Study: CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing. Image Credit: Dotted Yeti / Shutterstock
The monkeypox virus (MPXV), which was first isolated in 1958, is a double-stranded DNA (dsDNA) virus. It was recognized as a zoonotic disease when the first human case was detected in the Democratic Republic of the Congo in 1970. There is currently a global concern over MPXV due to its wider transmission from Central and West Africa.
Rapid, ultrasensitive, and specific detection is essential to curb this virus's spread. In a new study posted to the medRxiv* preprint server, researchers in China developed an MPXV assay combining clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/Cas) and recombinase-aided amplification (RAA) for the first time. The assay exhibited high selectivity and could distinguish between MPXV and other orthopoxviruses.
Background
Currently, MPXV testing uses methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP). PCR is the gold standard and is accurate and sensitive but challenging to deploy in low-resource areas. ELISA could provide false positive results for recent or remote vaccination, and LAMP’s disadvantages are related to a complicated primer design, poor quantitative performance, etc. Rapid, ultrasensitive, and low-cost methods facilitating on-site and facile MPXV detection have remained absent.
About the Study
CRISPR/Cas was first discovered in the adaptive immune system of prokaryotes. CRISPR/Cas12a system integrates signal transduction, and biorecognition has been used to detect several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has many advantages related to mild conditions, high sensitivity, operational ease, and powerful signal amplification.
To date, critical issues related to using CRISPR technology in MPXV detection, such as probe screening, analytical performance, pre-amplification, and point-of-care testing (POCT), have not been reported. Therefore, the current study proposed a rapid and ultrasensitive assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a, i.e., the RAA-Cas12a-MPXV assay, for the first time. The principle comprised three steps: RAA amplification, CRISPR/Cas12a cleavage, and signal output.
Key Results
In the first step, RAA selecting the DNA template produced a large number of amplicons. This augmented the sensitivity of the assay significantly. In the second step, the trans-cleavage activity of Cas12a was activated, which resulted in numerous ssDNA reporters being cleaved. The last step produced two different signal output modes: fluorescence assay for FQ reporters and lateral flow strip, which improved the usability and suitability of the RAA-Cas12a-MPXV assay.
Fluorescence results with or without DNA templates and the RAA product were compared to evaluate the feasibility of the assay. The no DNA group was the control, which showed no significant fluorescence change. This indicated that, in the absence of DNA templates, no RAA amplicon was generated for it to be recognized by subsequent CRISPR/Cas12a.
To ensure greater selectivity and sensitivity, three pairs of primers that bound to various sites of the MPXV-specific F3L gene were designed. To screen the optimal primers for future experiments, the RAA-Cas12a-MPXV fluorescence assay and the agarose gel electrophoresis (AGE) were performed. The third pair (F2+R2) achieved the brightest specific amplification band.
Many important experimental conditions related to the CRISPR/Cas12a system and the RAA-Cas12a-MPXV assay were optimized, with RAA's temperature and reaction time being the first. Next, the CRISPR/Cas12a system with crRNA2 was optimized.
The RAA-Cas12a-MPXV fluorescence assay was deemed to be easy to operate and amenable to be used for rapid testing. Compared to the PCR-Cas12a-MPXV fluorescence assay, the RAA-Cas12a-MPXV fluorescence assay revealed outstanding sensitivity with a 1000-fold lower limit of detection (LOD).
The selectivity of the RAA-Cas12a-MPXV fluorescence assay was determined by comparing the degree of fluorescence produced by MPXV with other viruses, such as variola virus (VARV), cowpox virus (CPXV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Toxoplasma Gondii virus (TOXV), and African swine fever virus (ASFV). Both naked eye observation and fluorescence values indicated that only MPXV induced enhanced fluorescence, while other viruses did not.
The FAM-Biotin reporter (FB-reporter) reporter was designed as a substitute for the FQ reporter of the fluorescence assay, and subsequently, the RAA-Cas12a-MPXV lateral flow strip assay was established for POCT. The addition of a reaction solution containing intact FB reporters cracked FAMs and biotin in the sample pads caused rapid binding of the anti-FAM antibody/AuNP complexes with isolated FAMs and FB reporters as the sample migrated forward. Finally, streptavidin, immobilized at the control band, captured cracked biotins and the FB reporter/anti-FAM antibody/AuNP complexes.
Initially, the control band appeared red due to the aggregation of AuNPs. Subsequently, the rest of the isolated FAM/anti-FAM antibody/AuNP complexes traveled towards the test band and reacted with FAM antibodies appearing red. Finally, amplicons activated Cas12a to cleave all FB reporters. The change of color only remained at the test band.
In a nutshell, when the color change was observed only in the test band or both the control and the test band, it was considered a positive result. In contrast, if the color change was only found at the control band, it indicated a negative result, i.e., the absence of DNA templates led to no amplicons for Cas12a activation.
Conclusions
A vital advantage of the RAA-Cas12a-MPXV assay is that it can be conducted at a mild temperature compared to conventional processes. Importantly, this assay was a powerful MPXV diagnosis method with superior selectivity, sensitivity, and portability.
