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The real-time quantification of the number of amplified products is...

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Q4ddPCR: a flexible, 4-target assay for high-resolution HIV reservoir profiling.

Rachel Scheck^1,2, Mark Melzer³, Gregory Gladkov²

¹Laboratory of Translational Immunology of Viral Infections, Department of Infectious Diseases and Critical Care Medicine, Charité-Universitätsmedizin Berlin, Berlin Institute of Health, Berlin, Germany.

Nature Communications

|February 20, 2026

Summary

A new assay, Q4ddPCR, precisely quantifies the intact HIV reservoir, overcoming limitations of older methods. This advancement offers more accurate measurements of viral dynamics for HIV cure research.

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Area of Science:

Virology
Molecular Biology
Immunology

Background:

Accurate quantification of the intact HIV reservoir is crucial for developing HIV cure strategies.
Existing assays like the intact proviral DNA assay (IPDA) face challenges with quantification failures and misclassification of defective proviruses due to HIV genetic diversity.

Purpose of the Study:

To introduce and validate Q4ddPCR, a novel droplet digital PCR assay designed for precise and scalable quantification of the intact HIV reservoir.
To compare the performance of Q4ddPCR with existing methods and assess its ability to capture reservoir dynamics.

Main Methods:

Development of Q4ddPCR, a modular assay targeting four conserved HIV genome regions, with two configurations available.
Benchmarking Q4ddPCR against 3650 near full-length proviral sequences from virally suppressed people with HIV (PWH).
Evaluation of Q4ddPCR in four independent cohorts and longitudinal samples from participants on antiretroviral therapy (ART).

Main Results:

Q4ddPCR demonstrated close agreement with sequence-confirmed reservoir measurements and revealed clonal reservoir dynamics missed by IPDA.
The assay enabled intact reservoir quantification in 95% of samples and showed strong correlation with viral outgrowth.
Longitudinal analysis revealed lower proviral frequencies and a steeper decline in intact proviral DNA with Q4ddPCR compared to IPDA.

Conclusions:

Q4ddPCR offers improved specificity and reduced quantification gaps for intact HIV reservoir measurement.
The multi-target approach of Q4ddPCR more accurately captures intact reservoir dynamics, supporting its utility in HIV cure research.
Findings validate mathematical modeling predictions regarding the benefits of multi-target assays for reservoir quantification.