Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay

Zhao, Fei; Hu, Yamei; Fan, Zhangling; Huang, Baoying; Wei, Liang; Xie, Yu; Huang, Yu; Mei, Shan; Wang, Liming; Wang, Lingwa; Ai, Bin; Fang, Jugao; Liang, Chen; Xu, Fengwen; Tan, Wenjie; Guo, Fei

doi:10.1016/j.crmeth.2023.100620

Cited by 6 publications

(6 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a result, RPA has been used in the detection of MPXV coupled with the CRISPR/Cas12a system. 20,21,33 Yet, this assay still requires a multistep procedure involving column or magnetic beads to prepare DNA samples before starting the RPA and CRISPR/Cas12a assays. To simplify the detection system and aim for field testing, we have developed an one-step sample preparation method and coupled RAA with lateral flow for visual readout, without the need of any laboratory instruments for MPXV detection.…”

Section: Discussionmentioning

confidence: 99%

“…Compared with other isothermal amplification techniques including loop‐mediated isothermal amplification (LAMP) and helicase‐dependent amplification (HDA) nucleic acid sequences‐based amplification (NASBA), the RAA/RPA method has a shorter reaction time, a simpler primer design and much less dependence on special equipment. As a result, RPA has been used in the detection of MPXV coupled with the CRISPR/Cas12a system 20,21,33 . Yet, this assay still requires a multistep procedure involving column or magnetic beads to prepare DNA samples before starting the RPA and CRISPR/Cas12a assays.…”

Section: Discussionmentioning

confidence: 99%

“…One pool targets viral genes D6R and E9L that are conserved among orthopoxviruses including MPXV, the other pool targets regions in the N3R and N4R genes that are nearly 100% conserved among multiple species of MPXV but exhibit low homology with other orthopoxviruses, which is consistent with previous studies. 20,26 The D6R and E9L genes were amplified from vaccinia virus (VACV) and cloned into plasmid vectors. The N3R and N4R genes were synthesized.…”

Section: Design and Test Primers And Probes Targeting Mpxvmentioning

confidence: 99%

“…Isothermal amplification methods have been explored as an alternative to real‐time PCR. Recombinase polymerase amplification (RPA) coupled CRISPR/Cas12 facilitates the rapid and sensitive diagnosis of mpox within 20 min 20,21 . However, the need of laboratory instruments and the complexity of sample extraction and manipulation remain a great challenge for field use.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A field diagnostic method for rapid and sensitive detection of mpox virus

Zhao,

Xu,

Wang

et al. 2024

Journal of Medical Virology

Self Cite

View full text Add to dashboard Cite

The mpox outbreak has subdued with fewer reported cases at the present in high‐income countries. It is known that mpox virus (MPXV) infection has been epidemic for more than 50 years in African countries. The ancestral MPXV strain has changed into multiple clades, indicating the ongoing evolution of MPXV, which reflects the historical neglect of mpox in Africa, especially after smallpox eradication, and bestows the danger of more severe mpox epidemics in the future. It is thus imperative to continue the development of mpox diagnostics and treatments so we can be prepared in the event of a new mpox epidemic. In this study, we have developed an MPXV detection tool that leverages the recombinase‐aid amplification assay by integrating lateral flow strips (RAA‐LF) and one‐step sample DNA preparation, with visible readout, no need of laboratory instrument, and ready for field deployment. The detection limit reaches 10 copies per reaction. The performance of our RAA‐FL assay in diagnosing mpox clinical samples is on par with that of the quantitative polymerase chain reaction (PCR) assay. Taken together, we have developed a point‐of‐care RAA‐LF method of high accuracy and sensitivity, readily deployable for field detection of MPXV. This diagnostic tool is expected to improve and accelerate field‐ and self‐diagnosis, allow timely isolation and treatment, reduce the spread of MPXV, thus effectively mitigate MPXV outbreak in the future.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Design and Test Primers And Probes Targeting Mpxvmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A field diagnostic method for rapid and sensitive detection of mpox virus

