Analytical Performances of Human Immunodeficiency Virus Type 1 RNA-Based Amplix® Real-Time PCR Platform for HIV-1 RNA Quantification

Figure 1

(a) Workflow for HIV-1 RNA load measurement using the Amplix platform combining automated station for nucleic acids extraction (Amplix station 16 Dx, Biosynex) and real-time PCR amplification (Amplix NG, Biosynex) and lyophilized HIV-1 RNA-based Amplix real-time PCR kit (Biosynex) targeting gag and LTR genes. (1) Samples preparation and preextraction processing, 190 µL of plasma, positive control (PC), weak positive control (WPC), negative control (NC), and calibration samples (CS) 1 and CS2, in which 10 µL of internal control (IC) is added, are subjected to DNA/RNA extraction, using the Amplix viral extraction kit (Biosynex). (2) Automated silica magnetic bead-based extraction (92 min). (3) Real-time PCR amplification (2 h 30) of extracted nucleic acids in 50 µL of elution buffer is deposited in ready master mix tubes containing all reagents for reverse transcription and PCR. RSC: recovery solution for control samples. (b) Typical amplification curves of controls and calibrators given by the Amplix DTmaster software (Biosynex) showing the duplex detection of fluorescence labelled hydrolysis probes (5′-fluorescein carboxylic acid [FAM] and 3′-black hole quencher-1 [BHQ1]) of PC (A), CS1 (B), CS2 (C), WPC (D), and NC (E), giving Ct values of 16.0, 21.3, 26.6, 30.4, and 33.0, respectively. (c) Amplification curves given by the Amplix DTmaster software (Biosynex) showing the duplex detection of fluorescence labelled hydrolysis probes (5′-carboxy-rhodamine-X [ROX] and 3′-black hole quencher-2 [BHQ2]) of the HIV-1-infected Patient #LEM (F) showing Ct at 36.7 and plasma HIV-1 RNA load of 7,700 copies/mL and of the NC (E′).

(a) Workflow

(b) Controls and calibrators [FAM]

(c) Patient #LEM [ROX]