Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

Edgü, Güven; Freund, Lena Julie; Hartje, Stefanie; Tacke, Eckhard; Hofferbert, Hans-Reinhard; Twyman, Richard M.; Noll, Gundula A.; Muth, Jost; Prüfer, Dirk

doi:10.3390/ijms21228741

Cited by 14 publications

(9 citation statements)

References 38 publications

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“…For RT-LAMP reactions run on the Axxin T8-ISO (T8), assay components described above were doubled to achieve 50 μL reaction volumes with two exceptions [36]: fluorescent dye was used at a 5X concentration instead of 50X [37], and 0.2 μL of Tte UvrD helicase (NEB) was included with each reaction [38,39]. Reactions contained 10 μL of sample unless otherwise noted.…”

Section: Rt-lamp Reactionsmentioning

confidence: 99%

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

et al. 2022

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The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69–91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

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Section: Rt-lamp Reactionsmentioning

confidence: 99%

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

et al. 2022

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“…Early detection of a pathogen can give growers enough time to devise a disease prevention strategy. Molecular techniques such as PCR and RT-PCR and field-based isothermal techniques such as LAMP, RT-LAMP, RPA, and NASBA are commonly used to detect phytopathogens ( Szemes et al, 2002 ; Lu et al, 2018 ; Wilisiani et al, 2019 ; Edgü et al, 2020 ; Wang et al, 2020b) . However, these techniques are time-consuming, expensive, have a high ratio of nonspecificity or low sensitivity, and cannot be applied to bulk plant samples in the field.…”

Section: Crispr/cas and Viral Diagnostics In Plantsmentioning

confidence: 99%

CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond

Ali

Mahfouz

2021

Plant Physiology

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Molecular engineering of plant immunity to confer resistance against plant viruses holds great promise for mitigating crop losses and improving plant productivity and yields, thereby enhancing food security. Several approaches have been employed to boost immunity in plants by interfering with the transmission or lifecycles of viruses. In this review, we discuss the successful application of CRISPR/Cas (clustered regularly interspaced short palindromic repeats [CRISPR]/CRISPR-associated protein [Cas]) systems to engineer plant immunity, increase plant resistance to viruses, and develop viral diagnostic tools. Furthermore, we examine the use of plant viruses as delivery systems to engineer virus resistance in plants and provide insight into the limitations of current CRISPR/Cas approaches and the potential of newly discovered CRISPR/Cas systems to engineer better immunity and develop better diagnostics tools for plant viruses. Finally, we outline potential solutions to key challenges in the field to enable the practical use of these systems for crop protection and viral diagnostics.

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“…Different methods have been developed for determining amplicons from isothermal nucleic acid amplification technologies, such as electrophoresis gels, fluorescent dyes, and lateral flow dipsticks (LFD). Among these methods, the LFD method, which based on lateral flow immunoassay can visually determine double-labeled amplicons within the short timeframe of 5-10 min 31 , has a great advantage in detecting amplicons. In this study, we aimed to establish an RT-CPA-LFD method combined with reverse transcription (RT) to amplify BPMV RNAs and evaluate the potential of this method for detecting BPMV in soybeans.…”

mentioning

confidence: 99%

A reverse transcription-cross-priming amplification method with lateral flow dipstick assay for the rapid detection of Bean pod mottle virus

Yang

Zhao

Wang

et al. 2022

Sci Rep

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Bean pod mottle virus (BPMV) is a destructive virus that causes serious economic losses in many countries every year, highlighting the importance of its effective detection. In this study, we developed a fast reverse transcription-cross-priming amplification (RT-CPA) coupled with lateral flow dipstick (LFD) diagnostic method for BPMV detection. The RT-CPA-LFD assay that targets the coat protein gene of BPMV was highly specific against diagnosing four other common viruses transmitted by soybean seeds, i.e., Southern bean mosaic virus (SBMV), Tomato ringspot virus (ToRSV), Arabis mosaic virus (ArMV), and Tobacco ringspot virus (TRSV). The sensitivities of the real-time fluorescent RT-CPA and the RT-CPA-LFD assay were at least 50 pg/μl and 500 pg/μl, respectively. Despite a compromise in the limit of detection of the RT-CPA method compared with TaqMan-MGB real-time RT-PCR, our results demonstrated a notably better performance in the detection of field samples of BPMV-infested soybean seeds. With the advantages of efficiency and convenience by visual determination, the RT-CPA-LFD assay presents a potential application for the rapid and accurate detection of BPMV in routine tests.

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Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

Cited by 14 publications

References 38 publications

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing

CRISPR/Cas systems versus plant viruses: engineering plant immunity and beyond

A reverse transcription-cross-priming amplification method with lateral flow dipstick assay for the rapid detection of Bean pod mottle virus

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