SYBR® Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

Poojari, Sudarsana; Alabi, Olufemi J.; Okubara, Patricia A.; Naidu, Rayapati A.

doi:10.1016/j.jviromet.2016.05.013

Cited by 31 publications

(31 citation statements)

References 26 publications

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“…Primer pairs for reverse transcription (RT)‐PCR detection of viruses are listed in Table . F2/R2, F3/R3 and F4/R4 described by Poojari et al () were used for detection of GLRaV‐3, GFLV and GVA, respectively. Coat protein (CP) gene‐specific primer pairs F1/R1, F5/R5, F6/R6 and F7/R7 were used for detection of GLRaV‐2, GRSPaV, GPGV and GFkV, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Primer amplification efficiency and specificity of qPCR (Fig. ) were determined according to methods described previously (Poojari et al , ). Vitis vinifera actin (XM_002282480.4) and N. benthamiana glyceraldehyde‐3‐phosphate dehydrogenase ( GAPDH ; AB937979.1) mRNA served as internal controls with the primer pair F12/R12 and F19/R19 (Table ).…”

Section: Methodsmentioning

confidence: 99%

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Crude garlic extract significantly inhibits replication of grapevine viruses

et al. 2019

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Efforts to control viral diseases of grapevine include the production of certified material and development of virus‐resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll‐associated virus 2 (GLRaV‐2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV‐2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro‐grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV‐2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting‐associated virus (GRSPaV) in field‐grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV‐3, GLRaV‐2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot‐grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis‐related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Crude garlic extract significantly inhibits replication of grapevine viruses

et al. 2019

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“…The minor groove binder (MGB) in the TRSV-P probe described in Yang et al (2007) was replaced with a BHQ1 quencher, and the hybridization temperature for F/R primers described in Jossey & Babadoost (2006) was set to 55°C according Sneideris et al (2012). The Poojari et al (2016) primer pair was tested in both conventional and real-time RT-PCR conditions. A total of five tests were assessed during this study.…”

Section: Rt-pcr Tests For the Selection Stepmentioning

confidence: 99%

“…The Poojari et al (2016) primer pair was tested in both conventional and real-time RT-PCR conditions. The conventional RT-PCR tests described in Jossey & Babadoost (2006), Fuchs et al (2010) and Poojari et al (2016) were tested using the SuperScript TM III One-Step RT-PCR System with Platinum TM Taq DNA Polymerase (Invitrogen, Carlsbad, CA, US) according to the manufacturer's recommendations: 10 lL of 29 reaction mix, 0.4 lL of a 10 lM stock solution of each primer, 0.8 lL of RT-Taq polymerase, 2 lL of RNA extract and molecular-grade water for a final volume of 20 lL. The conventional RT-PCR tests described in Jossey & Babadoost (2006), Fuchs et al (2010) and Poojari et al (2016) were tested using the SuperScript TM III One-Step RT-PCR System with Platinum TM Taq DNA Polymerase (Invitrogen, Carlsbad, CA, US) according to the manufacturer's recommendations: 10 lL of 29 reaction mix, 0.4 lL of a 10 lM stock solution of each primer, 0.8 lL of RT-Taq polymerase, 2 lL of RNA extract and molecular-grade water for a final volume of 20 lL.…”

Section: Rt-pcr Tests For the Selection Stepmentioning

confidence: 99%

“…In the EPPO region TRSV is recommended for regulation as a quarantine pest (EPPO A2 List) (EPPO/CABI, 1997) and is also specifically regulated in the European Union through Directive 2000/29/CE (Annex I, A1). Generic RT-PCRs for group A Nepoviruses (Digiaro et al, 2007;Wei & Clover, 2008) can detect TRSV and TRSV-specific RT-PCRs have been developed (Jossey & Babadoost, 2006;Yang et al, 2007;Martin et al, 2009;Fuchs et al, 2010;Poojari et al, 2016). Many detection methods are available such as biological indexing, immunoelectron microscopy (IEM), enzyme-linked immunosorbent assay (ELISA) and molecular methods such as reverse-transcription polymerase chain reaction (RT-PCR).…”

Section: Introductionmentioning

confidence: 99%

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Selection, optimization and characterization of molecular tests for the detection of Tobacco ringspot virus (TRSV)

Renvoisé¹,

Chambon²,

Gleize³

et al. 2019

EPPO Bulletin

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The aim of the study was to select, optimize and characterize RT‐PCR tests for the detection of Tobacco ringspot virus (TRSV) in post‐entry quarantine. Among five different tests, the Poojari et al. (Journal of Virological Methods 235, 112–118) and Jossey & Babadoost (Plant Disease 90, 1361) conventionnal RT‐PCR tests were chosen and optimized for this purpose. Ten TRSV isolates, 17 healthy plants from the genera Vitis, Prunus and Malus, four other Nepoviruses or Secoviridae isolates and serial dilutions of three TRSV isolates were used for the complete characterization of the optimized tests. In the tested conditions, the Poojari and Jossey & Babadoost tests respectively showed 100% and 95% inclusivity, 100% and 100% exclusivity, 100% and 96.4% analytical specificity, 100% and 100% diagnostic specificity, [10−7; 10−1] and [10−8; 10−2] limit of detection dilution factors (analytical sensitivity), 91.1% and 90.0% repeatability, 90.3% and 91.1% reproducibility and 100% and 100% selectivity. Due to its higher inclusivity, the optimized Poojari test is recommended for the detection of TRSV isolates for post‐entry quarantine purposes, but can also be used for global surveys. The optimized Jossey & Babadoost test showed the best analytical sensitivity, suggesting that combining both tests can further enhance detection.

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Grapevine leafroll-associated virus 1

Naidu¹

2017

Grapevine Viruses: Molecular Biology, Diagnostics and Management

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Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics.The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

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SYBR® Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses

Cited by 31 publications

References 26 publications

Crude garlic extract significantly inhibits replication of grapevine viruses

Crude garlic extract significantly inhibits replication of grapevine viruses

Selection, optimization and characterization of molecular tests for the detection of Tobacco ringspot virus (TRSV)

Grapevine leafroll-associated virus 1

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