Compact Rapid Detection Kit for Testing for Monkeypox Virus Shows Promising Results in New Research

Compact Rapid Detection Kit for Testing for Monkeypox Virus Shows Promising Results in New Research

In a recent report in the magazine Travel medicine and infectious diseases, researchers introduced a compact rapid detection kit that tests for monkeypox viruses using a combination of recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology.

Lernen: Taschenlabor zum schnellen Nachweis des Affenpockenvirus. Bildquelle: Tatiana Buzmakova/Shutterstock

background

The monkeypox outbreak has spread far beyond the endemic countries of West and Central Africa in 2022, and the World Health Organization (WHO) has now declared it a public health emergency of international concern. The etiological pathogen of monkeypox is the monkeypox virus, which belongs to the genus of orthopoxviruses.

The test currently used to detect monkeypox is the polymerase chain reaction (PCR) test, which amplifies the deoxyribonucleic acid (DNA) of orthopoxvirus or specific monkeypox viruses. However, PCR is a lengthy process that requires a thermal cycler, trained personnel and a laboratory.

Early detection and containment of infected individuals is essential to controlling a disease outbreak. Immunoassays such as lateral flow strips are simpler and faster than PCRs, but have higher cross-reactivity for orthopoxviruses, making accurate monkeypox detection difficult. Therefore, technology that detects monkeypox quickly and provides accurate and reliable results is critical.

Detection mechanism

In the present study, researchers developed a rapid detection kit or pocket laboratory for testing for monkeypox virus. The detection kit uses the principles of RPA and CRISPR technology to increase the specificity and sensitivity of the analysis. RPA amplifies monkeypox virus target genes that are scanned by the CRISPR guide ribonucleic acid (RNA) and the CRISPR-associated protein 12a (Cas12a) enzyme.

CRISPR/Cas12sa-mediated cleavage of the sequence occurs only when the monkeypox target amplicons are recognized, thereby eliminating the possibility of nonspecific signals. The cleavage results in fluorescence, indicating monkeypox-positive samples.

Pocket lab

The kit weighed 500g and the case was waterproof and compact enough to fit in a bag. The components consisted of two three-dimensionally printed heating blocks – Block A, which heats the samples to 80 °C for viral envelope lysis, and Block B, which heats the samples to 40 °C for DNA amplification.

The kit included tubes containing trehalose-protected lyophilized enzymes and sample tubes containing sodium chloride and magnesium acetate solution. A rehydration buffer containing primers and the fluorescence reporter as well as an ultraviolet (UV) flashlight to detect fluorescence were also included in the kit.

During the testing process, the sample is placed in the sample tube and the virus envelope is lysed using a heating block A. The heated sample is added to the tube containing the lyophilized enzymes and the rehydration buffer is added. Heating block B is then used to reinforce the target region. Finally, the fluorescence is detected with the UV flashlight.

During the experimental test, researchers conducted pseudotyped monkeypox virus samples, the total time was 25 minutes, and samples containing only ten virus particles were detected with accuracy.

Conclusions

In conclusion, the researchers presented a novel rapid detection kit that was compact and could be used to detect monkeypox virus without the use of complicated instruments, trained technicians, or a laboratory setup.

The detection kit uses the principles of RPA and CRISPR to amplify target regions of monkeypox DNA and cleave them using the CRISPR/Cas12a complex, with a fluorescent reporter signaling the detection of monkeypox virus DNA.

The tests with the pseudotyped virus showed successful and rapid detection of the monkeypox virus, suggesting that the kit could potentially be used for rapid testing of travelers and in areas that lack adequate medical infrastructure and trained personnel.

Reference:

• Wei, J., Meng, X., Li, J. & Pang, B. (2022). Taschenlabor zum schnellen Nachweis des Affenpockenvirus. Reisemedizin und Infektionskrankheiten. tue ich: https://doi.org/10.1016/j.tmaid.2022.102478 https://www.sciencedirect.com/science/article/pii/S1477893922002241?via%3Dihub

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