NEW YORK – Dutch company Molecular Biology Systems is in the process of validating its shoebox-sized NextGenPCR instrument for rapid SARS-CoV-2 diagnostic testing, claiming the system could potentially process more than 22,000 samples in a day by performing plate-based thermal cycling in less than eight minutes.

Research-use only instruments are now being recruited for SARS-CoV-2 testing under rapidly changing regulatory guidelines worldwide. US Food and Drug Administration guidance, for example, has evolved to specify that that test developers are allowed to create Emergency Use Authorized workflows on instruments that have not yet been cleared for diagnostic testing.

Gert de Vos, CEO and founder of MBS, said in an interview that similar guidance is now being issued in Europe for RUO instruments.

“The world has changed completely,” de Vos said. High-complexity labs are eminently qualified to do the required validation testing to verify RUO instruments, he added.

The NextGenPCR system can perform 30-cycle PCR in two minutes, de Vos said.

The instrument contains six blocks with three fixed temperatures. Sealed microwell plates containing the reagents are shuttled between temperatures for thermal cycling. This obviates the temperature ramping required with a standard thermal cycling block because temperature changes on NextGenPCR takes a tenth of a second, de Vos said.

The system uses its own consumable plates, which are the size and shape of standard 384-well plates with a very different middle section featuring a polypropylene sheet with indentations to create the wells. These plates come in 5-µl and 20-µl 96-well formats, but the firm has also developed a 384-well plate for the instrument that can be used with volumes from 2 µl to 5 µl.

Aliquoting sample to the plate can be done manually or with a robotic liquid-handling system, as the wells are in the same positions as standard 96- and 384-well plates.

When MBS learned that RUO instruments could be used for COVID-19 testing, the firm used the fast turnaround time of its platform to its advantage, obtaining reagents from Integrated DNA Technologies overnight and performing about 1,000 PCR runs in a few days.

The firm has now been working on SARS-CoV-2 testing for about one month.

It has run a dilution series of virus-containing plasmids from Integrated DNA Technologies on the NextGenPCR instrument and on a Bio-Rad CFX qPCR system. In the MBS lab, the limit of detection of the CDC kit on the Bio-Rad platform was 10 positive plasmid particles per sample, de Vos said, while the NextGenPCR system could detect down to one plasmid per sample. “It’s safe to say that we are at least as sensitive as a standard real-time cycler,” he said.

The NextGenPCR system is endpoint PCR, and the firm has validated it with the US Centers for Disease Control and Prevention protocol to perform 40 cycles in less than eight minutes. The reverse transcription step can be done outside the system, or on the instrument, de Vos said.

In addition, in a research evaluation of the NextGenPCR instrument published in The Journal of Microbiological Methods in February, a group at The University of Groningen found equivalent sensitivity compared to a standard thermal cycler, the Eppendorf Mastercycler Nexus PCR instrument.

The group was evaluating the MBS NextGenPCR instrument for detection of E. coli strains that cause urinary tract infections, so was looking for bacterial DNA in urine samples. It adapted a fast extraction method to take only 5 minutes.

The technique was then validated on 128 clinical urine samples from patients with UTIs, and all target gene regions were amplified similarly across the two machines. Amplification in the standard thermal cycler took more than three times longer than with the NextGenPCR instrument, however.

Jolanda Brons, the first author of the JMM study, commented in an email that overall the instrument “turned out to be just as good as a regular thermal cycler,” but with the benefit performing a run in a much shorter time period.

“Our research paper showed that this machine is very fast and accurate, [and] we believe it can be a very reliable machine in the detection of COVID-19,” she said.

Endpoint benefits

The original rationale for using quantitative PCR testing for COVID-19 is unclear, although there may be some indication that people with higher viral loads can have more severe illness. But for simply finding positive cases, endpoint PCR could be sufficient, de Vos said. The MBS instrument could also theoretically be used upfront, to quickly find positive cases, which could then be quantitated if needed.

Extraction could be a rate-limiting step in a rapid testing workflow, but de Vos said there are rapid sample preparation methods that take around five minutes. These essentially perform a lysis step and do not concentrate the RNA, “But in an endpoint PCR, that is not really necessary – it’s all about ‘Is it positive or negative?'”

The samples are sealed in the plate with a clear film, and because the CDC kit uses a fluorescent dye, a virus-positive well will light up.

The fact that the CDC test is not multiplexed may mean more pipetting is required per sample, but this also enables using a scanner to read out results, which obviates the need to run a gel, de Vos suggested.

“We simply take the plate out of our system and put it upside-down on a gel scanner, and that’s it – in a few seconds you have your answer,” de Vos said.

The CDC test requires three wells per patient sample, so using that assay an MBS 384-well plate can process 127 samples in a plate, in less than 8 minutes. Thus, in 24 hours the system can theoretically process 22,860 patient samples, de Vos said. This throughput can scale up, such that with 10 instruments, it would be possible to test a quarter of a million people a day, he added.

It can also be scaled down to run a single test, for example to run testing to mitigate exposures in other hospital procedures, like surgeries, de Vos said.

The instrument costs less than €11,000 ($11,938), de Vos said. The 96-well consumable plates are €4 each, and the 384-well plates cost €5, he added.

MBS is also evaluating a testing method that uses a single viral target based on an open-source protocol developed in the Czech Republic, de Vos said. This assay could be run with a single control in a plate, expanding the capacity to 383 patient samples per 8-minute run.

The company is also testing out other enzymes and reagents, including a colorimetric version of the assay which could be visually read.

“Any lab that has our system can do their own validation, and because the PCR doesn’t take 90 minutes but seven-and-a-half minutes, the validation doesn’t take a week, but instead takes a few days,” de Vos said, adding,

There are now several labs in Europe and the US validating the MBS NextGenPCR system for COVID-19 testing, de Vos said, but he declined to name them at this time. And, the firm has also been working on modifying the system to enable running real-time PCR as well.

The firm has also had a few recent business developments. It hired Ronald Burggrave as business development director for Europe, the Middle East, and Africa, and it brought on Brian McNally as chief commercial officer in November last year. McNally is based in the US, and de Vos noted that the MBS instrument is distributed in the US by Velocidy Bio.

The firm currently counts sequencing platform developers and forensics firms among its customers, de Vos said, but he was not at liberty to name them.

Manufacturing capacity has been a bottleneck for other companies during the COVID-19 pandemic, but de Vos doesn’t think MBS will have any issues. The company has approximately 30 instruments in stock and 100 on the way in the coming two weeks, he said. It could also manufacture up to 500 more in the next six weeks, and has an ample stock of consumables.

“As soon as we saw this coming, we started making plates in double shifts,” he said.