Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5 0 LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes.
DNA-Srand Breaks, Alpha-Particles, Bragg-MaximumThe yield of single-strand breaks (ssb) and double-strand breaks (dsb) produced by alphaparticles at the end of their track in DNA-films was determined experimentally. Helium nuclei were accelerated to 600 keV in the 400 kV ion accelerator and scattered at a carbon target. The elastically scattered alpha-particles with energies of 344 keV and 485 keV were used to irradiate supercircular plasmid DNA in vacuo.For the dosimetry of the alpha-particles a surface barrier detector was used and the energy distribution of the alpha-particles determined. The energy loss of the particles in the DNAlayer was calculated. DNA samples were separated into the three conformational isomers using agarose gel electrophoresis. A fter fluorochromation the number of ssb and dsb per plasmid DNA molecule was established from the band intensities assuming the validity of Poisson statistics.Linear dose effect correlations were found for ssb and dsb per plasmid molecule. In the case of 344 keV-alpha-particles the yield of dsb was (8.6 ± 0 .9 )x l0~n breaks/G yxdalton. The ratio of ssb/dsb was 0.5 ± 0.2. This is at least a factor of six larger than the ratio found in experiments with higher energy alpha-particles and from model calculations. Similar experiments with protons yielded a relative biological effectiveness (rbe) value of 2.8 for the induction of double-strand breaks by track end alpha-particles.
This report describes a rapid convenient screening system with improved sensitivity to detect mutations in the cystic fibrosis transmembrane regulator (CFTR) gene based on nonisotopic SSCP analysis. Because conventional SSCP analysis is often hampered by poor yield of single-stranded DNA, we applied the well-established solid-phase technique in which streptavidin-coated magnetic beads are used to immobilize biotinylated polymerase chain reaction (PCR) products. High yield of single-stranded DNA can be eluted from the solid phase by denaturation and used for SSCP analysis. An additional advantage of this procedure is that the immobilized single strand is available without any further purification steps for solid-phase sequencing.
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