Polymerase Chain Reaction (PCR)
In 1989, Science selected the polymerase chain reaction (PCR) as the major scientific development of the year. Polymerase chain reaction (PCR) is a method of in-vitro replication of target nucleic acid sequences. PCR amplifies a short nucleic acid sequence that is specific to the organism being detected. DNA from the PBMCs is released by enzyme lysis and mixed with a mixture consisting of DNA polymerase enzyme, deoxynucleoside triphosphates and primers that bind to specific areas of HIV gene. The deoxynucleoside triphosphates are the building blocks for new strands of DNA. There are various primers available that bind to gag region (SK 19 or SK-102) or envelope region (SK-70) of the HIV gene. The DNA to be tested will bind to new matching bases in PCR solution. Binding requires the help of primers to start the process. Primers are small sequences of DNA complementary to the target DNA sequences and will bond only to their matching target sequences. If no sequences match, PCR will not occur and a negative result will be recorded. If binding occurs, the deoxynucleoside triphosphates bind to the primer and extend the DNA strand till a complete copy of the target sequence is produced resulting in one copy of original. Multiple PCR cycles occur to yield millions of copies. This material is enough to detect presence of HIV DNA. The product is then subjected to agarose gel electrophoresis or tested by colorimetric ELISA like assay. On agarose gel, the amplified positive HIV DNA will be visualized after ultraviolet light illumination. With colorimetric assay, positive tests will give a colour to solution. In a quantitative assay, the intensity of the colour is related to the amount of DNA copies present and thus the original viral load can be calculated.
HIV PCRs are of 2 types: Qualitative PCR and Quantitative. The potential utility of DNA PCR for the >diagnosis of vertical HIV infection soon became readily apparent in infants. HIV DNA PCR has been found to be highly sensitive and specific for early diagnosis of pediatric HIV infection. Sensitivity of HIV PCR is less at birth and sensitivity increases rapidly to 95% at 4 weeks and to 99% at 6 months of age. Thus, in non-breast fed infants. HIV PCR can be done at 4-6 weeks after birth. In breastfeeding populations, HIV PCR should be done after one or two months after cessation of breastfeeding.
HIV PCR can be done on dried blood sample transported to a laboratory on a filter paper or on whole blood
However, false positive and false negative results with HIV DNA PCR may occur. The authors have reported a very high incidence of false positive HIV DNA PCR (75%) especially in younger infants in their study (Shah I et al, 2004). Repeating the PCR on independent samples may be required to reduce the test errors. One of the reasons stated for the false positive results is contamination. Optimal PCR conditions, inclusion of control samples, strict rules on sample preparation, pre-and post- PCR handling, repetition of results, confirmation of specificity by hybridization, choice of material from which HIV-1 is amplified and the primers used for amplification will all predict the reliability of HIV DNA PCR.
Infants who have HIV DNA PCR positive during the first days of life are conventionally assumed to have been infected during pregnancy (in-utero). Infants who test HIV negative in the first days of life but later are found to be positive are presumed to have been infected during labor and delivery (intra-partum) or through breast feeding