PCR need some help - (Jul/21/2010 )

I need some help Understanding the pcr . This is not a medical health forum but i think the people here can help me better so i am posting my query . Sorry if i post in the wrong place . I am very dejected due to my pcr results.

I was afraid of the HCV exposure but my doctor said that the risk was almost zero in the exposure i described.

However i underwent a PCR test at 26 day and 33rd day after exposure .

PCR at 26 day came as HCV RNA DETECTED and on 33rd day came back as HCV RNA NOT DETECTED. ( both qualitative)

so i further enquired about the testing procedure at the respective lab and got this info .

The first lab PCR test was done by REAL TIME RT PCR by flouresence method and they said that lower limit was 50 copoies per ml .

and second pcr was "nested rna pcr" the second lab mailed me their testing procedure as i asked them , their reply was

"For the HCV qualitative assay viral RNA is isolated from serum/plasma. For Reverse Transcription and PCR amplification, nested PCR is being employed. In this PCR system 2 sets of primers-outer set targeting bigger fragment of viral genome (5'NCR) and the inner set complimentary to regions inside the bigger fragments are used for amplification. Thus the amplified product of first step acts as a template to the nested step or second step. Detection of amplified sequence is done by 2% agarose gel electrophoresis."

which test should i belive ? the first one or the second one ? i dont know the lower limit of second test .

anyone help me please , i am so dejected ..

what are you trying to ask about PCR???

If you want to re-confirm whether you are infected with HCV, do a serology testing.

p/s: check your PM

I think you have to talk with your doctor and do another test. If I had to tests with contradictori results I would like to contrast with another one.
I have found this article:http://www.biomedcentral.com/1471-2334/4/55. It talks about the comparison of both techniques you talk about, anyway errors exists, so talk with doctor. He has to do the diagnosis!

Hope everything goes well

Thanks for the replies.

I would like to know one thing , Can nested PCR be used to detect HCV virus after 4 weeks ?


What is the sensitivity of nested pcr ?

hello there,

go to your physician, he may ask you to do other liver function tests as ALT/AST ... etc to check your liver function.


HCV :
acute infection : HCV RNA is positive 1-2 weeks after infection with HCV antibodies developing after some what 12 weeks.

taken from : oxford handbook of clinical and laboratory investigation.
p. 462.

so, if the virus is really there do antibody test, and if unfortunately gave you a positive test, do HCV genotype test to know which HCV infected you.

wish you all the good health, calm down ...
best wishes.