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 now global concern about MPXV due to its wider transmission from central and western Africa.
Rapid, ultra-sensitive and specific detection is critical to slowing the spread of this virus. In a new study published on the medRxiv* preprint server, researchers in China developed an MPXV assay that combines clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/Cas) and recombinase-assisted amplification (RAA) for the first time. The assay exhibited high selectivity and was able to distinguish between MPXV and other orthopoxviruses.
Study: CRISPR/Cas12a-mediated Ultrasensitive In Situ Monkeypox Viral Test. Image Credit: Dotted Yeti / Shutterstock
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 difficult to implement in low resource areas. ELISA could give false positive results for recent or remote vaccinations, and disadvantages of LAMP are related to complicated primer design, poor quantitative performance, etc. Rapid, ultrasensitive, low-cost methods that facilitate on-site detection of MPXV remain lacking.
About the study
CRISPR/Cas was first discovered in the adaptive immune system of prokaryotes. The 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 smooth conditions, high sensitivity, ease of operation, and powerful signal amplification.
To date, no critical issues related to the use of CRISPR technology in MPXV detection, such as probe detection, analytical performance, pre-amplification, and point-of-care testing (POCT), have been reported. Therefore, the current study proposed for the first time a rapid and ultrasensitive assay combining recombinase-assisted amplification (RAA) and CRISPR/Cas12a, namely the RAA-Cas12a-MPXV assay. The principle consisted of 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 significantly increased the sensitivity of the assay. In the second step, the trans cleavage activity of Cas12a was activated, resulting in the cleavage of numerous ssDNA reporters. The last step produced two different signal output modes: fluorescence assay for CF 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 RAA product were compared to assess assay feasibility. The DNA-free group was the control, showing no significant fluorescence change. This indicated that, in the absence of DNA templates, no RAA amplicon was generated to be recognized by subsequent CRISPR/Cas12a.
To ensure greater selectivity and sensitivity, three pairs of primers were designed that bind to various sites of the MPXV-specific F3L gene. To select the optimal primers for future experiments, the RAA-Cas12a-MPXV fluorescence assay and 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, the first being RAA reaction temperature and time. Next, the CRISPR/Cas12a system was optimized with crRNA2.
The RAA-Cas12a-MPXV fluorescence assay was considered to be easy to operate and suitable to be used for rapid testing. Compared to the PCR-Cas12a-MPXV fluorescence assay, the RAA-Cas12a-MPXV fluorescence assay revealed exceptional sensitivity with a lower limit of detection (LOD) of 1000-fold.
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 smallpox virus (VARV), bovine pox 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) was designed as a substitute for the FQ reporter of the fluorescence assay, and later the RAA-Cas12a-MPXV lateral flow strip assay for POCT was established. Addition of a reaction solution containing cracked FAM intact FB markers and biotin to the sample pads caused rapid binding of anti-FAM antibody/AuNP complexes with isolated FAM and FB markers as the sample migrated forward. Finally, streptavidin, immobilized on the control band, captured the disrupted biotins and the FB reporter/anti-FAM antibody/AuNP complexes.
Initially, the control band appeared red due to AuNP aggregation. Subsequently, the rest of the isolated FAM/anti-FAM antibody/AuNP complexes traveled towards the test band and reacted with the FAM antibodies appearing in red. Finally, the amplicons activated Cas12a to cleave all FB reporters. The color change was only maintained in the test band.
Briefly, when the color change was seen only in the test band or in both the control and the test band, it was considered a positive result. In contrast, if the color change was only found in the control band, it indicated a negative result, that is, the absence of DNA templates did not generate amplicons for Cas12a activation.
Conclusions
A key advantage of the RAA-Cas12a-MPXV assay is that it can be performed at a moderate temperature compared to conventional processes. Importantly, this assay was a powerful MPXV diagnostic method with superior selectivity, sensitivity, and portability.
*Important news
medRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice or health-related behavior, or be treated as established information.
Source: news.google.com