I'm  working on the multiplex assay for viral identification in patient nasal swab samples, using a Fast 7500 qPCR machine. I have noticed something anomalous in the raw fluorescent signals, and I would appreciate your input.

The quick summary: In an ideal situation, I tested 22 replicates of the same sample. I should have seen 22 of the same Ct value with pipetting errors. What I got was a curiously repeating pattern with radically different Cts based on well position in the plate. Huh?

In this experiment, I used a mixture of primers and only one probe (VIC) over two 12-well rows on a 96 well plate. I was concerned about contamination, and had rinsed the block and done background calibration readings. I put the same amount of primer, probe and template in each well except the first NTC column, which had no template. I asked the machine to read fluorescence on four channels per well - FAM, TAMRA, JUN, and VIC - even though there was only VIC probe in the wells.

In the first unknown well, we saw amplification of VIC fluorescence consistent with PCR. (Well G2, Fast7500.VIC.Positive.jpg) The other probe signals appear flat.

In the second unknown well G3, we see an exponential curve for VIC, but it is a decrease of signal. (Well G3, Fast7500.VIC.negative.jpg) It appears that JUN fluorescence is amplifying, even though no Jun probe is present. ???

In the third unknown well, we again see positive amplification of VIC fluorescence. (Well G4,  Fast7500.VIC.Positive.but.mirror.jpg) Curiously, here we observe the FAM signal decreasing in a mirrored fashion to the VIC signal. ???

In the fourth unknown well, we again see negative amplification of VIC fluorescent signal paired with an apparent increase in FAM fluorescent signal. (G5, Fast7500.VIC.negative.and.mirror.jpg) ???

In the fifth unknown well, the fluorescent signal pattern looks like the first picture I showed you, the sixth mirrors the second, the seventh the third, and the eighth the fourth. And so on.

Have you seen something like this before, singleplex OR multiplex?

Thank you,

Trista Robichaud