Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

Waliullah, Sumyya; Ling, Kai‐Shu; Cieniewicz, Elizabeth; Oliver, Jonathan E.; Ji, Pingsheng; Ali, Emran

doi:10.3390/ijms21051756

Cited by 34 publications

(22 citation statements)

References 52 publications

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“…Both LAMP and RT-LAMP use a specially designed four to six primer set as well as DNA polymerisation with strand-displacing activity to generate amplification products. These products can be visually detected using agarose gel electrophoresis or through colour changes visualised by adding fluorescent dyes to the reaction mixture (Notomi et al, 2000;Parida, Sannarangaiah, Dash, & Rao, 2008;Waliullah et al, 2020). Currently, the RT-LAMP technique has been successfully used in the detection of many RNA plant pathogens (Anandakumar et al, 2020;Bhat et al, 2013;He, Xue, Xu, & Wang, 2016;Liu et al, 2010;Zhang et al, 2020).…”

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confidence: 99%

Rapid visual detection of Japanese hornwort mosaic virus infecting Angelica sinensis by reverse transcription loop‐mediated isothermal amplification

Zhang

Wang

Xie

et al. 2020

Annals of Applied Biology

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Japanese hornwort mosaic virus (JHMV; genus Potyvirus, family Potyviridae) is a widespread virus that infects angelica (Angelica sinensis [Oliv.] Diels), an important Chinese herbal medicine plant grown in Gansu, China. JHMV infection has contributed to the deterioration in angelica quality and a reduction in yield. Consequently, there is a need to develop a reliable, simple and rapid detection method to accurately identify JHMV infection and help limit its spread. We describe here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) developed to detect the coat protein gene of JHMV. RT-LAMP amplification products were assessed through realtime fluorescence detection and by gel electrophoresis and SYBR Green I DNA staining for visual observation. This assay successfully detected JHMV in infected plants without cross reactivity recorded from six other plant viruses. Optimum LAMP reactions were conducted in betaine-free media with 6 mM Mg 2+ at 60 C for 60 min for JHMV. The detection limit was 0.28 pg/ml using RT-LAMP for JHMV plasmids. This detection limit for the RT-LAMP assay was 100 times lower than that of the conventional RT-polymerase chain reaction (RT-PCR) assay. Our field survey of angelica crops for JHMV using RT-LAMP further demonstrated a higher sensitivity than RT-PCR, detecting 78% versus 72%. Agreement (κ) between the results obtained from the two tests was 0.844. We found RT-LAMP is accurate and efficient in diagnosis and potentially improving JHMV disease management and forecasting in A. sinensis in China.

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confidence: 99%

Rapid visual detection of Japanese hornwort mosaic virus infecting Angelica sinensis by reverse transcription loop‐mediated isothermal amplification

Zhang

Wang

Xie

et al. 2020

Annals of Applied Biology

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“…In this study, we developed a LAMP-based quick detection protocol of M. partityla which can be done in on-site settings. This method can be run in the field using a colorimetric dye for visual confirmation or using a real-time amplification machine for results in less than one hour [34,35]. In this study, direct extraction of DNA from a single female isolated from root galls greatly improved the efficiency of the Extract-N-Amp™ Tissue PCR Kit.…”

Section: Plos Onementioning

confidence: 98%

Rapid detection of pecan root-knot nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

et al. 2020

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Meloidogyne partityla is the dominant root-knot nematode (RKN) species parasitizing pecan in Georgia. This species is known to cause a reduction in root growth and a decline in the yields of mature pecan trees. Rapid and accurate diagnosis of this RKN is required to control this nematode disease and reduce losses in pecan production. In this study, a loop-mediated isothermal amplification (LAMP) method was developed for simple, rapid, and on-site detection of M. partityla in infested plant roots and validated to detect the nematode in laboratory and field conditions. Specific primers were designed based on the sequence distinction of the internal transcribed spacer (ITS)-18S/5.8S ribosomal RNA gene between M. partityla and other Meloidogyne spp. The LAMP detection technique could detect the presence of M. partityla genomic DNA at a concentration as low as 1 pg, and no cross reactivity was found with DNA from other major RKN species such as M. javanica, M. incognita and M. arenaria, and M. hapla. We also conducted a traditional morphology-based diagnostic assay and conventional polymerase chain reaction (PCR) assay to determine which of these techniques was less time consuming, more sensitive, and convenient to use in the field. The LAMP assay provided more rapid results, amplifying the target nematode species in less than 60 min at 70˚C, with results 100 times more sensitive than conventional PCR (~2-3 hrs). Morphology-based, traditional diagnosis was highly time-consuming (2 days) and more laborious than conventional PCR and LAMP assays. These features greatly simplified the operating procedure and made the assay a powerful tool for rapid, on-site detection of pecan RKN, M. partityla. The developed LAMP assay will facilitate accurate pecan nematode diagnosis in the field and contribute to the management of the pathogen.

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“…This tool is extremely sensitive and specific due to amplification of nucleic acids accomplished by using up to six primers ( Becherer et al., 2020 ). Isothermal and energy efficient intensification requirements of LAMP technology makes it a prime candidate for rapid and inexpensive alternative assays ( Waliullah et al., 2020 ). Thus, LAMP is well established in several areas including medicine, agriculture, and food industries ( Mori and Notomi, 2009 ; Guan et al., 2010 ; Panno et al., 2020 ).…”

Section: Molecular Tools For Detection Of Fungimentioning

confidence: 99%

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

Hariharan¹,

Prasannath²

2021

Front. Cell. Infect. Microbiol.

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Phytopathogenic fungal species can cause enormous losses in quantity and quality of crop yields and this is a major economic issue in the global agricultural sector. Precise and rapid detection and identification of plant infecting fungi are essential to facilitate effective management of disease. DNA-based methods have become popular methods for accurate plant disease diagnostics. Recent developments in standard and variant polymerase chain reaction (PCR) assays including nested, multiplex, quantitative, bio and magnetic-capture hybridization PCR techniques, post and isothermal amplification methods, DNA and RNA based probe development, and next-generation sequencing provide novel tools in molecular diagnostics in fungal detection and differentiation fields. These molecular based detection techniques are effective in detecting symptomatic and asymptomatic diseases of both culturable and unculturable fungal pathogens in sole and co-infections. Even though the molecular diagnostic approaches have expanded substantially in the recent past, there is a long way to go in the development and application of molecular diagnostics in plant diseases. Molecular techniques used in plant disease diagnostics need to be more reliable, faster, and easier than conventional methods. Now the challenges are with scientists to develop practical techniques to be used for molecular diagnostics of plant diseases. Recent advancement in the improvement and application of molecular methods for diagnosing the widespread and emerging plant pathogenic fungi are discussed in this review.

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Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

Cited by 34 publications

References 52 publications

Rapid visual detection of Japanese hornwort mosaic virus infecting Angelica sinensis by reverse transcription loop‐mediated isothermal amplification

Rapid visual detection of Japanese hornwort mosaic virus infecting Angelica sinensis by reverse transcription loop‐mediated isothermal amplification

Rapid detection of pecan root-knot nematode, Meloidogyne partityla, in laboratory and field conditions using loop-mediated isothermal amplification

Recent Advances in Molecular Diagnostics of Fungal Plant Pathogens: A Mini Review

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