Loop-mediated isothermal amplification combined with lateral flow biosensor for rapid and sensitive detection of monkeypox virus

Abstract

The ongoing outbreak of the monkeypox, caused by monkeypox virus (MPXV), has been a public health emergency of international concern, indicating an urgent need for rapid and sensitive MPXV detection. Here, we designed a diagnostic test based on loop-mediated isothermal amplification (LAMP) and nanoparticle-based lateral flow biosensor(LFB)for diagnosis of MPXV infection, termed MPX-LAMP-LFB. A set of six LAMP primers was designed based the ATI gene of MPXV, and LAMP amplification of MPXV templates was performed at 63°C for only 40 min. The results were rapidly and visually decided using the LFB test within 2 min. The MPX-LAMP-LFB assay can specifically detect MPXV strains without cross-reaction with non-MPXV pathogens. The sensitivity of the MPX-LAMP-LFB assay is as low as 5 copies/μl of plasmid template and 12.5 copies/μl of pseudovirus in human blood samples. The whole process of the MPX-LAMP-LFB assay could be completed ~1 h, including rapid template preparation (15 min), LAMP reaction (40 min)and result reporting (<2 min). Collectively, MPX-LAMP-LFB assay developed here is a useful tool for rapid and reliable diagnosis of MPXV infection.

Rapid detection of monkeypox virus and differentiation of West African and Congo Basin strains using endonuclease restriction-mediated real-time PCR-based testing

“…When analyzing with a pseudotyped virus, the LoD level reached down to 12.5 copies per microliter. Although the sensitivity of multiplex ET-PCR assay was marginally lower than that of previous isothermal amplification-based studies, such as MCDA, 26 LAMP, 24 and RPA 36 techniques, as well as the established real-time PCR methods, 19,20 the newly developed method was able to identify generic, clade I and clade II MPXV strains in a single reaction rather than one or two of them. In addition, the combination of generic and clade-specific testing improves detection accuracy and aids in new clade identification.…”

“…9 Thus, NAATs are preferred laboratory tests to identify and characterize MPXV with rapidness, high sensitivity and specificity. 16 Recently, many NAATs have been developed to confirm MPXV infection, including conventional PCR, 17,18 real-time PCR, 19–21 recombinase polymerase amplification (RPA), 22,23 loop-mediated isothermal amplification (LAMP), 24,25 multiple cross displacement amplification (MCDA), 26 and restriction length fragment polymorphism (RFLP), 10,27 all of which commonly target one specific region of the monkeypox virus genome and possess varying sensitivities and limits of detection (LoD) for MPXV diagnosis. 28 Although the whole genome sequencing technique has also been reported to be deployed for MPXV strain identification, it mainly occurred in centralized and specialized laboratories, 29,30 which were usually not available for large-scale and timely testing.…”

Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing

“…By using LAMP technology, million-fold amplification products achieved within 15–60 min with two or three pairs of primers and the Bst DNA polymerase. Due to the simple amplification procedure and cost-effective instrument, LAMP technology has been utilized for multiple pathogens detection and exhibited extremely high analytical sensitivity and specificity ( Zhu et al, 2020 ; Si et al, 2021 ; Xiao et al, 2022 ; Huang et al, 2023 ). In addition, LAMP products could be analyzed with various formats, including turbidimeters, colorimetric regents, lateral flow biosensor (LFB), fluorescent dyes and more.…”

Electrochemical Paper-Based Nanobiosensor for Rapid and Sensitive Detection of Monkeypox Virus

“…The gold standard method widely used for the identification and diagnosis of Mpox infection is real-time polymerase chain reaction (qPCR) . Besides, other methods based on the immunoglobulins (IgG/IgM), such as lateral flow immunoassays (LFIAs), loop-mediated isothermal amplification (LAMP), and enzyme-linked immunosorbent assay (ELISA), have been already reported in the literature. However, the traditional instrumental methods (qPCR, LAMP, ELISA) require specialized labor to operate, expensive equipment and consumables, and long periods of analysis .…”