I am specifically interested in assay protocols that will work in a mobile lab.
Since it is a mobile lab, I assume it is a van or some kind of vehicle. If so, the thermocyclers should be light-weighted, and can analyze many Ebola samples at once. If you are going to use the mobile lab in places such as West Africa, an energy-saving one should be preferred. I am kind of worry the 'PCR contamination' problem, especially PCR a same gene repeatly in a closed environment (inside the vehicle). We were just discussing the PCR contamination issue at a RG site. See: https://www.researchgate.net/post/What_does_it_mean_if_the_negative_control_shows_bands_on_agarose_gel_after_PCR
Thanks. We have plenty of experience with the Bio-Rad MiniOpticon, and have run this in the field from the back of a 4x4 without contamination problems. But this model is no longer supported, so we're looking at a number of alternatives. I'm also keen to get the rtPCR chemistry optimised for field use. It's easier with bacteria when all we have to do is amplify a DNA target. The reverse transcriptase step has always been the challenge for our arbovirus field work. Ebola presents a similar challenge.
Check out Quanta Bioscience qRT-PCR products: the original scientists who invented things like SS I, II and III and the older/better "iScript" still reside at that company. Their products are preferred by the CDC etc. -- their one-step (RT and Taq-containing mixes) perform extremely well. Mikael Kubista's TATAA Biocenter is the other place to check out. 1 copy of something (at high efficiency) generally appears at a Cq of ~37. One of the dilemmas with finding low copies: e.g., if you have 1 mL of sample extract in which only 1 copy of Ebola exists, that entire 1 mL needs to be examined. If one only examines 50 uL of that 1 mL by qRT-PCR, the target has a better chance of being missed than detected since the remaining 950 uL has been left uninvestigated, and so on. It is better to entirely exhaust the sample into wells for RT-qPCR analysis than conclude a negative result in such cases. Concentrating samples up-front is always an option - but many times, RT and Taq inhibitors too are co-concentrated.
Prof Tim, I am glad that it is possible for detection of bacterial pathogens using mobile rt PCR in the fields especially in remote areas . Similar protocol can be applied in viral pathogen detection in the field with various optimization in different climate and environmental condition. We will work on further action plan at least for bacterial pathogens detection.
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