I am working on diagnosis of tuberculosis with Taqman Probe in RT PCR. But sometimes negative also comes positive & sometimes positive also comes negative. I am using Primer & Probe concentration 3 micromol (working stock). In 25 microliter reaction mixture, 1 microliter primers & Probe each.
Hello:
Think about the possibility to have cross contamination. Change all the reagents and do small aliquot of each.
Do a calibration curve, 1fg to 10 ug of DNA, to optimize you RT-PCR, using the expected PCR product as standard.
I used primers stock 10 uM each and 1 ul to 20 ul rxn.
Good luck.
In PCR primers are used and in hybridisation, nucleotide probes are used.
Reasons for false positives may be due to the presence of contamination or atypical mycobateria sharing the same nucleotide sequence.
False negatives may be due to improper specimen collection or denaturation of the organism disturbing the nucleotide sequence.
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