Maximizing viral detection with SIV droplet digital PCR (ddPCR) assays

Area of Science:

• Virology
• Molecular Biology
• Immunology

Background:

• Accurate detection of viral nucleic acids is vital for evaluating HIV-1 treatments.
• The simian immunodeficiency virus (SIV) in rhesus macaques serves as a key model for human HIV-1 infection.
• Assessing low-level viral replication and residual virus is essential for eradication strategies.

Purpose of the Study:

• To develop and validate highly sensitive droplet digital PCR (ddPCR) assays for simian immunodeficiency virus (SIV) detection.
• To evaluate the performance of the RainDance ddPCR platform for SIV DNA and RNA quantification.
• To assess the utility of these assays in the context of combination antiretroviral therapy (cART).

Main Methods:

• Development of two ddPCR assays: one for SIV DNA and one for SIV RNA (RT-ddPCR).
• Utilized the RainDance ddPCR platform for high-throughput nucleic acid quantification.
• Tested assay performance with varying cell DNA input and in the presence of significant inhibition.

Main Results:

• The RainDance ddPCR platform demonstrated tolerance to higher cell DNA input compared to qPCR and Bio-Rad ddPCR.
• The combined use of a high processivity RT enzyme and RainDance ddPCR successfully overcame severe inhibition in viral RNA samples.
• The developed assays provide highly sensitive detection of SIV nucleic acids.

Conclusions:

• The developed ddPCR assays are valuable tools for sensitive SIV detection, aiding HIV-1 research.
• The RainDance ddPCR platform offers advantages in handling high sample input and overcoming inhibition.
• These methodologies can be adapted for sensitive nucleic acid detection in various research applications.