Abstract:
Early detection of mother-to-child transfer of Human Immunodeficiency Virus (HIV-1) is of the utmost importance for monitoring the success of intervention strategies, as well as for optimal treatment of HIV-positive children. Serology can only be used confidently after 18 months, as remaining antibodies from the mother may give false positive results. This leaves only molecular methods for early detection of the virus; unfortunately, the technology is still too expensive for general use.
The aim of this project was to develop and validate a cost-effective, fast, early detection method for HIV infection in infants. PCR was chosen as the developmental method, a technique that amplifies proviral sequences of HIV DNA, detecting HIV infection in peripheral blood mononuclear cells (PBMC) from infants of seropositive women during neonatal (age less than 28 days) and post-neonatal periods.
A method based on the commercial Roche HIV-1 DNA assay was chosen for implementation on the Roche LightCycler instrument. The published primer set was used to detect both HIV-1 DNA and an internal control. The target DNA for use as internal control was constructed from the plasmid pBR322 so that an AT-rich part of the plasmid was flanked by the HIV-1 primer-binding sites. The resulting amplicon was cloned into a vector and multiplied in E. coli. Amplification of the plasmid by PCR in the Roche LightCycler in the presence of SYBR Green created an amplicon having a Tm different (81 ± 1°C ) from that of the HIV-1 amplicon (84 ± 1°C) so that post-amplification melting can be used to differentiate between HIV-1 and internal control.
After construction of the internal control, the reaction conditions were optimised so that the internal control would amplify strongly only in the absence of HIV-1 target DNA. Then 50 previously tested patient samples were analysed using the assay developed here. Only half of the known positive samples came up positive in the assay, indicating that it is not sensitive enough for diagnostic use in its current form. Various ways of improving the sensitivity are suggested for further development of the assay as described here.
