August 16th (Prof. Youssef)
@Adrian Villanueva @Yujeong Oh
Questions
- How are they working on detection of COVID (using fluidigm on RT-PCR)
- technique
- gene of choice
- reporting mechanism (sybr green?)
- how many sample at once?
- reaction time (because different PCR reagents require different reaction time)
- How sensitive is it?
- Why is it more sensitive than governmental/ official testing. How do you make it more sensitive? (primers?)
- Ask for whether we can work with them - their method on our few samples
- if he allows, ask for the reagents needed in their method
- if not, ask for pcr protocol they are using so we can use test it by ourselves following the protocol
*Fluidigm microfluidic chip (screenshot from fos lab ppt: detect SNP using PCR)
*This is the lab ppt explaining how IFC works!:
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Meeting notes
About our project:
- DNA extraction: need comparison between methods in implementing it without loosing sensitivity
- magnetic beads (becoming popular for purification?): enriching DNA w/o adding PCR inhibitors
- method that would skip extraction
- extract DNA from bird's feces:
- feces have many PCR inhibitors
- ratio between inhibitors (of downstream) and DNA
- skipping extraction?
- DNA is easier than RNA
- recommend to work with real samples —> synthetic DNA of target fragment
- serial dilution of DNA - use it for testing ; robustness of detection
- then, go back to actual sample
- team from erb can help us out with permits
- Get samples similar to Bd/Bsal species that can be found in the UAE.
- Do some benchmarking with PCR
- Spiking DNA (spike-in DNA?)
- Establishing detection limit is important
- Rafael is aware of concentrating DNA
- Not many enzymatic DNA as possible
- Biggest effort should be upstream; concentration of nucleic acids (enrich)
- talked to Prof Stephane: might have skin samples
- they had a plan to do skin microbiomes
- extract DNA from skin - sequencing the whole soup of DNA
- we can use some of those sample in our testing
- More DNA from the frog than pathogen DNA when we swab; frog DNA can be used as optimization (without inhibitors)
- spiking experiments; synthesized gene fragment
- actual test using standard PCR/ test; third benchmark
- we need to test false positive and false negative rate
- false negative rate would be key
- detection limit
- store sample in ethanol - and do test in lab
- silica matrices - go back to silica powder
- calex 100: one of basic extraction method
- magnetic beads: don't require centrifugation, only require magnetics
- What's really important is setting the benchmarks
- You can hypothesize which extraction method will work better. But at the end of the day, you'll only know which is better once you test it in the lab.
- positive control would be frog DNA: if not detected, invalid test
About COVID-19 project:
- improved version of RT-PCR
- most lab, extract RNA from the sample - one-step detection method (one reaction does RT-PCR and quantitative realtime PCR; same time converting RNA to cDNA and detect)
- RT-PCR of whole RNA using random hexamers ; convert RNA to cDNA → pre-amplification step of gene that you want to detect: three fragments; 1 human gene, two viral genes → fluidigm chip; nano-scale PCR test
- volume is small in fluidigm chip - increase sensitivity compare to tubs
- not expensive; small volumes
- Taqman uses primers and specific probe
- down to 1 copy of viral RNA/ microliter
- developing other method that would skip extraction
- smart pooling: one reaction from many samples- combination of samples - can deconvolute
- commonly used extraction medium: triton-X
- 20% false negative rate in UAE official testing
Future direction
- Add positive control to our test: amphibian's specific target gene in our test
- Order reagents in prof Youssef's protocol & test in lab
- Reach out to Prof Rafael about concentrating of nucleic acid
- Reach out to Prof Stephane about frog sample
- Get the sample import permit with help from ERB
- DNA extraction method