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

2014 ◽  
Vol 40 (01) ◽  
pp. 1-9 ◽  
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.

scholarly journals Simultaneous detection of porcine pseudorabies virus, porcine parvovirus and porcine circovirus type 2 by multiplex real-time PCR and amplicon melting curve analysis using SYBR Green I

2018 ◽  
Vol 63 (No. 8) ◽  
pp. 358-366
Author(s):  
LL Zheng ◽  
XH Jin ◽  
FS Wei ◽  
CQ Wang ◽  
HY Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pseudorabies Virus ◽  
Simultaneous Detection ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Porcine Parvovirus ◽  
Porcine Pseudorabies Virus

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.

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

2011 ◽  
Vol 60 (4) ◽  
pp. 459-466 ◽  
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.

Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii

Nematology ◽  
2017 ◽  
Vol 19 (7) ◽  
pp. 789-804 ◽  
Author(s):  
Sylvie Gamel ◽  
Aude Letort ◽  
Didier Fouville ◽  
Laurent Folcher ◽  
Eric Grenier
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Simultaneous Detection ◽  
Nematode Species ◽  
Globodera Pallida ◽  
Heterodera Schachtii ◽  
Cyst Nematodes ◽  
Detection And Identification ◽  
Template Dna

Considering the growing trade of seed potato, reliable diagnostic protocols are required for the detection of regulated nematode species. In this study, a specific and sensitive multiplex Taqman-based real-time PCR method was developed in order to detect and identifyGlobodera pallida,G. rostochiensisandHeterodera schachtii. The newly designed primers and probes enabled the detection of all the target populations tested and with no cross-reaction for closely related non-target species (55 populations tested). The limit of detection (LOD) was one juvenile forG. rostochiensisandG. pallidaand five juveniles forH. schachtii. For monitoring potato cyst nematodes, this analytical tool would extend the number of cyst investigated as five juveniles can be detected among 50 cysts in a sample. Furthermore, this multiplex assay detects DNA of the three targeted species in template DNA obtained directly from float material after nematode extraction from soil.

scholarly journals Establishment of Simultaneous Detection and Quantitation of HCVRNA by Third Generation Intercalating Dye Real-time PCR

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Akrahm M. Saleh Habil ◽  
Hairul Aini Hamzah ◽  
Muhammad Imad Al-Deen Mustafa ◽  
Norlelawati A. Talib ◽  
Siti Nurul Fazlin Abdul Rahman
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
False Negative ◽  
Simultaneous Detection ◽  
Pcr Amplification ◽  
Serum Samples ◽  
Negative Results ◽  
Broad Dynamic Range

Introduction: Rapid quantification of hepatitis C virus is helpful in determining and monitoring of the disease progression and nature of the virus replication. The aim of the present study was to establish a fast, specific and sensitive tool for HCVRNA quantification. Materials and Methods: A total of 50 serum samples, comprising of 40 HCV-positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to real-time PCR amplification. Real-time PCR using EvaGreen dye and primers targeting a 5’UTR was carried out. Reference samples with known viral load were treated similarly to the unknown samples and used to create the standard curves. Results: Our method showed a high level of analytical specificity and accuracy, with a low limit of detection (~2 IU/ml). It yielded repeatable results with less than 4% of intra- assay variation. The assay covered a broad dynamic range of quantification, ranging from 0.34 to 6 log IU/ml. The diagnostic sensitivity, specificity, and accuracy were all 100%, indicating neither false positive nor false negative results were obtained. Conclusion: The developed real time PCR using EvaGreen dye has demonstrated a highly analytical and diagnostic performance for HCV quantification, suggesting its potential in clinical diagnosis and management.

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