Nested PCR-false positives - (Nov/01/2006 )
Dear all,
I have been working with nested pcr for infectious diseases.My first round product is 396bp,and second one is 293bp. After the first pcr, i take 1 in 100 dilution of the first product and then perform the second pcr.
My problem is that whenever I perform true negative disease controls,I get positive in the second round pcr.I checked its first round product, they are negative.To rule out contamination I changed everything,but iam getting false positives in all cases. Positive cases when used i got 3/5 positives also,which is acceptable.
Please help me,why i am getting such precise band in nested pcr,while these same sample give negative result in ordinary pcr.If it is something with sensitivity,why should all negative control patients show disease bands?
Hoping for any reply
niveda
I have been working with nested pcr for infectious diseases.My first round product is 396bp,and second one is 293bp. After the first pcr, i take 1 in 100 dilution of the first product and then perform the second pcr.
My problem is that whenever I perform true negative disease controls,I get positive in the second round pcr.I checked its first round product, they are negative.To rule out contamination I changed everything,but iam getting false positives in all cases. Positive cases when used i got 3/5 positives also,which is acceptable.
Please help me,why i am getting such precise band in nested pcr,while these same sample give negative result in ordinary pcr.If it is something with sensitivity,why should all negative control patients show disease bands?
Hoping for any reply
niveda
What is the basis of disease? Is it an etiological agent, or a mutation of some sort? Is it the *presence* of the 296 bp fragment that is supposed to be diagnostic? Or do you then look for an RFLP or some such on the product to diagnose disease?
It sounds like you are trying to PCR up from an infectious agent (bacterium, virus, or some sch.) Can you tell us the organism? Many infectious bacteria have noninfectious relatives living on us. You could be amplifying a homolog from a non-pathogen.
You can also try a blast with your pimers, and see if something in the database hits weakly, that might be the basis of your problem.
Just to be sure it's not contamination, and you are probably already doing this, but do you have a no DNA control in addition to negative controls and is it clean? You should make a no DNA for first round PCR and then dilute it 1/100 as with your samples and use it for second round PCR.