The SARS-CoV-2 pandemic has brought with it a variety of new (and newly-applied) approaches in science and public health. mRNA vaccines have been given to over 160 million US residents – a “technology” (mRNA isn’t exactly technology, but the delivery method is certainly novel) which has not been widely applied in the past. While there were several iterations of qRT-PCR based approaches to detect SARS-CoV-2 in the early phase of the pandemic, testing via antigens, rapid qRT-PCR, and by reverse-transcriptase loop-mediated isothermal amplification (RT-LOOP) mean that from nasal swab or saliva sample to positive/negative result is down to a mere hour or two. This latter method, which has been in use for some time to detect several human pathogens, is gaining prominence since it doesn’t require expensive, temperature-changing thermocyclers (rather, just a water bath at 65 degrees Celsius) and is very rapid (~ 30 mins vs 60 mins plus in a thermocycler). It’s also incredibly inexpensive and, importantly, can be colorimetric, where a positive reaction turns a certain color compared to positive samples and negative controls.
In the last few months, the lab has centered on using qRT-PCR as a tool for detection of aquatic invertebrate flaviviruses (aiFVs) – this is a multi-step process whereby RNA in tissue samples is extracted, then the RNA is converted to cDNA by reverse transcriptase (RT), then amplified in quantitative PCR (qPCR). We are using this as a way to identify samples positive for aiFVs and then follow this with reverse transcriptase PCR using degenerate primers to study the diversity of aiFVs within positive samples. But as a preliminary step this was proving cumbersome; and our ultimate goal is to develop a field-deployable assay which could be used by partner citizen scientists, high schools, Alaskan tribal groups and fishers to monitor for the presence of these viruses.
So one day two weeks ago, Ian consulted with New England Biolabs who offer not only a very competitive/inexpensive RT-LAMP kit (which comprises one doubly-concentrated (2X) master mix reagent, which includes a color-changing indicator for positive detection, but also an online design tool for the 6 primers needed to perform the assay. Ian thus designed the primers, ordered them in, along with the NEB kit. Our expectation was that it “probably wasn’t going to work” since our experience has been somewhat meagre with amplification of aiFVs by qRT-PCR. So Jay took 8 samples of sea cucumber extracted RNA (one of which had come up by qRT-PCR), our synthetic aiFV nonstructural gene 5, and several negative (nuclease free water) controls, added them to the reagents and primers, and sat them in a water bath at 65 degrees for 30 mins. To our great surprise, the positive came up positive, the negatives did not change color, and… the qRT-PCR positive sample also changed color. Did this work? Well, it was a good start.
So next, Jay ran more and more samples, including a wider suite of qRT-PCR positive samples, and those came up positive – along with the standard. Hooray!
We still have much work to do. First, what is the limit-of-detection (sensitivity) of the approach? Can we abbreviate our RNA extraction protocol which normally calls for some specialized lab equipment (like micro centrifuges) to become field-deployable? And what is our overall RNA extraction efficiency for these viruses? We’ll be looking at these in the next few weeks.
But all going to plan (which I guess it never does…), we soon will have a field-deployable, simple and inexpensive protocol to screen huge numbers of samples which we can then investigate for aiFV diversity. This represents somewhat a quantum leap in our abilities – circumventing many of the early expensive steps for viral detection!
We’ve also been making ardent stides with seagrass. Jordan returned from the West Falmouth Harbor with seagrass cores for our aquarium system – at this stage it’s about learning how to keep these happy and healthy in an artificial lab setting, so that he can use these for experiments in the fall when the site is inaccessible and weather does not permit easy access. While the aquarium room setup is promising, we’re still working on some details, like how to regulate light conditions on a day: night cycle – so unfortunately we’ve experienced a huge algal bloom in our aquarium… so it is definitely a learning process. Jordan’s also working on cultivating bacteria and algae from these seagrass blades. He’ll be heading out to Cape Cod another few times this summer – when hopefully our aquarium keeping improves!
Stay tuned for more from the team in coming weeks…