A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 proviral DNA by real-time PCR

Abstract

Early diagnosis and access to treatment for infants with human immunodeficiency virus-1 (HIV-1) is critical to reduce infant mortality. The lack of simple point-of-care tests impedes the timely initiation of antiretroviral therapy. The development of FINA, filtration isolation of nucleic acids, a novel DNA extraction method that can be performed by clinic personnel in less than 2 min has been reported previously. In this report, significant improvements in the DNA extraction and amplification methods are detailed that allow sensitive quantitation of as little as 10 copies of HIV-1 proviral DNA and detection of 3 copies extracted from 100 μl of whole blood. An internal control to detect PCR inhibition was also incorporated. In a preliminary field evaluation of 61 South African infants, the FINA test demonstrated 100% sensitivity and specificity. The proviral copy number of the infant specimens was quantified, and it was established that 100 microliters of whole blood is required for sensitive diagnosis of infants.

Rapid and sensitive detection of HIV-1 p24 antigen by immunomagnetic separation coupled with catalytic fluorescent immunoassay

“…PCR-based nucleic acid testing (NAT) is sensitive but expensive and complex, thus limiting its application [10][11][12]. HIV-1 p24 antigen in serum or plasma is one of the surrogate biomarkers for early diagnosis of HIV-1 infection, because it appears at an earlier stage of HIV infection than antibodies and remains at a high level during the early, acute phase of the infection [13][14][15].…”

A simple and rapid DNA extraction method for Chlamydia trachomatis detection from urogenital swabs

“…Filtration isolation of nucleic acids (FINA) is a simple, rapid DNA extraction process (<2 minutes) developed for proviral HIV-1 detection from blood (1, 2). Genomic DNA is trapped in a glass-fiber disk, washed to remove inhibitors, and qPCR amplified from filter.…”

“…The assay targets parA located on the multi-copy cryptic plasmid found in all CT serovars including the Swedish nvCT serovar (8, 9) plus the hydroxypyruvate reductase gene of Cucurbita pepo (pumpkin) , which is unlikely to cross-react with urogenital pathogens as a non-competitive internal control (IC) (2, 10). McCoy cells infected by CT [ATCC®VR-346™ Serovar F; (American Type Culture Collection; Manassas, VA)] were used as the model system for infected urogenital epithelial cells to demonstrate that plasmid DNA can be co-isolated with gDNA.…”

“…DNA was extracted from CT-infected McCoy cells or swabs by FINA method (1, 2, 11) with minor modifications. A DNA capture membrane (D=5.1-mm; Fusion 5, GE Healthcare, Pittsburgh, PA) was sandwiched between 707 blotter pad (25X25-mm; VWR International, West Chester, PA) and PARAFILM (Bemis Flexible Packaging; Neenah, WI) with a 4.3-mm-diameter hole exposing the center of DNA capture membrane (11) (Figure 1A).…”

“…To verify true negative results, 500 copies of IC plasmid (2) was included in each reaction. Negative CT samples with no IC amplification are considered invalid.…”