Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for detection of beet necrotic yellow vein virus

Almasi, Mohammad; Almasi, Galavizh

doi:10.1007/s00705-016-3116-0

Cited by 9 publications

(4 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In addition, for confirmatory checking of the RT-RPA product, optimized conditions for product visualization and downstream sequence analysis revealed the legitimacy of the RT-RPA product, thus validating the method for BNYVV detection. Previously developed molecular methods for BNYVV detection include regular RT-PCR, nested RT-PCR, and IC-RT-LAMP assays ( Henry et al, 1995 ; Morris et al, 2001 ; Almasi and Almasi, 2017 ). However, these methods require gel electrophoresis for downstream product analysis, which hinders high-throughput application.…”

Section: Discussionmentioning

confidence: 99%

“…Later, molecular biology-based standard reverse transcription (RT)-PCR and nested RT-PCR methods were developed to increase the sensitivity of BNYVV detection ( Henry et al, 1995 ; Morris et al, 2001 ). An immunocapture loop-mediated isothermal amplification (IC-RT-LAMP) method subsequently was reported to be more sensitive ( Almasi and Almasi, 2017 ). Hitherto, it has been a challenge developing an ideal assay that can be simple to perform, sensitive, specific, rapid, and at the same time cost-effective and tractable to high-throughput diagnostics.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

2021

View full text Add to dashboard Cite

Rhizomania is a disease of sugarbeet caused by beet necrotic yellow vein virus (BNYVV) that significantly affects sugarbeet yield globally. Accurate and sensitive detection methods for BNYVV in plants and field soil are necessary for growers to make informed decisions on variety selection to manage this disease. A recently developed CRISPR-Cas-based detection method has proven highly sensitive and accurate in human virus diagnostics. Here, we report the development of a CRISPR-Cas12a-based method for detecting BNYVV in the roots of sugarbeet. A critical aspect of this technique is the identification of conditions for isothermal amplification of viral fragments. Toward this end, we have developed a reverse transcription (RT) recombinase polymerase amplification (RPA) for detecting BNYVV in sugarbeet roots. The RT-RPA product was visualized, and its sequence was confirmed. Subsequently, we designed and validated the cutting efficiency of guide RNA targeting BNYVV via in vitro activity assay in the presence of Cas12a. The sensitivity of CRISPR-Cas12a trans reporter-based detection for BNYVV was determined using a serially diluted synthetic BNYVV target sequence. Further, we have validated the developed CRISPR-Cas12a assay for detecting BNYVV in the root-tissue of sugarbeet bait plants reared in BNYVV-infested field soil. The results revealed that BNYVV detection is highly sensitive and specific to the infected roots relative to healthy control roots as measured quantitatively through the reporter signal. To our knowledge, this is the first report establishing isothermal RT-RPA- and CRISPR-based methods for virus diagnostic approaches for detecting BNYVV from rhizomania diseased sugarbeet roots.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

2021

View full text Add to dashboard Cite

show abstract

“…HNB dye can change from purple to sky blue during reactions. HNB detection for viruses [164][165][166][167][168], bacteria [169,170], and fungi [171][172][173][174] has also been developed. There is only a prototype of direct detection of plant pathogen via magnesium pyrophosphate precipitation [175].…”

Section: Colorimetric Detection Of Lamp Ampliconsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

View full text Add to dashboard Cite

Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

show abstract

“…According to previous reports, LAMP method has been widely used in the detection of various diseases in people [10,11]. Moreover, the diagnosis of plant pathology and food quality control can be well achieved by LAMP [12–15]. LAMP technique has also solved the problems of complicated procedures, expensive equipment, and time-consumption in the past.…”

Section: Introductionmentioning

confidence: 99%

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification

Huang

Li²,

et al. 2019

Bioscience Reports

View full text Add to dashboard Cite

Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

show abstract

Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for detection of beet necrotic yellow vein virus

Cited by 9 publications

References 30 publications

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

CRISPR-Based Isothermal Next-Generation Diagnostic Method for Virus Detection in Sugarbeet

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification

Contact Info

Product

Resources

About