Real-Time LAMP amplification results from Pocket qPCR and open qLAMP.
Note: Before arriving in Cameroon Fran prepared 200 lyophilized reactions of GMO detective. However, the intercalant dye added was not optimized yet for GMO detective and it come out to be too small to produce a proper signal. Furthermore, the huge humidity of Cameroon, together with a not optimized sealing process generated the reactions to be gradually rehydrated, losing their efficiency over the residency. After a few days of the residence, the signal was not even visible to the naked eye.
We use two kinds of primers for the amplification:
We prepare two positive controls for each primer set with a sample previously quantified in the nanodrop and a negative control without a sample. Two duplicates for each condition. Eight tubes in total. In Open qLAMP, the initial well was not responding so we skipped one negative control.
The experiment was conducted as part of a 30-person workshop. Tube preparation was chaotic and false positives abounded during the experiment. Both the GMO amplification and the plant control come positive. Because this amplification was performed outside of the lab in a desk we were using previously with GMO controls, it is quite probable that the GMO detection is a false positive.
A third amplification was performed to compare the efficiency of two different enzymes to amplify a Tuberculosis synthetic DNA sample (No pathogenic, just synthetic DNA). The two enzymes used were the in-house produced Beneficial Bio purified enzyme versus the commercial Bst 2.0 enzyme from New England Biolabs.
The signal/noise ratio was too big showing the need to increase the concentration of intercalant dye (We used EvaGreen which is also not the best LAMP dye). The Mboalab enzyme performed quite well against the commercial one.
During an electrical power out we tested running the qPocketPCR from a USB power bank with promising results.
