Is anyone implementing this now?

[daniel...@gmail.com]

Hi,
Is anyone implementing this entire workflow now? Would like to know what the friction points are if this is something my company would like to participate in implementing - I work for illumina.
Daniel

[Mikolaj Slabicki]

Hi Daniel,

In the upcoming few days, this protocol will be tested on clinical samples. We hope that sensitivity and test robustness will allow moving to the implementation phase. Fingers crossed.

Best,

Mikolaj

[daniel...@gmail.com]

please keep me posted if you think Illumina can play a part in this. my work mail is dbrami followed by at followed by illumina dot com.

[Fernando Rivadavia]

Hi, I work for ILMN and am following this discussion in the hopes that it works. I've already brought the protocol to the attention of internal teams. Our customers are begging for a high throughput NGS diagnostic solution, due to increasing backlog of qPCR testing in many countries. Feel free to write to me if there's anything you think we can do to make this protocol a reality: friva...@illumina.com . 

Thanks!

Fernando

[Jonathan Schmid-Burgk]

Hi Daniel and Fernando,

Thanks for offering support from Illumina. We are currently optimizing and testing the LAMP-Seq protocol, and will reach out to you as soon the molecular biology is finalized.

Best,

Jonathan

[daniel...@gmail.com]

Fantastic, does this mean the clinical sample data came back? 

What about Aaron request for access to FASTQ data? 

As stated, we have a lot of eager resources here that can assist with increasing this protocol's amazing scalability. My VP is aware of this effort so please get us involved as soon as it makes sense.

Keep it up!

Daniel

 

[yannic...@upmc.fr]

here we try this RT-LAMPseq protocol on real saliva sample (4 plex with 6 positives and 4 négatives on each). for the first experiment, nothing usable in fastq.

We Don't know if the LAMP rxn work or if it's because of later PCR.

We use B set of primers and follow the protocol…

We prepare 200µL RT-LAMP rxn and add 300µL of Saliva (500µL final with paper component concentration) --> 65 °C for 30 minutes to react, and to 95°C for 10 minutes to sterilize

1- we do not see the expected smear at the end of LAMP. can we view it on a bioAnalyzer device? if not, should you make an agarose gel (what conditions and how many inputs on the gel)?

2- should we purify the LAMP before the 1st PCR? what are the PCR cycling conditions (time and temperature) for the first and second PCR?

3- how we can control the first and second PCR? what should we observe on a gel ?

does anyone have answers to these questions or can help us?

Yannick

[Jonathan Schmid-Burgk]

Hi Yannick,

we are validating and optimizing protocol iterations since a few weeks, and made similar observations like you using high volumes of unpurified biosamples. We will share an updated protocol as soon as we are confident in it.

The below protocol works better than the one suggested in the pre-print on clean RNA:

Starter Kit protocol (2020-04-16)

Thanks,

Jonathan

[David Li]

Daniel: we have recently updated the preprint with validation on 26 oropharyngeal swab patient samples.

Yannick: the updated preprint contains the latest protocol, which works on OP swabs. We have yet to try to this on saliva.

[yannic...@upmc.fr]

many thanks… we 're going to test the latest protocol with our saliva samples.