Development and Validation of LAMP Assays for Distinguishing MPXV Clades with Fluorescent and Colorimetric Readouts

Abstract

Human monkeypox (Mpox) is a zoonotic disease caused by the Monkeypox virus (MPXV). As of 14 August 2024, the World Health Organization (WHO) has declared it a global health emergency. For Mpox, this was the second public health emergency of global significance in the past two years. MPXV belongs to the Poxviridae family and is phylogenetically and epidemically divided into two clades: the Congo Basin (Clade-I) and the West African (Clade-II) clades. Clade-I has been associated with more severe disease progression and higher mortality compared to Clade-II, and thus the differentiation between clades can play an important role in predicting disease prognosis. The LAMP technique has the advantages of not requiring thermal cycling and achieving higher amplification in a shorter time compared to qPCR. Different types of LAMP assays were developed in this study to benefit from these advantages. We report the development of LAMP-1 and LAMP-2 assays using the LAMP method to detect MPXV Clade-I and Clade-II, respectively. The LAMP-1 assay includes both fluorescence and visible colorimetric readout tests developed with sensitivities of 10³ and 10⁷ copies, respectively. For the LAMP-2 assay, a probe-based test utilizing the Novel R-Duplex DARQ probe was developed, offering fluorescence detection at a sensitivity of 10³ copies. As a result, we successfully developed three highly specific molecular diagnostic tests that distinctly differentiate between MPXV clades, delivering essential tools for the precise diagnosis and effective control of Mpox.

Keywords: loop-mediated isothermal amplification technology; monkeypox virus clade detection; novel reverse-duplex detection of amplification by release of quenching probe.

Figure 1

Three different molecular tests were developed using the LAMP method for MPXV Clade detection and are shown in the figure. Schematic diagram showing the workflow of LAMP-1 and LAMP-2 for Clade-I and Clade-II detection. LAMP-1 assay was used to detect Clade-I. The amplification was verified based on fluorescent emission and a colorimetric change from pink to yellow. LAMP-2 assay was used to detect Clade-II. The probe-based LAMP method can achieve fluorescence detection by using the R-Duplex DARQ probe for Clade-II detection.

Figure 2

A scheme depicting primers and target gene for Clade-I detection, specificity, and sensitivity results. (A) Location of LAMP-1 assay primers on D14L sequences. (B) LAMP-1 fluorescence reading results for clade detection. The successful amplification of Clade-I within approximately 18 min with no Clade-II amplification indicates high specificity of the assay. (C) LoD analysis of template DNA with 10-fold serial dilution samples for LAMP-1 assay. The sensitivity of the tests is 10³ copies. PTC: positive control. NTC: negative control.

Figure 3

LAMP-1 assay colorimetric reading results. (A) Screenshot of the developed MATLAB-based GUI. The interface displays sections for image upload, vial count entry, and sample selection through diagonal corner coordinates. The GUI measures average sample intensities and displays them in a bar plot, with options to export the bar plot values as a text file. (B) Screenshot of the MATLAB-based GUI displaying an image with twelve samples, including the negative control tube (Sample 1), with specified corners and the resulting bar plot of average sample intensities. The GUI accurately captures the relative difference in sample intensities, highlighting samples 10 and 11 in comparison to the control sample, which signifies the functionality of the app.

Figure 4

A scheme depicting primers and target gene for Clade-II detection and specificity results. (A) Location of LAMP-2 assay primers on target gene sequences. (B) LAMP-2 fluorescence reading results for Clade detection. The higher fluorescence intensity and earlier amplification of Clade-II indicate the successful detection of Clade-II. PTC: positive control. NTC: negative control.

Figure 5

A schematic illustration of the structure and working principle of the fluorescent probe designed for Clade-II. (A) R-Duplex DARQ probe structure. (B) Working principle of R-Duplex DARQ probe in the LAMP reaction.

Figure 6

(A) Fluorescence readout of LAMP assay with new R- Duplex DARQ probes: In reactions performed with Clade-I DNA, no fluorescent signal was observed when using the R-Duplex DARQ-1 probe (red line) or the R-Duplex DARQ-2 probe (blue line). In contrast, in LAMP reactions performed with Clade-II DNA, fluorescent signal was detected using both the R-Duplex DARQ-1 probe (black line) and the R-Duplex DARQ-2 probe (orange line). This indicates that the probe-based LAMP-2 assay successfully achieved amplification in Clade-II DNA and enabled fluorescence detection. (B) LoD analysis of R- Duplex DARQ-1 probe. (C) LoD analysis of R- Duplex DARQ-2 probe. Both probes showed a sensitivity value of 10³ copies. NTC: negative control.