Using highly multiplexed ddPCR method to better detect patients in vivo
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Cell: Using highly multiplexed ddPCR method to better detect patients in vivo. Quantitative determination of HIV virus pools with replication capacity is essential for evaluating treatment strategies.
Quantitative determination of HIV virus pools with replication capacity is essential for evaluating treatment strategies.
The viral outgrowth assay (VOA) underestimates the HIV virus pool because it fails to induce all replication-competent HIV proviruses (provirus, that is, HIV in which the viral genome is integrated into the host cell DNA).
HIV DNA tests for a single area or two areas of HIV (called single-test and double-test, respectively) overestimate the HIV virus pool because these tests fail to exclude many defective HIV proviruses.
PCR-based testing methods are highly sensitive and require less blood than VOA. However, in the PCR test method, people usually test for a single conservative target, which will greatly overestimate the size of the HIV virus pool that has the ability to replicate.
This is because most of the HIV integrated into the host cell DNA Proviruses are all defective, and PCR testing for a single target does not distinguish between intact HIV proviruses and HIV proviruses with DNA deletion and/or inactivation mutations.
Reliable estimation of a genetically complete HIV provirus requires verification of the existence of multiple regions on the HIV genome and whether the target sequence in each region tested comes from the same HIV provirus.
Droplet digital PCR (ddPCR) provides an alternative option. In ddPCR, the PCR reaction solution (including template DNA) is divided into thousands of individual droplets, and the PCR detection results of each droplet are reported separately. A ddPCR assay protocol reported in 2019 uses such multiple methods to detect two regions of the HIV-1 genome in each droplet.
In a new study, American researchers used two ddPCR assay methods (hereinafter referred to as assay method 1 and assay method 2) targeting three regions (triple) of the HIV genome to develop a 5-region test method (two These triple ddPCR assays each target two unique HIV genome regions, but collectively target 1 overlapping region, allowing inter-batch quality control).
By combining these two parallel triple ddPCR assays, these authors are confident that the truly complete HIV-1 virus genome can be quantitatively determined. As a further improvement, they optimized one of the multiple ddPCR assay methods for specific quantitative determination of T cells in order to accurately normalize the quantitatively determined number with the number of HIV target cells to be studied.
This extra step is particularly useful for tissue biopsy, because the cell population in the tissue is difficult to separate and purify compared to blood. The relevant research results were published in the journal Cell Reports Medicine on April 20, 2021, with the title of the paper “A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir”.
Specifically, the three HIV targets of the measurement method 1 are located at the 3’end of the HIV pol gene, the tat gene and the env gene, while the three HIV targets of the measurement method 2 are located in the long terminal repeat (LTR)/gag region, The 5’end of the pol gene and env. In each assay method, specific primers and probes are designed for each HIV target.
Of the three HIV targets in each assay method, two use the same dye for probe detection, but at different concentrations, so that different HIV targets can be distinguished on the X/Y graph of fluorescence amplitude. This allows these authors to quantitatively determine droplets containing different combinations of targets.
The primers and probes for env are the same in Assay 1 and Assay 2. In 201 clinical samples, the env performance of these two assays is almost the same. In addition, in these clinical samples, the failure rate of five primer/probe pairs to detect the target is extremely low: gag 0.5%; 3’pol 1%; env 3.1% (both measurement methods are the same); tat 3.6%; 5 ‘pol 6.3%.
Design of two HIV-1 triple ddPCR assay methods, the picture is from Cell Reports Medicine, 2021, doi:10.1016/j.xcrm.2021.100243.
These authors used this 5-region test method to assess the number of HIV proviruses contained in these 5 regions. The number of HIV proviruses they estimated was 12.1 times higher than the matched quantitative VOA method and correlated with the latter (Spearman’s ρ = 0.48), but their estimated HIV virus pool was significantly less than the previous DNA measurement method.
The authors used their test methods on longitudinal blood samples and mucous membrane samples from HIV-infected individuals who were treated with antiretroviral drugs (ART).
In these patients, intact HIV provirus decays faster in blood CD4+ T cells than defective HIV provirus, and the frequency of intact HIV provirus in mucosal T cells and circulating T cells is similar.
(source:internet, reference only)
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