Plant Virus Diagnostics: Traditional to Recent and Emerging Advances

Nabi, Sajad Un; Yadav, Manoj Kumar

doi:10.1007/978-981-15-6275-4_5

Cited by 6 publications

(4 citation statements)

References 81 publications

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“…), chemical methods are rarely used to control plant viruses; hence, robust, timely and precise detection and diagnosis is the only option to check the spread of viruses in apples. Protein-based diagnostic assays, such as ELISA, are relatively less sensitive than PCR and are time-consuming for testing of multiple virus infections (Baranwal et al 2021). Several diagnostic procedures and assays have been optimized and finally developed for the concurrent detection of number of viruses/viroids infecting apples (Kumar et al 2014).…”

Section: Introductionmentioning

confidence: 99%

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

Nabi

Madhu

Rao³

2022

Letters in Applied Microbiology

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Significance and Impact of the Study: In India, a new virus, apple necrotic mosaic virus (ApNMV), has been found to infect the apple trees along with ApMV, apple stem pitting virus (ASPV) and apple stem grooving virus (ASGV). This study was designed to detect the ApNMV along with three other viruses (ApMV, ASPV and ASGV) simultaneously using multiplex RT-PCR in a single tube. The assay developed in this study will be an effective detection tool for large-scale surveys and indexing of large apple germplasm for the production of virus-free planting material in temperate fruits. This will also help in studying the distribution of all the four viruses in apple growing regions of India and abroad.

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Section: Introductionmentioning

confidence: 99%

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

Nabi

Madhu

Rao³

2022

Letters in Applied Microbiology

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“…Our objects were three plant viruses, namely potato virus Y (PVY), potato virus S (PVS), and potato leafroll virus (PLRV), which are of interest for crop protection in agriculture. 22,23 Choosing regimes for LFT, we used our experience in the realization of RPA-LFT for single potato virus X (PVX) detection 10 that did not include ssDNA interactions. In this study, we designed and selected bifunctional oligonucleotides with different ssDNA tags and primers specific to viruses.…”

Section: ■ Introductionmentioning

confidence: 99%

“…For this study, we chose RNA viruses for the target because they are the most difficult natural case for multiplex assay due to the need for an additional reverse transcription (RT) reaction, potentially leading to cross-reactivity and additional nonspecific reactions. Our objects were three plant viruses, namely potato virus Y (PVY), potato virus S (PVS), and potato leafroll virus (PLRV), which are of interest for crop protection in agriculture. , Choosing regimes for LFT, we used our experience in the realization of RPA-LFT for single potato virus X (PVX) detection that did not include ssDNA interactions. In this study, we designed and selected bifunctional oligonucleotides with different ssDNA tags and primers specific to viruses.…”

Section: Introductionmentioning

confidence: 99%

Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode—Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay

et al. 2021

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A multiplex assay based on recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a desirable tool for many areas. This multiplex assay could be efficiently realized using single-stranded (ss) DNAs located in separate zones on the test strip and bound complementary ssDNA tags of doublestranded (ds) DNA amplicons. Here, we investigate how to enrich multiplex assay capabilities using ssDNAs. Bifunctional oligonucleotide probes integrating (1) a forward primer for RPA, (2) a C9 spacer to stop polymerase, and (3) a ssDNA tag for binding at test strip are developed. The amplicons have a unique individual ssDNA tag at one end and a universal label of fluorescein introducing through a reverse primer at the other end. A conjugate of gold nanoparticles (GNP) with antibodies to fluorescein is used to detect all amplicons. The remainder of primers after RPA interacting with GNP conjugate was found to be a limiting factor for sensitive and specific multiplex assay. The addition of anti-RPA-primers before the use of test strips was proposed to simply and effectively eliminate remaining primers. This approach was successfully applied for the detection of three priority plant RNA viruses: potato virus Y (PVY), −S (PVS) and potato leafroll virus (PLRV). The total time of the assay is 30 min. The multiplex RPA-LFT detected at least 4 ng of PVY per g of plant leaves, 0.04 ng/g for PVS, and 0.04 ng/g for PLRV. The testing of healthy and infected potato samples showed concordance between the developed assay and reverse transcription-polymerase chain reaction. Thus, the capabilities of the proposed universal modules (ssDNA anchors, bifunctional probes, and blocking anti-primers) for multiplex detection of RNA analytes with high specificity and sensitivity were demonstrated.

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“…Detection and diagnosis are therefore the most critical part of the management of plant viruses. The basis for detecting and identifying viruses are the biological, morphological, and intrinsic properties, which include serological and molecular techniques (Baranwal et al, 2021). Enzyme-linked immunosorbent assay (ELISA) is considered a sensitive and specific method for virus detection (Zahn et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Pathological Changes in Sweet Basil (Ocimum basilicum L.) Caused by Mixed Infection of Tobacco Mosaic Virus and Phytoplasma

et al. 2021

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Egyptian Journal of Botany http://ejbo.journals.ekb.eg/ 59 O CIMUM basilicum L. (a medicinal plant known as sweet basil) was investigated and showed disease symptoms, including mosaic mottling, leaf distortion, blistering, witches' broom, and phyllody. The plants were found to be infected by both tobacco mosaic virus (TMV) and phytoplasma. Positive TMV was indicated by serological identification using direct antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Phytoplasma infection was identified molecularly using the phytoplasma P1/P7 primer pair and the nested primer, R16F2n/R16R2, which successfully generated a DNA band at ~1200 bp. The virus was successfully transmitted mechanically to Nicotiana tabacum, Datura metal, Datura stramonium, and Phaseolus vulgaris. Light and transmission electron microscopy showed pleomorphic phytoplasmas and rod-shaped particles (300 nm) of TMV, respectively. Histopathological and cytopathological studies revealed changes in the cell behavior of the infected leaves of sweet basil due to virus and phytoplasma infection. Hypoplasia and hyperplasia were observed in the mosaic areas of the mesophyll. Alterations in the vascular tissues and degeneration of the organelles were observed in ultrathin sections of the infected mesophyll. The presence of phytoplasma cells and crystalline bodies were indicative of the mixed infection. To our knowledge, this is the first report of a mixed infection of TMV and phytoplasmas in sweet basil.

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Plant Virus Diagnostics: Traditional to Recent and Emerging Advances

Cited by 6 publications

References 81 publications

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

Development of multiplex RT-PCR assay for simultaneous detection of four viruses infecting apple (Malus domestica)

Multiplex Assay of Viruses Integrating Recombinase Polymerase Amplification, Barcode—Anti-Barcode Pairs, Blocking Anti-Primers, and Lateral Flow Assay

Pathological Changes in Sweet Basil (Ocimum basilicum L.) Caused by Mixed Infection of Tobacco Mosaic Virus and Phytoplasma

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