Qiaamp viral rna extraction kit

Extraction of sample RNA from the FTA card samples was done according to the procedure developed by NICD. Briefly, twelve 4 mm discs were punched out of the FTA cards using a new biopsy disposable punch (Kai Europe) per card, and placed into two sterile and appropriately labeled 1.5 ml microfuge tubes (six discs per tube). A total of 200 µl of RNA processing buffer (80 µl of glycogen [5 mg/ml], 4 µl of 1 M dithiothreitol and 1.916 ml of Tris‐EDTA‐1 solution [10 mmol/L TRIS‐HCL/0.1 mmol/L EDTA, pH 7.6]) was added to the tubes containing the discs. The tubes were briefly vortexed and then heated at 70°C for 5 min. They were briefly spun and the supernatant transferred into another sterile microfuge tube containing 620 µl of buffer AVL mixed with carrier RNA. Viral RNA was then extracted using the QIAGEN QIAamp Viral RNA extraction kit (QIAGEN) following the manufacturer's guidelines¹⁵ and stored at −20°C until required for subsequent laboratory analyzes. Sterile, unspotted FTA cards were punched out and extracted alongside the FTA discs as RNA extraction controls.

Viral RNA was extracted from the samples using the QIAGEN QIAamp Viral RNA extraction kit (QIAGEN, Hilden, Germany) following the manufacturer’s guidelines.

Viral RNA was isolated from cryopreserved plasma samples and purified using the QIAamp Viral RNA Extraction Kit (Qiagen) followed by reverse transcription using the SuperScript III Reverse Transcriptase System (Invitrogen). The Env gp120 C2-V5 (approximately 799 bp, HXB2 [accession number: K03455] positions 6883–7681) was amplified by nested touchdown PCR (primers listed in Table S1). Amplified DNA was purified using the MinElute Gel Extraction Kit (Qiagen), and ligated into a pCR4-TOPO sequencing vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen). Chemically competent One Shot MAX Efficiency DH5α E. coli (Invitrogen) were transformed with the prepared plasmids, and cultured overnight at 37 °C. Eighteen colonies were selected for colony PCR (M13F and M13R primers, Table S1), and the resulting products were purified using ExoSAP-IT (Affymetrix) and sequenced to generate forward and reverse reads (Source BioScience). Contigs were assembled and controlled manually using Geneious v9.0.5^{61 (link)} (HXB2 gp120 positions 661–1455, accession number K03455). Sequences were multiple aligned using MUSCLE^{62 (link)}, and then manually edited in MEGA v6.06^{63 (link)}. Sequences were controlled for intra-patient clustering by maximum-likelihood phylogenetics.