Increased Threshold Sensitivity of Viral Load Determinations Using an Optimized Sample Processing Method

Abstract

A new sample processing method was developed to support quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) assays to determine plasma viral load in SIV-infected (Simian Immunodeficiency Virus) Rhesus macaques, an important model for HIV-1 (Human Immunodeficiency Virus Type 1) infection in humans. The method allows for up to 1.5 milliliters of plasma to be tested resulting in an assay threshold sensitivity as low as 20 copies of SIV RNA (ribonucleic acid) equivalents per milliliter of plasma. The method yields high and consistent recoveries of high quality RNA, demonstrated for a range of samples containing from 3 to 10,000 nominal virus particles. An inter-assay percent coefficient of variation of 36% and an intra-assay percent coefficient of variation of 19% were demonstrated, both consistent with the inherent variations in the RT-PCR assay itself and suggestive that the processing method does not contribute any additional, significant variation to the testing. It is also able to accommodate over 70,000 'contaminating' cells per sample with no effect on the RNA yield and amplification efficiency of the assay. Any carryover and concentration of potentially inhibitory material is negligible, as no effect on the efficiency and performance of the RT-PCR assay was noted for any cell-free sample. The method is convenient, with all operations performed in a single microcentrifuge tube, which also avoids any loss of RNA due to sample transfers as is common with other methods. This new processing method represents a significant improvement over that previously applied to quantitative RT-PCR assays for SIV load determinations in cell-free samples and can be readily extended to assays for other retroviral targets in clinical specimens, such as HIV-1.