CPV 2, FPV and PPV DNA, with a reproducible limit of detection of as few as 10 copies/μL 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 quantification of parvovirus infection in canines, felines and swine.

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