Development of a Loop-Mediated Isothermal Amplification Method for Rapid and Visual Detection of Monkeypox Virus

Abstract

Monkeypox virus (MPXV) is a human pathogenic virus that belongs to the genus Orthopoxvirus. In 2022, MPXV caused an unprecedented number of infections in many countries. As it is difficult to distinguish MPXV from other pathogens by its symptoms in the early stage of infection, a rapid and reliable assay for MPXV detection is needed. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of MPXV and evaluated its application in simulated clinical samples. The A27L-1 and F3L-1 primer sets were identified as the optimal primers, and 63°C was the most appropriate reaction temperature for sequence amplification. The detection limits of the LAMP assay using primer sets A27L-1 and F3L-1 were both 20 copies/reaction mixture, which were >100-fold higher in terms of sensitivity, compared with conventional PCR. The LAMP assay findings were negative for all 21 non-MPXV pathogens, confirming the high specificity of our assay. All three types of simulated clinical samples were clearly identified by our LAMP assay, and the detection limits were consistent with the sensitivity results, indicating efficient clinical sample identification. Our rapid and reliable MPXV LAMP assay could be useful for MPXV detection and on-site diagnosis, especially in primary hospitals and rural areas. IMPORTANCE MPXV outbreaks rapidly grew in the first half of 2022, and this virus has been recognized as an increasing public health threat, particularly in the context of the COVID-19 pandemic. Thus, developing reliable and fast detection methods for MPXV is necessary.

Keywords: loop-mediated isothermal amplification; monkeypox virus; orthopoxvirus; rapid detection; visual detection.

FIG 1

Primer sequence alignment with orthopoxvirus DNA. The primer sets A27L-1 and F3L-1 were aligned with the targeted sequence of DNA within several orthopoxvirus species. Virus strains: monkeypox (MPXV) West African strain MPXV-Singapore 2019 (MT250197), MPXV Congo Basin strain MPXV-Zaire-96-I-16 (GenBank AF380138); MPXV isolate Monkeypox/PT0001/2022 (GenBank ON585029); vaccinia virus Acambis/2019 (GenBank MT227314); cowpox virus CPXV/Cepad 332 (GenBank MK035759); variola major virus Bangladesh-1975 (GenBank L22579); variola minor virus (GenBank Y16780). (A) Sequence alignment of primer set A27L-1 with orthopoxvirus DNA; (B) Sequence alignment of primer set F3L-1 with orthopoxvirus DNA. CB, Congo Basin; WA, West African; F3, outer forward primer; B3, outer backward primer; FIP, forward inner primer F1c-F2; BIP, backward inner primer B1c-B2; LF, loop forward primer; LB, loop backward primer.

FIG 2

Analysis of optimal primers and reaction temperature for LAMP assay. (A) The best primer set for amplification of the A27L gene in the LAMP assay. (B) The optimal reaction temperature for the LAMP assay with the A27L-1 primer set. (C) The best primer set for amplifying F3L in the LAMP assay. (D) The optimal reaction temperature for the LAMP assay with the primer set F3L-1.

FIG 3

LAMP assay sensitivity for MPXV. (A and B) The sensitivity of LAMP reaction with primer set A27L-1, detected by turbidity and visual observation. (D and E) The sensitivity of LAMP reaction with primer set F3L-1, detected by turbidity and visual observation. (C and F) The sensitivity of the conventional PCR assay using primers that target the A27L and F3L genes of MPXV. The turbidity was measured using a Loopamp real-time turbidimeter, and the result also was detected by visual observation according to the color change from orange to green. Five-microliter volumes of PCR products of conventional PCR were separated by 1% agarose gel electrophoresis, and samples positive for the MPXV A27L gene showed a DNA fragment at 206 bp, while those positive for the F3L gene showed a DNA fragment at 193 bp. Lanes: 1, 10⁷ copies/μL; 2, 10⁶ copies/μL; 3, 10⁵ copies/μL; 4, 10⁴ copies/μL; 5, 10³ copies/μL; 6, 10² copies/μL; 7, 10¹ copies/μL; 8, 10⁰ copies/μL.

FIG 4

LAMP assay specificity for MPXV. (A) The specificity of the LAMP reaction with primer set A27L-1, detected by turbidity. Color key: PC-1, positive control (MPXV pUC-A27L recombinant plasmid); PC-2, positive control (pseudovirus MPXV-A27L); NC, negative control (sterile water); 1, variola virus plasmid containing A27L fragment; 2, cowpox virus plasmid containing A27L fragment; 3, vaccinia virus plasmid containing A27L fragment; 4, human coronavirus; 5, influenza B virus; 6, respiratory syncytial virus A; 7, respiratory syncytial virus B; 8, parainfluenza virus; 9, human metapneumovirus; 10, human enterovirus 71; 11, coxsackievirus A; 12, coxsackievirus B; 13, herpes simplex virus 1; 14, herpes simplex virus type 2; 15, varicella-zoster virus; 16, adenovirus; 17, human bocavirus; 18, rhinovirus; 19, Klebsiella pneumoniae; 20, Mycoplasma pneumoniae; 21, Streptococcus pneumoniae; 22, Haemophilus influenzae; 23, Stenotrophomonas maltophilia; 24, Staphylococcus aureus. (B) The specificity of the LAMP reaction with primer set F3L-1, detected by turbidity. The turbidity was measured using a Loopamp real-time turbidimeter. Color key: PC-1, positive control (MPXV pUC-F3L recombinant plasmid); PC-2, positive control (pseudovirus MPXV-F3L); NC, negative control (sterile water); 1, variola virus plasmid containing F3L fragment; 2, cowpox virus plasmid containing F3L fragment; 3, vaccinia virus plasmid containing F3L fragment (remaining color assignments 4 to 24 are those listed for panel A).