Analyses of 11 commercially available PCR kits to detect monkeypox virus DNA

In a recent study posted to the medRxiv* server, researchers in Germany and Switzerland evaluated 11 commercial reverse transcription-polymerase chain reaction (RT-PCR) kits (A to L) for Monkeypox virus (MPXV) detection.

Study: Evaluation of eleven commercially available PCR kits for the detection of Monkeypox virus DNA. Image Credit: Salov Evgeniy / Shutterstock

Background

From May 2022, cases of MPXV began increasing worldwide, particularly in Europe and the United States. Prior to this, this disease was endemic in Central and Western Africa for nearly 50 years. Consequently, the 2022 MPXV outbreak was declared a public health emergency of international concern on 23 July 2022.

Thus, before 2022, the availability of RT-PCR kits detecting orthopoxviruses (OPVs), especially MPXVs, was negligible. As a result, specialized laboratories relied on in-house protocols for their diagnosis. Only recently, ready-to-use RT-PCR test kits for MPXV diagnosis have become available.

About the study

In the present study, researchers evaluated 11 ready-to-use RT-PCR kits for MPXV diagnosis against the reference diagnostic workflow of the German Consultant Lab for poxviruses. The 18-specimen reference panel comprised deoxyribonucleic acid (DNA) from MPXV clade I, clade IIa and clade IIb, other OPV, and varicella-zoster virus (VZV). More specifically, it had one generic OPV PCR, one MPXV-specific PCR, and one MPXV clade II-specific PCR.

These kits analyzed all specimens in duplicates; additionally, the team incorporated an inhibition control into these specimens before viral DNA extraction. The team ensured proper sampling using a human DNA-specific RT-PCR reaction.

The researchers used the manufacturer’s manual to run these diagnostic kits, all set to obtain the lowest possible cycle threshold (C_T) values. Further, the researchers ensured the comparability of all 11 test kits. For this, they ran these kits on the BioRad CFX 96 real-time cycler, which is compatible with the fluorophores used by these tests.

For a more detailed characterization of the test kits, the team plotted the C_T values for each DNA sample relative to the number of genome equivalents (GE), determined using a plasmid standard curve. Finally, they examined the slope of the curve, reflecting RT-PCR efficiency, and the Y-intercept, which indicated the theoretically minimal C_T value obtained with an assay.

Study findings

For the 2012 clade II MPXV isolate, except kit F, all the kits showed CT values in the expected range. In addition, they exhibited good analytical sensitivity, with C_T values of 36, reflecting less than five GE per reaction. However, for a C_T value of 38, reflecting less than one GE per reaction and the lowest dilution, two kits and two generic reference PCRs for OPV and MPXV failed to detect both specimens. On the other hand, six kits could detect only one of the two duplicates; three kits and clade II-specific reference RT-PCR detected both, indicating high analytical sensitivity. Notably, the dropout of one duplicate does not necessarily reflect poor assay performance.

For clade IIb, these kits showed slightly better detection rates for samples with higher C_T values. Thus, kit L failed to detect samples with C_T values of 38, while most kits detected a single replicate. Again, kit K was unable to detect one replicate of a specimen with a C_T value of 32, while three kits could not even detect one replicate of the lowest concentration with a C_T value of 35 against clade I MPXV. These kits fetched similar CT values for most specimens, especially considering the varying sample volumes used per reaction. It might have contributed to nearly two-three C_T value shifts.

Some test manuals mentioned the limit of detection (LOD) in DNA copies/mL, which is not an optimal metric for crusts and dry swab samples. Encouragingly, all controls included in the kits performed as expected. Also, these test kits performed within the range specified by their respective manuals.

Conclusion

Overall, the 11 kits evaluated in the current study showed comparable and high detection sensitivity for MPXV clade I and II DNAs. Therefore, these kits seemed apt for determining clinically-relevant MPXV DNA loads from properly-sampled skin lesions. Adhering to their intended purpose, these kits showed superior analytical sensitivity towards MPXV or OPVs and used samples with less than approximately five GE per reaction or C_T values of 36.

The researchers cautioned that these kits and others entering the commercial space are intended for research purposes currently. More data is needed before they can be used to detect MPXV reliably in clinical specimens. Only then would these kits empower communities globally in MPXV diagnoses.

*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.