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

Martínez, E.; Riera, Pere; Sitjà, Marta; Fang, Yuan; Oliveira, Sara; Maldonado, Jaime

doi:10.1016/j.rvsc.2007.10.003

Cited by 35 publications

(21 citation statements)

References 41 publications

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“…Consequently, SYBR Green RT-PCR is considered a flexible and cost-effective approach that can be directly applied without the need to design and synthesize fluorescently labeled target-specific probes (Martínez et al 2008), and is less dependent on sequence variations. In addition, the post-amplification melting curve analysis provides highly specific detection (Kong et al 2009), although further research is needed to ensure the specificity of the Tm.…”

Section: Discussionmentioning

confidence: 99%

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

López-Vázquez¹,

Bandı́n²,

Dopazo³

2015

Dis. Aquat. Org.

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In the present study, 2 systems of real-time RT-PCR -one based on SYBR Green and the other on TaqMan -were designed to detect strains from any genotype of viral haemorrhagic septicaemia virus (VHSV), with high sensitivity and repeatability/reproducibility. In addition, the method was optimized for quantitative purposes (qRT-PCR), and standard curves with different types of reference templates were constructed and compared. Specificity was tested against 26 isolates from 4 genotypes. The sensitivity of the procedures was first tested against cell culture isolation, obtaining a limit of detection (LD) of 10 0 TCID 50 ml −1 (100-fold below the LD using cell culture), at a threshold cycle value (Ct) of 36. Sensitivity was also evaluated using RNA from crude (LD = 1 fg; 160 genome copies) and purified virus (100 ag; 16 copies), plasmid DNA (2 copies) and RNA transcript (15 copies). No differences between both chemistries were observed in sensitivity and dynamic range. To evaluate repeatability and reproducibility, all experiments were performed in triplicate and on 3 different days, by workers with different levels of experience, obtaining Ct values with coefficients of variation always < 5. This fact, together with the high efficiency and R 2 values of the standard curves, encouraged us to analyse the reliability of the method for viral quantification. The results not only demonstrated that the procedure can be used for detection, identification and quantification of this virus, but also demonstrated a clear correlation between the regression lines obtained with different standards, which will help scientists to compare sensitivity results between different studies.

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Section: Discussionmentioning

confidence: 99%

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

López-Vázquez¹,

Bandı́n²,

Dopazo³

2015

Dis. Aquat. Org.

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“…MCA can be useful to detect nucleotide variability because PCR products with different lengths, nucleotide composition and guanine-cytosine (GC) content showed different melting temperatures (T m ) (Herrmann et al, 2006). For this reason, MCAbased SYBR Green qPCR methods have already been successfully used to detect, quantify and genotype various human and veterinary pathogens (Hays et al, 2011;Liu et al, 2006;Martínez et al, 2008;Maurelli et al, 2009;Payungporn et al, 2008;Tanriverdi et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2

Balboni¹,

Dondi²,

Prosperi³

et al. 2015

Journal of Virological Methods

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Canine adenovirus type 1 (CAdV-1) and canine adenovirus type 2 (CAdV-2) cause infectious canine hepatitis (ICH) and infectious tracheobronchitis (ITB) in dogs, respectively. Cases of ICH have been documented in recent years and recent surveys have demonstrated a wide percentage of asymptomatic CAdV-1 infection in the canine population. Since both CAdV types are detectable in the same biological matrices, and viral coinfection with CAdV-1 and CAdV-2 are reported with high frequency, it is urgent to have available a rapid, highly sensitive and specific assay for the diagnosis of CAdV infection and distinction between CAdV-1 and CAdV-2. In order to detect canine adenovirus in biological samples and to rapidly distinguish the two viral types, a SYBR Green real-time PCR assay was optimized to discriminate CAdV-1 and CAdV-2 via a melting curve analysis. The developed assay showed high sensitivity and reproducibility and was highly efficient and specific in discriminating the two CAdV types. This reliable and rapid technique may represent a simple, useful and economic option for simultaneous CAdV types detection, which would be feasible and attractive for all diagnostic laboratories, both for clinical purposes and for epidemiological investigations.

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“…This method is very useful for the detection of viral infections, especially for the early detection of infection, and has been used to detect several different pathogens in recent years [6][7][8][9][10]. Current RT-qPCR methods for the detection of TGEV have focused on the TGEV N gene.…”

Section: Discussionmentioning

confidence: 99%

Development and primary application of the SYBR Green I RT-qPCR assay for the detection of the transmissible gastroenteritis virus (TGEV) S gene

Yue

Yuan

et al. 2011

2011 4th International Conference on Biomedical Engineering and Informatics (BMEI)

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This study established a SYBR Green I real-time quantitative PCR detection assay (RT-qPCR) for the detection of the porcine transmissible gastroenteritis virus (TGEV) using specific primers designed to amplify the highly conserved porcine TGEV S gene sequence. The threshold cycle (Ct) and the log plasmid copy numbers had a good linear relationship with an efficiency of 1.05 (R 2 = 0.999). The advantages of utilizing this approach for the rapid detection of TGEV include excellent sensitivity, reproducibility, and low cost. Ninety-six porcine fecal samples were tested in this study, and 7 samples more than PCR assay were detected by this assay.

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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

Cited by 35 publications

References 41 publications

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

Real-time RT-PCR for detection, identification and absolute quantification of viral haemorrhagic septicaemia virus using different types of standards

Development of a SYBR Green real-time PCR assay with melting curve analysis for simultaneous detection and differentiation of canine adenovirus type 1 and type 2

Development and primary application of the SYBR Green I RT-qPCR assay for the detection of the transmissible gastroenteritis virus (TGEV) S gene

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