The exquisite sensitivity of the polymerase chain reaction means DNA contamination can ruin an entire experiment. Tidiness and adherence to a strict set of protocols can avoid disaster.

Avoiding false positives with PCR

PRIMARY infection with the human immunodeficiency virus (HIV) is generally followed by a burst of viraemia with or without clinical symptoms1–3. This in turn is followed by a prolonged period of clinical latency. During this period there is little, if any, detectable viraemia, the numbers of infected cells in the blood are very low, and it is extremely difficult to demonstrate virus expression in these cells4. We have analysed viral burden and levels of virus replication simultaneously in the blood and lymphoid organs of the same individuals at various stages of HIV disease. Here we report that in early-stage disease there is a dichotomy between the levels of viral burden and virus replication in peripheral blood versus lymphoid organs. HIV disease is active in the lymphoid tissue throughout the period of clinical latency, even at times when minimal viral activity is demonstrated in blood.

HIV infection is active and progressive in lymphoid tissue during the clinically latent stage of disease

THE development of molecular genetics, both as a self-contained field and as a body of techniques broadly useful in biologic investigation, has had a profound influence on medical research. The beneficiaries include every discipline in basic science and, at least indirectly, most clinical and applied medical disciplines. Certain technical milestones can be identified over the past several decades that have been particularly important in the progress of the field. One is the discovery of restriction endonucleases, which together with the development of DNA ligation and transformation procedures, led to the ability to clone and thus propagate genes of any organism . . .

https://www.nejm.org/doi/pdf/10.1056/NEJM199001183220307

The polymerase chain reaction. A new method of using molecular genetics for medical diagnosis.

A blood donor infected with human immunodeficiency virus-type 1 (HIV-1) and a cohort of six blood or blood product recipients infected from this donor remain free of HIV-1-related disease with stable and normal CD4 lymphocyte counts 10 to 14 years after infection. HIV-1 sequences from either virus isolates or patient peripheral blood mononuclear cells had similar deletions in the nef gene and in the region of overlap of nef and the U3 region of the long terminal repeat (LTR). Full-length sequencing of one isolate genome and amplification of selected HIV-1 genome regions from other cohort members revealed no other abnormalities of obvious functional significance. These data show that survival after HIV infection can be determined by the HIV genome and support the importance of nef or the U3 region of the LTR in determining the pathogenicity of HIV-1.

https://science.sciencemag.org/content/sci/270/5238/988.full.pdf

Genomic Structure of an Attenuated Quasi Species of HIV-1 from a Blood Transfusion Donor and Recipients

Primary effusion lymphoma: a distinct clinicopathologic entity associated with the Kaposi's sarcoma-associated herpes virus.

By means of a selective DNA amplification technique called polymerase chain reaction, proviral sequences of the human immunodeficiency virus (HIV-1) were identified directly in DNA isolated from peripheral blood mononuclear cells (PBMCs) of persons seropositive but not in DNA isolated from PBMCs of persons seronegative for the virus. Primer pairs from multiple regions of the HIV-1 genome were used to achieve maximum sensitivity of provirus detection. HIV-1 sequences were detected in 100% of DNA specimens from seropositive, homosexual men from whom the virus was isolated by coculture, but in none of the DNA specimens from a control group of seronegative, virus culture-negative persons. However, HIV-1 sequences were detected in 64% of DNA specimens from seropositive, virus culture-negative homosexual men. This method of DNA amplification made it possible to obtain results within 3 days, whereas virus isolation takes up to 3 to 4 weeks. The method may therefore be used to complement or replace virus isolation as a routine means of determining HIV-1 infection.

http://science.sciencemag.org/content/sci/239/4837/295.full.pdf

Sheila W. Mitchell

Papers

DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells