PCR is6110 Tuberculosis - Non specific amplification (Jul/24/2004 )

Hi! Im student from Guatemala City.

We are having problems trying to standarize the detection of MTB from sputum samples with a PCR-Is6110.
We have to obtain a PCR product of 300 pb, but with several sputums we obtain bands of 400 pb, with other we do obtain the band in 300 pb. What can cause that? And more important, how we can get the band in 300 pb?

Thanks a lot! And sorry for the spelling!

Hi,

I think the most possible cause is contamination. contamination control is very important for MTB PCR. There is a paper dealing a lot with contamination problem (Full text is available for this paper and free)

http://www.pubmedcentral.nih.gov/articlere...ertype=abstract

Hi!
I'm not familiar with MTB PCR, however from general PCR I'd say that it could be
1) contamination as postdoc said. Do your negative controls come up with any bands? If so throw out all solutions, start again.
2) Seeing you are getting some correct fragments, but not all, could you have polymorphisms of your gene? Therefore giving you different sizes? If these facilities are avaiable try sequencing your 400bp product and examine the sequence.
3) As I said I'm not sure what MTB PCR is, so disregard this point if I'm off track- is it a DNA, RNA PCR or both? You could possibly be amplifying a larger fragment off DNA.
Hope some of the above helps