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TaqMan qPCR low-template issues - (May/31/2012 )

ViiA7 Machine
TaqMan® Universal Master Mix II
FAM reporter/NFQ-MGB probe
Quantification of pathogen genomic DNA

Hi Folks,

I really need some help figuring this problem out. The issue in a nut shell is that I get weird curves for low-template standards and sometimes the NTC, which looks like some sort of cross amplifcation. This was not happening before. I have changed literally everything - new probe, new primers, new water, new master mix, different machine, different plates, tried BSA, took extra precautions to prevent contamination. ABI even sent me new MM, but nothing seems to work.

I would appreciate any suggestions or advice.
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I am using ABI 7500 for multiplex qPCR. Our multiplex panel is designed to detect 3-4 pathogens per reaction (total of 8 reaction to detect 33 pathogens). In one reaction we are detecting S aureus (FAM), S pneumoniae (CY5), C. pneumoniae (VIC), Hemophilus influenzae B (ROX). I do not know why we are getting low positive curves (high ct value) for S. aureus even in NTC.

We have done everything that we could. We have changed pipettes, working area, ordered new enzymes, primer-probes and even requested others to do the PCR for me. But the S. auresu problem is there. We don't think there is any problem with dye FAM. because in the same panel there are other targets with FAM dye but they are not showing any problem.

I just do not know, could it be intrinsic problem of Real Time PCR? I mean any template independent event?

Please share your experience.