PCR, Cloning, Sequence, Repeat

Wasn’t there some statement about doing the same thing over and over again and expecting a different result…? In early March we reported on the exciting application of a new primer set targeting the non-structural (NS5; also known as the RNA-dependent RNA polymerase) gene of a cluster of aquatic invertebrate flaviviruses (aiFVs). These were designed in early January around several flaviviruses in the one somewhat conserved genome region between sea cucumber, shark (yes, I know not an invertebrate – but the shark was an almost 100% match to a crab virus), and nudibranch aiFV. PCR using these primers (and other PCR primers designed around the sea cucumber flavivirus alone) generated amplicons of about the right, expected, size. However, upon cloning and sequencing these amplicons it was clear they were not coming from aiFVs, but rather from bacteria which were probably in the sea cucumber microbiome. We then re-designed these around a different set of sequences, and repeated the exercise. Unfortunately, while these generated amplicons of expected size, these too did not match flaviviruses. And so for a third time we tried this again, and same result. Notwithstanding technical issues in the lab unrelated to the project, this has set us back a bit in our efforts to study aiFV diversity generally!

Why has this been such a challenge? When designing what we consider ‘highly specific’ PCR primers (and probes… see next section), it is very difficult to know what the pool of DNA that a metazoan comprises – it’s made up of both the metazoan host, but also a myriad of other microorganisms living on and within tissues. Which is why we are testing PCR products thoroughly to ensure what we’re amplifying really is what we are targeting with our approach!

So it’s back to the drawing board again. However, we have a contingency plan. Ian has designed two pairs of TaqMan real-time PCR primers/probes, one targeting the NS5 gene of the sea cucumber aiFV, and the other targeting the Envelope region of the FV polyprotein. This approach is the same as used for detection of COVID-19 in clinical samples, though clinical labs running COVID tests do so with robots and are able to screen thousands of samples per hour. We’ll be running only a fraction of that speed since humans are running the reactions!

When the primer sets come in this week, we hope to apply these broadly to cDNA from a huge number of samples. This is a major advantage of qPCR – you can screen hundreds of samples for a gene of interest with real-time detection of products (c.f. running on gel, imaging, etc). Plus, through our work in the last 7 years on echinoderm disease, we have accumulated a library of 88 sea cucumber tissue samples; roughly half are Apostichopus californicus (from southeast Alaska and the Salish Sea), while the other half are various other species from the Salish Sea, Australia and New Zealand. Hopefully we get some hits this time.

The idea behind using a TaqMan probe is that in addition to a forward and reverse primer needing good sequence match to target DNA, the chemistry includes another 20nt long probe which has to match the amplified sequence to report detection. Nevertheless, experience has shown us that even with well-designed TaqMan primer/probe pairs, there is the potential for false-positives when applied to tissues of unknown DNA sequence (e.g. our early sea star associated densovirus work in sea star wasting disease turned out to amplify many different densoviruses, not just the one we suspected was involved in disease). Hence, if we do detect any aiFVs in any sample, we’ll clone and sequence these to ensure what we are detecting really is what we are hoping for!

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