Most pcr still uses taq I believe. I recall playing with a couple of other polymerases back in the dark ages. That was mostly about circumventing Perkins Elmer’s licensing.
I’m sure there are other options, I haven’t heard of anything radically better.
Real-time or quantitative PCR was was only just becoming reliable when I last played with the dna thermal cycling…I spent way too many hrs banging my head against a first gen qPCR machine…the concept was good, but the early implementation I got to service/maintain was a finicky wee beast.
Fortunately the concepts haven’t changed.
From a price of admission standpoint, qPCR is gonna set you back more, but day to day use is going to be less time consuming.
Not sure reaching back 10-15years for hardware is appropriate without actually digging in, but the machine I was beating my head against back in 2001 was already several years old…it was still in use in 2010 .
5-10years younger should presumably have all the necessary features and be 1/10th the price of a brand new rig
We used to perform the trick by removing tubes from the cycler at various points and comparing them on a gel…
I was considering the real-time PCR (qPCR) QuantStudio 3 from Applied Biosystems (Thermo Fisher Scientific). It’s what LeafWorks uses; a 96-site well is more than we need for in-house testing. I have a call set up with a Fisher Sci sales rep next week to discuss suitable models. The QuantStudio 3 is about $34K, so I’d prefer to find a newer used model or a 48-well model instead.
Also, it seems like reverse transcription PCR (RT-PCR) or reverse transcription real-time PCR (RT-qPCR) would be the best option to test for a greater number of viroids and viruses. What do you think?
For example, here is an RT-qPCR multiplex assay for Hop latent viroid (HLVd), Lettuce Chlorosis Virus (LCV), and Cannabis Cryptic Virus (CanCV) from MedicalGenomics for ~$2.5K for 200 tests. But at that cost, I’d rather create assays in house if it makes sense.
And now I think about it; you’re correct that it makes more sense to set up PCR and do in-house testing from the jump. So we will plan to set up a small lab where our mothers will be temperature staged and test them before brinding plants to the new facilty.
Thanks for the paper, I’ll review it later. I have found a few other and more recent papers as well. This is interesting, too:
Multiplexing is easy and extremely powerful, in the case of qPCR your limited by your fluorophores or the detection on your rig. Pretty sure we only had a single channel on the last one I used, but the sequencers in the next room certainly used four at the time, so I may be misremembering.
You should be able to put together your own assays for less per test, but that may depend on what dye modified primers are going for these days (should be way less than I recall).
If you’ve got $35k to throw at the problem, making sure you’re not wiped out by something like this viriod is priceless. We
Yeah, I could work in ~$50K for a PCR budget. But I’d rather spend less on the PCR if feasible. And I would use it for many things, including sex identification and cannabinoid ratios of seedlings when pheno hunting, early detection of PM, detection of latent bud rot before hanging up to dry, edible and beverage QA program, etc.
While I could plate out and grow samples to identify various pathogens, using qPCR is so much faster and ensures the PCR isn’t acting like a giant paperweight.
Good read here about sanitizing tools. I’ve been using straight 12% bleach for a while now then rinsing in ro water but sounds like it’s a bit overkill. Tool sterilization comprehensive report.pdf (1.2 MB)
I soak my shears in simple green to get off the stickiness, ph of about 9. I still use peroxide after that, but I would be impressed if the viroid would not die or fall off in the simple green.
I bought a 3 station dishwahing sink for prepping trays and domes. All get washed in dish soap, soaked in 5% bleach, then rinsed. Since each of my strains comes from a common mom, we only clean tools between strains. Seems to work well enough, roughly 2k cuts get taken every 5 days at this point.
The key is not bringing it in. Lately I’ve been quarantine testing in a 100 degree, 2000ppfd environment, that really brings it out
I think I paid about thirty cents for the seed, so a test seems like overkill. The plant was not a runt, which is how I had thought the viroid would affect seeds, making them easy to cull.
I want to learn how to spot it through the ways it affects the plant. That is much more important to me than this particular plant.
Hard to tell by just looking at one symptom….testing is always the most effective way to tell. there would need to be many more symptoms to even have anywhere close to an idea to try to diagnose it visually……even then It’s only a hunch. It can transfer through seed so it’s possible….just test it to be 100% sure or if you continue to run it just be on the look out for the signs keep it away from other healthy plants (don’t cross contaminate plants with run off, utensils for clean up/or taking clones, sap from plants can carry the viroid)horizontal growth, weak brittle branches that fall to the touch, tacoing/ curling/ twisting, throwing off 1 to 3 bladed leaves, plants dying out of nowhere, overall plant health hard to maintain and cloning success % way down are all things I’ve noticed but it can also be a silent killer and you don’t know before it’s too late so testing before anything if you can afford it if not plenty of bleach and being mindful of cross contamination by quarantining plants until you know for sure.
These are plants that are tested positive that I’ve noticed had symptoms maybe it will help or not but at least it’s a start none of them look normal these are all signs that the viroid is affecting the plants overall health
Should be obvious which is the viroid in each photo. These are old photos of apple fritter and cereal milk. The weird branching is the dead giveaway to me.
