Who has HLVd testing in-house?
How are you performing it?
RT-qPCR?
RT-LAMP?
What’s your consumables cost per test?
Who you got running it?
Some background reading:
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I’ve tried hitting these folks up, no response yet…
hoping they know what they’re doing and are actually ready to ship.
then there is
which looks to use RT-LAMP as well. they claim they were first to market with in-house testing. owned by Kaycha labs, for what that’s worth…
…and if you want to dig in, and design your own LAMP based detection, I’d start here: https://scholar.google.com/scholar?q=LAMP+Hop+latent+viroid
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Medicinal Genomics goes into the reasons one might want to use qPCR rather than LAMP
Seems like sound reasoning given they’ve developed LAMP based kits for other screening questions.
Given the two orders of magnitude difference in equipment costs, I’ll likely be running with LAMP based to start…
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Agdia offers a kit based different flavor of isothermal amplification (RPA) with a battery powered fluorometer that even @thumper’s wook might be able to figure out.
$10k for the fancy fluorometer, and ~$15 a test for consumables.
technique only uses two primers, compared to LAMP’s 6-8 primers, so you can multi-plex and/or run an internal amplification control. which the Medicinal Genomics article above will tell you are both advantages of qPCR over LAMP.
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Actually….you ought start here. Primers and all
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if you have a clean garden and dont contact other grows whats the main vector for this virus?
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IF you have a clean garden, then keeping it isolated should do the trick.
that means if your WOOK also has a garden, they are your primary vector.
it is possible that insects may also act as vectors, but that seems an unlikely source for introduction into an indoor grow.
the problem is that not all plants show sysmptoms, or don’t show symptoms until flower, so it’s easy to contaminate your entire garden before you even realize there is an issue.
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i thought the main obv thing was a certain curled looking leaf. That may be for a tobacco mosaic though I generally always started from seed so havent dealt with this shit until likely my clones had it. I know it can spread in a cloning machine but how else does it spread? via air? contacting an infected plant with a watering wand and then touching an uninfected plant? I know scissors obv.
Agriscience in Colorado does this testing.
I’m not looking to mail test samples (we’re in OR).
I’m looking to bring testing in house.
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I hope someday there is research about transmission via seeds. Right now the answer is a theoretical yes, but not much more than that. I suspect any seed that pops a positive sprout will be a runt that most people would cull anyway, and likely before it was mature enough to spread the disease. In wild cannabis, that runt would get grown over by other plants and not get to reproduce. That’s nature’s disease control.
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Seed coat. And yeah. 
I can attest to finding it in seed multiple times. It’s very real. @cyclopath good on you for striving to test in-house brother. Thanks for making this thread. Stuff is fucking nightmare sauce.
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Can you use microscopy and look for viroid’s or a change in the chromatin pattern?
If you want to save some $, but a gel doc and a UV lamp and you should be able to see a band corresponding to the different PCR products. I always thought qPCR was pretty inaccurate unless you are really skilled and familiar with the equipment and primers, and on top of it you don’t really learn anything by quantifying the viroid since if something is replicating in a tissue there should be many copies of it.
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This will absolutely work for the PCR and RPA based methods, with LAMP based methods you’d need to add a restriction digest first because of the nature of the amplified product.
You would of course want to get reagent kits that didn’t include the fancy fluorescent trickery (FAM) you wouldn’t be using.
You could absolutely use FISH to diagnose plants with a ‘scope, but it’s generally performed on really expensive gear.
You should be able to see changes in the chromatin by looking at the polytene chromosomes, you probably would need to look pretty carefully until you got familiar with the banding pattern, and if you have a powerful enough scope and the right stains, see the viroid’s.
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in plants, chromosome visualization is usually done using meristematic tissue, so that you have (the required) dividing cells/condensed chromosomes. eg see bio 101 level visualization in onion: https://www.bio.miami.edu/dana/151/151F21_chromosome_squash.pdf
polytene chromosomes are not universal, and are only found in certain organs in the organisims that have them. see: Polytene chromosome - Wikipedia or SciELO - Brazil - Plant polytene chromosomes Plant polytene chromosomes if you prefer.
if one is to argue that meristem culture can help you escape the virus, how can one also use meristematic tissue as the diagnostic?
absolutely.
I’d just argue that “the right stain” was a fluorescently labeled anti-sense probe to the viroid genomic RNA.
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The viroid might for occlusion bodies which you can stain for. The viroid might also change the cellular structure. These things are pretty easy to see.
I’m confused about the whole tissue culture being used to escape viruses, especially if you can use that tissue for diagnostics. Tissue culture is a very artificial environment for the plant and you can try and treat the tissues with stuff so maybe this combination works. I do not like tissue culture, because it causes changes in the chromatin. I prefer sexual reproduction to try and get rid of diseases, but you say its passed through the seed. This tells me it integrates into the genome. Polyploidy can result in incomplete replication, and these new incomplete genomes are recombined by the cell. I always wondered if there were viruses hidden in the heterochromatin which can be unleashed if a cell is unlucky enough or stressed out enough to have the latent and suppressed viral information which is then now open and available to replicate in… or if the viruses are digging into the heterochromatin for some ancient suppressed transposase. The heterochromatin is mostly repetitive sequences, and viruses, transposons and other wierd nucleotide beings.
I wouldn’t be surprised if polytene chromosomes are present in some tissues of the plant, my guess is they will be present if they are in cannabis, in the flowers around a seed. The developing seed looks exactly like the eggs in a fly ovary. They have nurse cells feeding an egg which is reprogramming it’s chromatin, and follicle cells surrounding the nurse cells and egg forming the egg shell. Both the Nurse and follicle cells are polytene in flies, and even if they aren’t as clear as in the fly, you will probably still see some polytenization.
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yep. if you’re good at it and have the gear, it is absolutely worth exploring.
not something I would expect to be able to hand-off the average grow team member. which is my long term goal assuming I can get this dialed in and useful.
Now you’re speaking my language…
an ancient viroid on its intergenomic voyage, rummaging amongst the intergenic wastelands for a coat protein to call its own.
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Chaibio and minipcr have some decent open source options, and they both will have a kit you can build an sop for I’m sure.
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