Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

Tomás, Gonzalo; Hernández, Martı́n; Marandino, Ana; Panzera, Yanina; Maya, Leticia; Hernández, Diego; Pereda, Ariel; Banda, Alejandro; Villegas, Pedro; Aguirre, Sebastián; Pérez, Ruben

doi:10.1016/j.jviromet.2012.06.012

Cited by 24 publications

(19 citation statements)

References 43 publications

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“…Each sample was stored at -80°C until analysis. IBDV presence was confirmed in 15 samples (Table 1) with real-time PCR, as previously described (Tomás et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

“…Genetic analysis has been the most widely used technique to characterize and classify IBDVs (Cao et al, 1998;Banda & Villegas, 2004;Hernández et al, 2006;Jackwood & Sommer-Wagner, 2007;Yamaguchi et al, 2007;Tomás et al, 2012). Studies have been mainly focused on the hypervariable domain of VP2 (hvVP2) because it codes for the most important immunogenic regions of the protein (Azad et al, 1987;Bayliss et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

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Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

et al. 2015

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Infectious bursal disease virus (IBDV) is one of the most concerning health problems for world poultry production. IBDVs comprise four well-defined evolutionary lineages known as classic (c), classic attenuated (ca), variant (va) and very virulent (vv) strains. Here, we characterized IBDVs from South America by the genetic analysis of both segments of the viral genome. Viruses belonging to c, ca and vv strains were unambiguously classified by the presence of molecular markers and phylogenetic analysis of the hypervariable region of the vp2 gene. Notably, the majority of the characterized viruses (9 out of 15) could not be accurately assigned to any of the previously described strains and were then denoted as distinct (d) IBDVs. These dIBDVs constitute an independent evolutionary lineage that also comprises field IBDVs from America, Europe and Asia. The hypervariable VP2 sequence of dIBDVs has a unique and conserved molecular signature (272T, 289P, 290I and 296F) that is a diagnostic character for classification. A discriminant analysis of principal components (DAPC) also identified the dIBDVs as a cluster of genetically related viruses separated from the typical strains. DAPC and genetic distance estimation indicated that the dIBDVs are one of the most genetically divergent IBDV lineages. The vp1 gene of the dIBDVs has non-vvIBDV markers and unique nucleotide and amino acid features that support their divergence in both genomic segments. The present study suggests that the dIBDVs comprise a neglected, highly divergent lineage that has been circulating in world poultry production since the early time of IBDV emergence.

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“…Each sample was stored at -80°C until analysis. IBDV presence was confirmed in 15 samples (Table 1) with real-time PCR, as previously described (Tomás et al, 2012).…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

et al. 2015

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“…In order to discriminate geno-groups of IBDV strains, an rRT-PCR run in two stages has been developed by Tomás et al [17]. In the first stage, RT was performed on RNA producing complementary DNA (cDNA) by the activity of reverse transcriptase and in the second one, the cDNA obtained was amplified by PCR under the activity of the DNA polymerase using primers and probes which included out with the VP5/VP2 overlying region of segment A of IBDV.…”

Section: Diagnosis Of Clinical Cases Of Infectious Bursal Disease Usimentioning

confidence: 99%

“…The primers and probes used for rRT-PCR amplification of IBDV were designed by Bioneer, Korea and targeting the VP5/VP2 overlapping region of segment A as described by Tomás et al [17]. The forward primer F 178 matched positions 178-198 (5'-GAGCCTTCTGATGCCAACAAC-3'); the probes positions 222-236 (FAM-5'-ACACCCTAGAGAAGC-3'-MGB) for detection of vvIBDV, (VIC-5'-ACACCCTGGAGAAGC-3'-MGB) for detection of non-vvIBDVs and the reverse primer was located at positions 272-248 (5'-TCAAATTGTAGGTCGAGGTCTCTGA-3').…”

Section: Primer and Probe Designmentioning

confidence: 99%

Diagnosis of Clinical Cases of Infectious Bursal Disease Using a Modified Rapid Taq Man-MGB Real-Time RT-PCR Assay

Cheggag¹,

Zro²,

Sebbar³

et al. 2018

JAST-A

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Infectious bursal disease (IBD) is an important contagious viral infection of immune system of poultry. This infection possesses a permanent threat to the profitability of poultry industry worldwide. The aim of this work was to modify the Taq Man-MGB real-time reverse transcription-polymerase chain reaction (rRT-PCR) in one step involving two fluorogenic Taq Man labeled probe and using this protocol for detection of infectious bursal disease virus (IBDV) collected from suspected cases distributed in different regions of the country during the period 2013-2016. The intralaboratory validation of modified method was realized for specificity, linearity, repeatability, sensitivity and reproducibility. It allowed reducing the test running time by six folds. This method was applied on 102 pools of bursa of fabricius (BF) samples collected from affected broiler farms suspected to be infected by IBDV. Birds showing macroscopic lesions including muscle petechial hemorrhages, hypertrophy and hemorrhage of BF, were subjected to molecular analysis using modified protocol "Taq Man-MGB rRT-PCR". The validation satisfied all criteria and the assay developed could be a useful tool for a very rapid diagnosis of IBDV and permit to detect and to discriminate in one-step very virulent (vv) from non-vv (classic and variant) IBDV strains. Out of 84 IBDV positive samples, a prevalence of 39% for vv strains and 61% for classical strains was noted. These results indicate that despite the vaccination against IBDV, the vv form of this pathologie continues to cause serious problems for Moroccan broiler chickens. The obtained results indicate the successfully detection of IBDV and differentiated all vvIBDV strains from non-vvIBDV strains; Avian infectious agent RNA viruses tested are negative, demonstrating great specificity of the assay. The results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of IBDV strains in samples of avian origin.

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“…Recently, Taqman-MGB probes are widely applied in pathogen detection and single nucleotide polymorphism (SNP) diagnosis. E.g., Taqman-MGB probes were used in the detection of equine herpes virus 5 (EHV-5) [31], infectious bursal disease virus (IBDV) [32], clostridium piliforme [33], coliforms [34], trisomy 21 [35] and differentiation of virulent and vaccine strains of avian paramyxovirus type 1 [29]. However, as far as we know, there are no reports associated with Taqman-MGB probe that targeting NP (nucleoprotein) gene of AIV despite of its high conservation.…”

Section: Introductionmentioning

confidence: 99%

Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

et al. 2017

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Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.

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Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

Cited by 24 publications

References 43 publications

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

Genetic characterization of South American infectious bursal disease virus reveals the existence of a distinct worldwide-spread genetic lineage

Diagnosis of Clinical Cases of Infectious Bursal Disease Using a Modified Rapid Taq Man-MGB Real-Time RT-PCR Assay

Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

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