I‘ve developed a rt-lamp system for detection of a relatively wide range of coronaviruses. Even with rather strict precautions（ always wear mask, clean my workspace, add my etc and nc samples before I touch my pc samples）, NTC and NC sometimes show unexpected amlification. And the unexpected amplification of my NTC and NC is quite inconsistent. I tried to read something from the melting curve since i am using the fluorescent lamp kit, but the tm values are unstable too, making it tricky to distinguish the non- specific products and the aerosol contamination？?
Currently im using fip/bip at 1.5um for each, loop primers lf and lb at 0.8um for each, and f3/b3 at 0.2um. I tried to lower the concentration of my primers but （FIP/BIP conc. 1.6um/for each to 1.5um for each）, which helped once but the same setup didnt go the other day. Im hesitant to reduce the primers concentration too drastically as it might weaken the amplification of my pc sample. is there anyone with experience who can give me some advices？ I want to make it stable and i am desperate💔
Contamination can be in the dust on the bench or in the pipettes used so 2 tries are....1 move to another working area and borrow pipettes that have never ben used for this reaction.
2 design primers for a larger target so these will not bind to the contamination amplimer
Paul Rutland Thank you so much for the helpful suggestions! 1. I usually move to another table to add my positive control (PC) samples, but I didn’t change the pipettes this time. I always use filter tips, but I’ll try to borrow pipettes that have never been used before next time — thank you for the advice.
2. I believe the primers I used in this case should not amplify my negative control sample, as the NC template lacks part of the target region. May I ask if lowering the primer concentration might help reduce non-specific amplification or possible contamination? I suspect that my current primer concentration might be a bit high. If so, which primers would you recommend adjusting first — FIP/BIP or the loop primers?
Thanks again for answering my silly question 😭
Filter tips are very good but some amplimer from loading gels or transferring product can still get into and onto the pipettes. A thorough strip down and clean of the pipettes is good. Contamination can also happen from aerosols caused by flipping open tube lids and if amplimer gets onto the dust in a lab then air movement and air con can spread it around. I really do not think that diluting primers further will work. The contamiantion is already amplified and will further amplify better than genomic template so may still be the major amplimer in the tube. Your question is a good and important one as contamination is a huge problem once it is established and a nightmare to get rid of. I have known people to move to another lab to run reactions that were contaminated (possibly airborne)
good luck paul
Paul Rutland Thanks so much again!! I’ve already aliquoted all my reagents before use, and I’m planning to work in the clean bench with some clean pipettes tomorrow. I’ll also try UV after each run and see if that helps.
I really can’t tell you how much I appreciate your advice. I didn’t know who else to ask, so your message really meant a lot. Wishing you all the best with your work too! Hayden
Thats good Rrr Rr . As your contamination is sporadic it would be a good idea to run 4 or 5 NTC samples to make sure that contamination is not an issue