Essentially every organization during this crisis has been faced with the intertwined decisions of what ongoing work to shut down and what COVID-19 related work to boot up. The former makes grading ONT's progress a bit tricky; it's unfair to expect them to live up to their pre-pandemic timelines. In the latter category, ONT has something very interesting cooking on the virus detection front.
ONT's premier bit of effort related to the pandemic is LamPORE, which combines Loop-Mediated Isothermal Amplification (LAMP) with readout by nanopore sequencing. As noted in the pseudoacronym (shouldn't it be LMIA?), this is an isothermal process, incubated at 60-65 degrees C. That eliminates the need for a thermocycler. LAMP also uses different polymerases than the RT-qPCR methods that are the workhorse of SARS-CoV-2 detection, so the supply chain is somewhat differentiated from that for RT-qPCR. Indeed, it is possible for LAMP to use polymerases with both reverse transcription and strand-displacing DNA polymerase activities, avoiding even the use of dedicated reverse transcriptases. Particularly in the early days of the pandemic, these supply chains were greatly stressed and it was sometimes impossible to get RT-qPCR mastermixes.
I've thought - and may still -- writing a whole piece on isothermal amplification, as it is interesting both for the sheer number of different methods and their relatively minimal usage. LAMP requires four primers which interact with each other in a specific way which correctly amplified by a strand-displacing polymerase. Once a certain amount of amplification occurs, the correct interactions are set up and what is akin to rolling circle amplification drives the generation of long multimers of the target, with multimers in alternating orientations. The exact layout of the primers never quite sticks in my head; after decades of designing PCR primers for minimal interaction with each other I think my mind reflexively rejects schemes that insist on it. Some of the initial LAMP patents reportedly expired relatively recently (I haven't verified this), which also makes it attractive.
There are now multiple SARS-CoV-2 assays given FDA Emergency Use Authorization (EUA). Color Genomics has one which detects amplification based on the pH change from polymerization shifting the color of a pH-sensitive indicator dye. As noted by ONT's Dan Turner, this style of LAMP assay can encounter difficulties from either sample contaminants interfering with the color change or non-specific amplification triggering a false positive. Sherlock Biosciences has a CRISPR-based kit which I wrote about in The CRISPR Journal; when correct amplification products are generated Cas13a is activated into a non-specific nuclease which in turn cleaves probes to release fluorescent reporters.
With LamPORE, a sequencing-based readout is employed. The long double-stranded products of LAMP are ideal targets for ONT's transposase-based rapid barcoding kit. By incorporating 8 barcodes within the amplification primers and 12 from the rapid kit, it is possible to have a combinatorial barcode space of 96. ONT has been screening barcodes to identify the best performers, and hopes to soon have 96. With an announced expansion of the rapid barcoding to 96, this will enable nearly 10,000 samples to be pooled into a single run. Each sample has four amplicons: three targeting viral genes plus one targeting a human mRNA. The latter serves as a check that negative samples really had biological input versus those from incompetent swabbing that retrieved nothing.
An ongoing question in the COVID-19 field concerns the required sensitivity of the diagnostic assays. The bias is towards very high sensitivity, so many of the RT-qPCR assays with EUA can detect significantly better than 1,000 viral copies per mL of swab fluid. If you work through the protocols and the subsequent math, many of these use concentration of sample in the RNA extraction step. With or without this, many of these tests are operating near the Poisson limit: at the limit-of-detection (LoD) the volume of analyte going into the amplification reaction may have 4-5 viral copies. Problems in comparing the sensitivities of these assays is a rant for another time, but this is generally where the bar lies -- though the FDA has granted EUAs to a few assays that are far less sensitive than this.
Turner showed data suggesting that LamPORE will be a serious contender on LoD. In one study 79 of 80 clinical positive samples were positive by LamPORE, with the outlier being the least concentrated (Ct=39) samples in the panel. These are for nasopharyngeal (NP) swabs, the workhorse so far of COVID-19 testing, with a sampling technique one colleague of mine compares to how the Egyptians pulled brains from mummies. More enticing for repeat testing is saliva, the use of which for diagnosis of COVID-19 remains a very hot topic. In saliva, Turner reports an LoD of approximately 2,500 copies per milliliter, which is worse than the best RT-qPCR for NP swabs but perhaps competitive in saliva space.
Turner also showed data with a different amplicon panel designed to detect six respiratory virus pathogens: COVID-19, influenza A, influenza B, RSV-A, a rhinovirus and parainfluenza 3. More widespread deployment and use of respiratory virus tests could be one thin silver lining from the dark cloud of the pandemic.
I think it was in the comments stream that someone pointed out that LamPORE fits nicely into ONT's overall strategy of supporting sequencing in remote and low resource environments. By trading a thermocycler for a constant temperature incubation, a key bit of bulky and expensive equipment is cast off. Simple water baths can support a 60C incubation. So LamPORE might show up post-pandemic in clinics across the world or out in farmers fields for use diagnosing agricultural diseases.
I wasn't sure how much I'd write on LamPORE, but it's more than I thought. Better to write the rest of the ONT announcements tomorrow. Stay tuned!