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Old 05-20-2012
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Virus HIV diagnosis: PCR vs RT-PCR?

In kaplan they say:

PCR is used in early testing after known exposure to HIV-positive blood
(ie. nurse is pricked by a needle from an HIV-positive patient, or a newborn born to an HIV-positive mother)
RT-PCR is used to determine viral load and monitor status of infection and response to drugs.

In FA they kinda mash the two together and say (pg. 173 FA 2011):

PCR/"viral load tests" allow monitoring of effect of a drug and is what should be used in a newborn born to an HIV-positive mother (source: DIT).


Can someone help me differentiate PCR vs RT-PCR in HIV diagnosis?
Which is checking viral load? When do we use either?
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Let me give this a shot..
PCR is basically using primers and heat stable DNA polymerases to amplify a specific segment of DNA. In the context of HIV, it's the best, most specific and earliest way to diagnose infection.

Basically if you have a pin prick, and some virus get in and infect the host. The first thing the HIV does is reverse transcribe it's RNA to a DNA provirus and integrate it into host cell DNA. At this early stage western blot or elisa won't help cuz the provirus is dormant and not expressing any viral proteins/antigens. But since PCR expands DNA, even if the HIV hasn't really had a chance to become an active infection, the presence of the provirus is enough to detect and confirm infection. So that's why it's the best choice for early detection of HIV infection. Also in infant's of HIV+ mothers, PCR is the gold standard since the mother's antibody (IgG) crosses the placenta, the western blot/ELISA will give false positives even when the infant is not infected.

As far as viral load is concerned, we use RT-PCR. This is simply using reverse transcriptase to make a cDNA copy of the viral RNA, and then PCR amplifying the cDNA. Here we're looking for the presence and amount of VIRAL RNA. If we take a standard amount of blood from a HIV+ patient, and use RT-PCR to amplify the RNA, the higher the amount of virus present in the blood (the viral load) the greater the number of copies of amplified cDNA you'll have. Basically if you have 100 virions in blood, and we use RT-PCR to amplify their RNA, it might yield 10,000 DNA copies. But if we get 500,000 DNA copies, we'll know the viral load is way more. So the greater the amplification, the greater the viral load.

that's the way I understood it, hope that helps, and as always please correct me if i made any mistakes
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Old 05-22-2012
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Quote:
Originally Posted by slowpoke View Post
Let me give this a shot..
PCR is basically using primers and heat stable DNA polymerases to amplify a specific segment of DNA. In the context of HIV, it's the best, most specific and earliest way to diagnose infection.

Basically if you have a pin prick, and some virus get in and infect the host. The first thing the HIV does is reverse transcribe it's RNA to a DNA provirus and integrate it into host cell DNA. At this early stage western blot or elisa won't help cuz the provirus is dormant and not expressing any viral proteins/antigens. But since PCR expands DNA, even if the HIV hasn't really had a chance to become an active infection, the presence of the provirus is enough to detect and confirm infection. So that's why it's the best choice for early detection of HIV infection. Also in infant's of HIV+ mothers, PCR is the gold standard since the mother's antibody (IgG) crosses the placenta, the western blot/ELISA will give false positives even when the infant is not infected.

As far as viral load is concerned, we use RT-PCR. This is simply using reverse transcriptase to make a cDNA copy of the viral RNA, and then PCR amplifying the cDNA. Here we're looking for the presence and amount of VIRAL RNA. If we take a standard amount of blood from a HIV+ patient, and use RT-PCR to amplify the RNA, the higher the amount of virus present in the blood (the viral load) the greater the number of copies of amplified cDNA you'll have. Basically if you have 100 virions in blood, and we use RT-PCR to amplify their RNA, it might yield 10,000 DNA copies. But if we get 500,000 DNA copies, we'll know the viral load is way more. So the greater the amplification, the greater the viral load.

that's the way I understood it, hope that helps, and as always please correct me if i made any mistakes
great explanation of both
i think its spot on

the only question now is, what do we use viral load for? just to track treatment progress?
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