Real-time reverse transcription polymerase chain reaction development for rapid detection of Tomato brown rugose fruit virus and comparison with other techniques

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Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials and Methods

Source of viral material

Screening of ToBRFV using end-point RT-PCR

Mechanical transmission

Full genome sequencing

Primer and Taqman® MGB probe design

ToBRFV genotype-specific real-time RT-PCR assay with TaqMan MGB probe

Standard curve

Different methods for sample preparation and comparison of different ToBRFV detection techniques

Immunocapture in real-time PCR tubes

Total RNA extraction

Direct crude extract

Leaf-disk crude extract

Results

Screening of ToBRFV using end-point RT-PCR

Mechanical transmission

Full genome sequencing

Primer, Taqman® MGB probe design and protocol optimisation

ToBRFV specific real-time RT-PCR assay with TaqMan MGB probe

Standard curve

Different methods for sample preparation and comparison of different ToBRFV detection techniques

Discussion

Conclusions

Supplemental Information

Sequencing of Real time RT-PCR products.

DOI: 10.7717/peerj.7928/supp-1

RAW_DATA_Standard_curve.

Standard curves prepared with 10-fold serial dilutions of in vitro-synthesized RNA transcripts from ToBRFV isolate ToB-SIC01/19 using RT-qPCR with TaqMan MGB probe.

DOI: 10.7717/peerj.7928/supp-2

Hybridisation percentage (%) of primer ToB5520F, ToB5598R and ToB-probe5558 against different tobamoviruses that affect tomato and pepper. In the table are reported only the data with the highest sequence homology inside each species.

DOI: 10.7717/peerj.7928/supp-3

Additional Information and Declarations

Competing Interests

Stefano Panno is employed by Molecular Dynamics Srl.

Author Contributions

Stefano Panno performed the experiments, analysed the data, authored or reviewed drafts of the paper, approved the final draft.

Susana Ruiz-Ruiz performed the experiments, analysed the data, authored or reviewed drafts of the paper, approved the final draft.

Andrea Giovanni Caruso performed the experiments, analysed the data, prepared figures and/or tables, approved the final draft, bioinformatic analysis.

Ana Alfaro-Fernandez analysed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, approved the final draft.

Maria Isabel Font San Ambrosio analysed the data, contributed reagents/materials/analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.

Salvatore Davino conceived and designed the experiments, contributed reagents/materials/analysis tools, authored or reviewed drafts of the paper, approved the final draft.

DNA Deposition

The following information was supplied regarding the deposition of DNA sequences:

The sequence data is available at GenBank: MN167466.

Data Availability

The following information was supplied regarding data availability:

Raw data are available in the Supplemental Files.

Funding

The authors received no funding for this work.

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