Journal: Scientific Reports

Article Title: Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC

doi: 10.1038/s41598-021-03016-1

Figure Lengend Snippet: Revised qPCR and RTqPCR quantification assay synthetic standard and formats. Created with BioRender.com. ( a ) Synthetic DNA plasmid template containing the 433–633 HIV-1 LTR insert was linearized by Sca I and used for standard curve generation in the HIV-1 LDAs. RNA transcripts from the linearized DNA plasmid were made using the T7 promoter site and were used as HIV-1 RNA controls. ( b ) Protocol for Non-nested qPCR on crude DNA lysates. ( c ) Protocol for semi-nested RT-qPCR on RNA extracts.

Article Snippet: To improve sensitivity and precision at the lowest target concentrations, DNA lysates were pre-digested with an EcoRI-HF enzyme (New England BioLabs, Ipswich, MA, USA), according to the manufacturer’s instructions, before amplification (Fig. b).

Techniques: Plasmid Preparation, Quantitative RT-PCR