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A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

ABSTRACT: Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assays are specific, sensitive, and reproducible. Together, they estimate the number of proviruses containing all five primer-probe regions. Our 5-target results are on average 12.1-fold higher than and correlate with paired quantitative VOA (Spearman's ρ = 0.48) but estimate a markedly smaller reservoir than previous DNA assays. In patients on antiretroviral therapy, decay rates in blood CD4⁺ T cells are faster for intact than for defective proviruses, and intact provirus frequencies are similar in mucosal and circulating T cells.

SUBMITTER: Levy CN

PROVIDER: S-EPMC8080125 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

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A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Levy Claire N CN Hughes Sean M SM Roychoudhury Pavitra P Reeves Daniel B DB Amstuz Chelsea C Zhu Haiying H Huang Meei-Li ML Wei Yulun Y Bull Marta E ME Cassidy Noah A J NAJ McClure Jan J Frenkel Lisa M LM Stone Mars M Bakkour Sonia S Wonderlich Elizabeth R ER Busch Michael P MP Deeks Steven G SG Schiffer Joshua T JT Coombs Robert W RW Lehman Dara A DA Jerome Keith R KR Hladik Florian F

Cell reports. Medicine 20210412 4

Quantifying the replication-competent HIV reservoir is essential for evaluating curative strategies. Viral outgrowth assays (VOAs) underestimate the reservoir because they fail to induce all replication-competent proviruses. Single- or double-region HIV DNA assays overestimate it because they fail to exclude many defective proviruses. We designed two triplex droplet digital PCR assays, each with 2 unique targets and 1 in common, and normalize the results to PCR-based T cell counts. Both HIV assa ...[more]

PMID: 33948574

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Project description:Foodborne illness attributed to enterohemorrhagic E. coli (EHEC), a highly pathogenic subset of Shiga toxin-producing E. coli (STEC), is increasingly recognized as a significant public health issue. Current microbiological methods for identification of EHEC in foods often use PCR-based approaches to screen enrichment broth cultures for characteristic gene markers [i.e., Shiga toxin (stx) and intimin (eae)]. However, false positives arise when complex food matrices, such as beef, contain mixtures of eae-negative STEC and eae-positive E. coli, but no EHEC with both markers in a single cell. To reduce false-positive detection of EHEC in food enrichment samples, a Multiplexed, Single Intact Cell droplet digital PCR (MuSIC ddPCR) assay capable of detecting the co-occurrence of the stx and eae genes in a single bacterial cell was developed. This method requires: (1) dispersal of intact bacteria into droplets; (2) release of genomic DNA (gDNA) by heat lysis; and (3) amplification and detection of genetic targets (stx and eae) using standard TaqMan chemistries with ddPCR. Performance of the method was tested with panels of EHEC and non-target E. coli. By determining the linkage (i.e., the proportion of droplets in which stx and eae targets were both amplified), samples containing EHEC (typically greater than 20% linkage) could be distinguished from samples containing mixtures of eae-negative STEC and eae-positive E. coli (0-2% linkage). The use of intact cells was necessary as this linkage was not observed with gDNA extracts. EHEC could be accurately identified in enrichment broth cultures containing excess amounts of background E. coli and in enrichment cultures derived from ground beef/pork and leafy-green produce samples. To our knowledge, this is the first report of dual-target detection in single bacterial cells using ddPCR. The application of MuSIC ddPCR to enrichment-culture screening would reduce false-positives, thereby improving the cost, speed, and accuracy of current methods for EHEC detection in foods.

Dataset Information

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

Publications

A highly multiplexed droplet digital PCR assay to measure the intact HIV-1 proviral reservoir.

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