Tuesday, 1 April 2014

All the dirt on digital PCR

Principles of Molecular Virology Principles of Molecular Virology, Chapter 1 (Introduction to Viruses), describes the techniques used to study viruses, including bioinformatics. But molecular techniques have moved on a lot in the last few years, so the new edition will have an update on this.



I'm updating the bioinformatics section of Chapter 1 and collecting resources to illustrate recent advances in how nucleic acid technologies have moved on in the last few years. One of those advances has been digital PCR (dPCR), an amazingly sensitive technique used to measure tiny quantities in nucleic acid in clinical samples, as in this example: Highly Precise Measurement of HIV DNA by Droplet Digital PCR. (2013) PLoS ONE 8(4): e55943. doi:10.1371/journal.pone.0055943

This new technology is sensitive enough to convince us that in some circumstances, people can be cured of HIV infection: Challenges in Detecting HIV Persistence during Potentially Curative Interventions: A Study of the Berlin Patient. (2013) PLoS Pathog 9(5): e1003347. doi:10.1371/journal.ppat.1003347
There is intense interest in developing a cure for HIV. How such a cure will be quantified and defined is not known. We applied a series of measurements of HIV persistence to the study of an HIV+ adult who has exhibited evidence of cure after a stem cell transplant. Samples from blood, spinal fluid, lymph node, and gut were analyzed in multiple laboratories using different approaches. No HIV was detected in blood cells, spinal fluid, lymph node, or small intestine, and no infectious virus was recovered from blood. However, HIV was detected in plasma (2 laboratories) and HIV DNA was detected in the rectum (1 laboratory) at levels considerably lower than those expected in antiretroviral treated patients. The occasional, low-level HIV signals might be due to persistent HIV or might reflect false positives. The sensitivity of the current generation of assays to detect HIV RNA, HIV DNA, and infectious virus are close to the limits of detection. Improvements in these tests will be needed for future curative studies. The lack of rebounding virus after five years without therapy, the failure to isolate infectious virus, and the waning HIV-specific immune responses all indicate that the Berlin Patient has been effectively cured.


This commercial video from Bio-Rad explains the dPCR procedure:



TaqMan PCR - commercial video from Life technologies:







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