Zhao,

Xu,

Wang

et al. 2024

Journal of Medical Virology

Self Cite

View full text Add to dashboard Cite

show abstract

“…In addition, the integration of an electrochemical biosensor for amplicon detection enhances the applicability of nucleic acid testing . To date, RAA-based viral assays have been developed for the detection of various viruses including HPV, mpox virus, and zika virus, among others. − However, it is difficult to realize quantitative or semiquantitative visual detection of pathogens through the interpretation of terminal data.…”

Section: Introductionmentioning

confidence: 99%

Sensitive, Semiquantitative, and Portable Nucleic Acid Detection of Rabies Virus Using a Personal Glucose Meter

Lu,

Bai,

Wang

et al. 2024

ACS Omega

View full text Add to dashboard Cite

Rabies is a zoonotic infection with the potential to infect all mammals and poses a significant threat to mortality. Although enzyme-linked immunosorbent tests and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) have been established for rabies virus (RABV) detection, they require skilled staff. Here, we introduce a personal glucose meter (PGM)-based nucleic acid (NA-PGM) detection method to diagnose RABV. This method ensures sensitive and convenient RABV diagnosis through hybridization of reverse transcription-recombinase aided amplification (RT-RAA) amplicons with probes labeled with sucrose-converting enzymes, reaching a detection level as low as 6.3 copies/μL equivalent to 12.26 copies. NA-PGM allows for the differentiation of RABV from other closely related viruses. In addition, NA-PGM showed excellent performance on 65 clinical samples with a 100% accuracy rate compared with the widely adopted RT-qPCR method. Thus, our developed NA-PGM method stands out as sensitive, semiquantitative, and portable for RABV detection, showcasing promise as a versatile platform for a wide range of pathogens.

show abstract

A clinically used anti‐human papilloma virus agent (3‐hydroxyphthalic anhydride‐modified bovine β‐lactoglobulin) has a potential for topical application to prevent sexual transmission of monkeypox virus

Sha,

Huang,

Hua

et al. 2024

MedComm

View full text Add to dashboard Cite

A global outbreak of monkeypox (mpox) caused by the mpox virus (MPXV) has posed a serious threat to public health worldwide, thus calling for the urgent development of antivirals and vaccines to curb its further spread. In this study, we screened 41 anhydride‐modified proteins and found that 3‐hydroxyphthalic anhydride‐modified β‐lactoglobulin (3HP‐β‐LG), a clinically used anti‐HPV agent, was highly effective in inhibiting infection of vaccinia virus Tiantan strain (VACV‐VTT) and MPXV. Mechanistic studies demonstrated that 3HP‐β‐LG bound to the virus, not the host cell, by targeting the early stage of virus entry, possibly through the interaction between the amino acids with negatively charges in 3HP‐β‐LG and the key amino acids with positive charges in the target region of A29L, a key surface protein of MPXV. A synergistic effect was observed when 3HP‐β‐LG was combined with tecovirimat, a small‐molecule antiviral drug approved by the United States Food and Drug Administration and the European Medicine Agency for the treatment of smallpox and mpox. Because of its clinically proven safety and stability, 3HP‐β‐LG shows promise for further development as a prophylactic agent to prevent the sexual transmission of MPXV.

show abstract

Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay

Cited by 6 publications

References 49 publications

A field diagnostic method for rapid and sensitive detection of mpox virus

A field diagnostic method for rapid and sensitive detection of mpox virus

Sensitive, Semiquantitative, and Portable Nucleic Acid Detection of Rabies Virus Using a Personal Glucose Meter

A clinically used anti‐human papilloma virus agent (3‐hydroxyphthalic anhydride‐modified bovine β‐lactoglobulin) has a potential for topical application to prevent sexual transmission of monkeypox virus

Contact Info

Product

Resources

About