Rapid, point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR

Abstract

PCR detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA is the method recommended for use for the diagnosis of HIV-1 infection in infants in limited-resource settings. Currently, testing must be performed in central laboratories, which are usually located some distance from health care facilities. While the collection and transportation of samples, such as dried blood spots, has improved test accessibility, the results are often not returned for several weeks. To enable PCR to be performed at the point of care while the mothers wait, we have developed a vertical filtration method that uses a separation membrane and an absorbent pad to extract cellular DNA from whole blood in less than 2 min. Cells are trapped in the separation membrane as the specimen is collected, and then a lysis buffer is added. The membrane retains the DNA, while the buffer washes away PCR inhibitors, which get wicked into the absorbent blotter pad. The membrane containing the entrapped DNA is then added to the PCR mixture without further purification. The method demonstrates a high degree of reproducibility and analytical sensitivity and allows the quantification of as few as 20 copies of HIV-1 proviral DNA from 100 microl of blood. In a blinded study with 182 longitudinal samples from infants (ages, 0 to 72 weeks) obtained from the Women and Infants Transmission Study, our assay demonstrated a sensitivity of 99% and a specificity of 100%.

FIG. 1.

(a) Schematic of DNA extraction by FINA; (b) the in-house FINA module; (c) the disk transferred to a tube for PCR.

FIG. 2.

(a) Amplification plots obtained with 100 μl HIV-negative blood spiked with 0.4 to 200 HIV-1 copies/μl blood after DNA extraction by FINA and qPCR. The arrows indicate the average C_T values obtained with each of the concentrations. NEG, negative control (HIV-negative blood). (b) Standard curve of average C_T value versus log(copy number) obtained with 100 μl HIV-negative blood spiked with 0.4 to 200 HIV-1 copies/μl blood after DNA extraction by FINA and qPCR. The slope of −3.296 indicates a PCR efficiency (Eff) of 101.1%.

FIG. 3.

Plots showing the effect of storage on HIV-1 proviral DNA. The graphs show the mean log(copy number) versus storage time (in weeks) when the starting concentration was 8 copies/μl (a total of 800 copies before extraction by FINA) (a) and 0.8 copies/μl (a total of 80 copies before extraction by FINA) (b). The bars indicate ±1 standard deviation.