Unlike the fourth-generation ELISA and
western blot, HIV PCR does not rely on the
presence of antigens or antibodies in the blood.
Instead, it identifies genetic material by highlighting sequences of HIV within a patient’s
DNA. This is done through nucleic-acid amplification testing to observe the resulting polymerase chain reaction (PCR). The HIV PCR
process is then further divided into two sub-categories that determine viral load in a serum
sample: RNA PCR and DNA PCR. To perform
the test, an enzyme called DNA polymerase is
used to pursue a pro-viral genome sequence
and amplify it. In addition to the enzyme, the
PCR-inducing cocktail also contains oligonu-cleotides and deoxynucleotide precursors, as
well as a cofactor called MgC12.
Trends toward more point-of-care PCR
technologies have pushed boundaries at univer-
sities. California Institute of Technology has
developed one such technology. The new PCR
machine is small enough to stow in a backpack
and operates at the push of a button. The diag-
nostic prototype runs off a rechargeable battery
and comprises a chip that can analyze blood
sample to spot different pathogens. The cost:
used to create a standardized, accurate assay for
persistent HIV DNA in infected patients. “This
is attributed to the fact that ddPCR is able to
reliably detect HIV DNA targets well below the
limit of quantification of qPCR,” says Bio-Rad’s
GXD Div., Hercules, Calif. When using identical
quantities of clinical samples from peripheral
blood, ddPCR’s precision was between four-fold
to more than 20-fold better than qPCR. More-
over, ddPCR reaction conditions can be tuned
to make it less sensitive to unknown mutations
within an HIV-positive sample, thereby increas-
ing its ability to detect and quantify the presence
of the virus.
These results suggest the ddPCR assay could
prove useful for clinical studies aimed at eradication of HIV from infected patients. In fact,
Dr. Richman worked with a team of researchers,
led by Dr. Deborah Persaud of Johns Hopkins
Children’s Medical Center, that showed, for the
first time, the functional cure of an HIV-infected
infant. The ddPCR assay was used to confirm the
findings, determining there was in fact no detectable virus in the baby.
under $1,000 and one scan only costs $5.
However, PCR doesn’t just serve as a diagnostic tool. The need to find a cure for HIV has
spurred demand for new assays that can more
precisely quantify reservoir HIV DNA. Bio-Rad’s
QX100 Droplet Digital PCR (ddPCR) system has
been used in a research setting for this purpose.
The ddPCR is a nucleic acid molecule counting method that works by partitioning the sample into droplets to an extent that either zero or
only a small number of DNA molecules of interest (targets) are present in each partition, with
thousands of reactions running in parallel. The
result gives users an absolute measurement that
doesn’t require the need for a standard curve.
Eliminating the standard curve reduces error
and improves precision, enabling reproducibility.
Because samples are partitioned and an endpoint analysis performed, the background DNA
is reduced, lessening competitive and inhibitor
effects and allowing for greater discrimination
between similar nucleic acid quantities.
A recent study in PLOS ONE led by Matthew
Stain and Douglas Richman of the Center for
AIDS Research at the Univ. of California, San
Diego found that Bio-Rad’s ddPCR can be
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