Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

Menting, Sandra; Erhart, Annette; Schallig, Henk D. F. H.

doi:10.3390/tropicalmed8060320

Cited by 2 publications

(1 citation statement)

References 21 publications

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“…During the past three years, the improvements and advancements in POC diagnostics research have highlighted the versatility, efficiency, and efficacy of isothermal amplification techniques, especially loop-mediated isothermal amplification (LAMP). Methods like LAMP [7,8] or reverse transcriptase LAMP (RT-LAMP) [9], recombinase polymerase amplification (RPA) [10,11], and helicase-dependent amplification (HDA) or reverse transcriptase HDA (RT-HDA) [12] have emerged as potential alternatives to RT-PCR. More recently, modified isothermal techniques, like SHARP [13] and CRISPR-based detection methods (SHERLOCK, DETECTR, etc.)…”

Section: Introductionmentioning

confidence: 99%

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

Agarwal,

Hamidizadeh,

Bier

2023

Biosensors

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This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

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Section: Introductionmentioning

confidence: 99%

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

Agarwal,

Hamidizadeh,

Bier

2023

Biosensors

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A Sequencing-Based Phylogenetic Analysis of Various Strains of Watermelon Silver Mottle Virus in Northern China and Their One-Step Detection Using Reverse Transcription Loop-Mediated Isothermal Amplification

Qiao,

Jiang,

Chen

et al. 2024

Plant Disease

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Watermelon silver mottle virus (WSMoV), a potentially invasive virus, is known to reduce the yield and degrade the quality of infected crops in Cucurbitaceae and Solanaceae families, resulting in significant economic losses in limited areas of several Asian countries. WSMoV previously detected on various crops in southern China has now become more prevail on watermelon and sweet pepper in northern cities of China for the first time. A sequencing-based phylogenetic analysis has confirmed that the viral strains infecting cucumber, watermelon and sweet pepper plants in Shandong Province are most closely related to those isolated from Guangdong, Guangxi and Taiwan, suggesting a farther and continuous spread of WSMoV throughout China. To develop a fast, accurate, and practical protocol for WSMoV detection, we designed a set of primers from the conserved sequence of WSMoV nucleocapsid protein (N) gene for a one-step assay based on the reverse transcription loop-mediated isothermal amplification (RT-LAMP). The RT-LAMP assay was performed successfully for 50 min at 61°C and exhibited a highly specific result without cross-reactions with other similar viruses and a sensitivity that is 100-fold higher than the traditional RT-PCR. The confirmation of 26 WSMoV suspect samples collected from various regions in Shandong through the RT-LAMP testing has demonstrated that the assay is suitable and practical for detection of WSMoV in both laboratory and field settings.

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Laboratory Evaluation of a SARS-CoV-2 RT-LAMP Test

Cited by 2 publications

References 21 publications

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay

A Sequencing-Based Phylogenetic Analysis of Various Strains of Watermelon Silver Mottle Virus in Northern China and Their One-Step Detection Using Reverse Transcription Loop-Mediated Isothermal Amplification

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