In a recent review posted to the medRxiv* preprint server, researchers provided an overview of existing molecular methods for mpox (MPX) virus (MPXV) detection.

Background

The ongoing 2022 MPXV outbreak has affected non-endemic nations across the globe. The swiftly increasing MPX case counts have warranted the development of rapid diagnostic technologies to timely identify and provide the most appropriate treatment to all affected individuals. Molecular techniques are reportedly the most accurate and safe for virus detection.

About the review

In the present study, researchers presented new perspectives and an overview of currently used molecular methods for the detection of MPXV.

Data were retrieved from databases such as Science Direct, PubMed, Scielo, Scopus, and VHL databases, in September 2022, of relevant articles from the period between 2012 and 2022. Only full-text articles, including polymerase chain reaction (PCR), real-time polymerase chain reaction (rt-PCR), and PCR-enzyme linked immunosorbent assays (PCR-ELISA), were analyzed. Case reports, editorial letters, and editorials were excluded, and duplicate articles were removed.

A total of 256 articles were initially identified. Of these, only 12 articles were eligible for further review, three articles were chosen to review MPXV detection by PCR, and nine articles were chosen to review rt-PCR for MPXV detection. Ten articles described PCR-ELISA but did not meet the eligibility criteria., and therefore, were excluded.

Real-time polymerase chain reaction

Real-time PCR is considered the gold standard of MPX diagnosis and differs from conventional PCR in the detection of the amplified product. In traditional PCR, detection is performed by gel electrophoresis, whereas real-time PCR utilizes the property of fluorescence for viral detection, yielding more agility in obtaining results with a lesser contamination risk and greater accuracy.

Further, multiplexed real-time PCR assays can be performed for simultaneous detection and differentiation of MPXV and varicella zoster virus (VZV) with high specificity, sensitivity, and reproducibility, by targeting the B6R gene to yield results with low detection limits.

PCR and PCR-ELISA

The PCR method comprises the artificial amplification of the genomic material of interest in a regulated manner, using primers responsible for the enlarged deoxyribonucleic acid (DNA). PCR assays can be performed for the identification of different lineages of a virus, such as differentiating the West African clade from the Congo Basin clade of MPXV from skin lesions and nasopharyngeal and oropharyngeal swabs.

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PCR assays are highly sensitive and specific but have a few limitations, including the need for a step to identify the amplified genetic products, which is usually performed using gel electrophoresis, increasing the assay duration. Therefore, PCR assays are not suitable for resource-limited regions. Further, only the presence/absence of a specific gene can be identified by gel electrophoresis, and the process exposes the personnel to health risks and increases environmental damage.

PCR-ELISA is a combination of PCR and ELISA techniques and utilizes ELISA as an alternative to the gel electrophoresis step. ELISA is an analytical and environment-friendly technique used for identifying antigen-antibody reactions based on the binding capacities and color changes observed after enzyme-substrate reactions. These are performed to identify and quantify specific molecules. MPXV DNA is labeled with biotin or digoxigenin and can be identified based on color changes.

PCR-ELISA is performed in three steps, including PCR amplification, amplified product immobilization on microplates, and highly sensitive colorimetric detection of the product, enabling the processing of multiple samples in a short period.

Other techniques for molecular diagnosis of MPX

Molecular diagnostic techniques include LAMP (loop-mediated isothermal amplification) analysis, a modification of the traditional PCR analysis utilizing enzymes for amplification at sustained temperatures, and highly specific MPXV detection, based on increased fluorescence post-replication. The assays have reportedly used F3L-1 and A27L-1 primer sets for the J2L and A26L genes at 63.0°C for rapid MPXV genome amplification and on-site MPX diagnosis, particularly in rural areas and primary hospitals.

Another method is the RPA (recombinase polymerase) amplification, a modification of the isothermal amplification technique, utilizing DNA polymerase, allowing thermally stable reactions (temperature ranging between 37.0°C and 42.0°C). The test is reportedly 100% specific and 95% sensitive and yields results within 10 minutes. 

CRISPR (clustered regularly interspaced short palindromic repeats)-based methods have also been documented. Among bacterial organisms, the CRISPR-Cas diagnostic system detects viruses by which the microbe has been priorly infected, generating RNA (ribonucleic acid) probes for viral genomic interactions.

Detection is based on fluorescence signals and is rapidly performed within minutes. Genomic sequencing is another technique for highly sensitive and specific viral detection, and the RANS (rapid amplicon nanopore sequencing system) has been developed for MPX diagnosis.

Conclusion

Overall, the review findings highlighted different molecular-based methods for MPXV detection, which are not yet adequate for the increasing global health burden of MPX.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.