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Fast and scalable extraction protocol from AusDx

by Dmitry Kuevda | Apr 12, 2020 | in Catalogue of Resources | RNA extraction: New methods
D1: Path Lab Bulletin
D3: Catalogue of Resources
We (AusDiagnostics) just finalized our formula of fast extraction of CoVid-19 RNA (along with fluA FluB snd RSV). So far the method works quite well giving no more than 2 cycles delay in amplification take off and deliver much greater sensitivity than direct PCR assays.

While it use the same principle as standard magnetic bead extraction protocols we have significantly reduce the use of raw materials and simplified procedure itself so it is manageable to perform this protocol manually. We believe this will help us significantly scale up the production and make much more extraction tests from the same source of raw materials. We minimized a list of raw materials as well. The simplicity of protocol (combined lysis and precipitation followed by just one washing step) also eliminates the mandatory need of automatic extraction machines. Minimum equipment needed could be reduced down to manual pipettes (we use 8-channel and 96-channel to speed the process up) and simple 3D printed magnetic rack.

Have you validated this method, if so, how and what were the results of the validation?​

The method so far was validated in-house on modeled CoVid-19, RSV and FluA samples against full extraction kit (MT-Prep, AusDiagnostics). The fast extraction has shown the delay of take off (Ct) in the range between 1 to 2.5 cycles. All modeled low positive samples were detected though. We also did reproducibility study between two types of magnetic racks and two operators. Variation of take off values in case of extraction of the same sample was +/-1 cycle

How quickly could this be deployed and what are the dependencies?​

First trial batches planned in production in one week. We need 2-3 weeks to fill us the stock of raw materials and prepare production for a bigger scale.

What is the likely production volume?​

Initial production capacity estimated at the level of 10,000 tests per day with potential rise to 30,000 tests per day (Sydney manufacturing site). It is also possible and planned to make a technology transfer to our UK manufacturing site and further increase the production scale.

What are the risks and barriers to using this at scale?​

Supply of raw material is still limiting the production abilities the most. supply of raw materials for the production scale listed above has been secured through contracts. We are working on possible increase of supply 

Who are you already partnering with on this?

A few Australia labs will start validations in a week

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Sue Hill Apr 12, 2020

Really interesting. What is the potential throughput within the laboratory setting given the move to a manual process from automated ?

Dmitry Kuevda Apr 16, 2020

Hi Sue, We try to make this protocol accessible for different equipment settings. At the moment we are actively testing extraction of 96 samples in deep well plates on a magnetic rack. We tried manual 8-channel pipettes and automatic 96 channel pipette station. Extraction time with 8-channel pipette was around 35-40 min for 96 samples and less than 20 min with 96 channel pipette station. We also tried this protocol in tubes with standard single channel pipette. It roughly takes 30 min for 24 samples. Having all these results in mind the total turnaround time for Covid-19 testing is at least half an hour shorter (but more hands on). We also speculate that as long as reagents could be preloaded into tubes/plates samples could be loaded into lysis buffer in the dedicated lab area without preliminary inactivation. The nature of lysis buffer containing guanidine and alcohol will inactivate virus immediately. This will save some time for inactivation process which take place when automatic machines are used.

Rob King Apr 13, 2020

Status label added: D

Kyle Beacham Apr 14, 2020

The idea has been progressed to the next milestone.

Bev Matthews Apr 23, 2020

Status label added: D1

Status label removed: D

Tom Jordan Apr 28, 2020

Thank you for sharing this protocol with us Dmitry. I am pleased to let you know that we have shared your suggestion with the diagnostics community via a weekly Testing Methods bulletin, to encourage labs to test this and scale up. The bulletin directs readers to this page to consider the protocol in detail.

As one of the earlier proposals we reviewed, your suggestion went out in issue 1 on the 16 April. You can access this from the COVID-19 resources hub on the RCPath website: https://www.rcpath.org/profession/coronavirus-resource-hub.html

Kind regards,
Tom Jordan
On behalf of the Testing Methods team

Tom Jordan Apr 28, 2020

The idea has been progressed to the next milestone.

Dmitry Kuevda Apr 28, 2020

Current status update: the method was widely tested on manual extraction protocols. Using of a deep well plate and a 8-channel pipette allows to extract 24 samples in less than 15 minutes. We also successfully programmed and tested 96-channel electronic pipette from Integra. The protocol time for 96 extractions lasts 15-20 min. The protocol runs at room temperature. The plate of extracts with magnetic beads could go straight to the robot for automatic liquid handling without reformatting (our magnet is compatible with standard SBS positions of most liquid handling robots)

Charlotte Cookson May 7, 2020

Hi Dmitry, we are trying to make contact to see if you would like to join a conversation we are planning for next week. Charlotte

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Charlotte Cookson May 7, 2020

Status label added: Requires more information

Bev Matthews Jun 10, 2020

Status label added: D3: Catalogue of Resources

Status label removed: More information requested

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