Testing Cannabis for the Hop Latent Viroid
Follow us as we test for HLV at the Summit Genetics facility
The Hop Latent Viroid is a growing virus known to cause a myriad of symptoms in Cannabis, including stunting, chlorosis, leaf deformation, brittle stems, reduced flower mass and trichome number, and overall alteration of typical plant morphology. This is becoming a massive problem for grows around the world, and as with all viruses, there’s no eliminating it once a plant is infected.
Separating infected plants from healthy Cannabis plants is the only true way to stop the spread of HLV, a process that requires diagnostic testing to confirm the viroid is present. Though this new virus is only now coming to mainstream attention, the science pros at Agdia Inc. have already developed a system for testing HLV in Cannabis. The AmplifyRP XRT for HLVd is a kit made specifically for testing cannabis against the hop latent viroid. The system uses the AmpliFire Isothermal Fluorometer, GEB-filled mesh extraction bags, PD1 diluent-filled test tubes, and reaction pellets to prepare samples for rapid testing and detection.
Using the AmplifyRP XRT for HLVd, the growers at Summit Genetics are already in the process of testing all our plants for HLV, in order to identify and exclude affected plants from our facility. As part of our mission to educate the industry on this problematic virus, we’re eager to share this testing process with you. Today we’re going to walk you through the process of testing, but you can learn more about HLV on our blog if you’re not yet familiar.
Preparing the Cannabis Samples
The first step to testing a cannabis plant for the hop latent viroid is preparing your leaf samples for use in the Extract Pouches. They need to be made smaller (so as to fit the pouch), so you only want to keep the area of the leaves containing the highest concentration of hormones for maximum accuracy. Note that older plants contain more hormones, and therefore provide a more accurate reading. Using a clean pair of clippers, trim each sample down to the basal portion of the leaf; you want about a nickel-sized circle from the center of each sample.
Be sure to thoroughly clean your clippers, or use a new pair, between each new clipping. The moisture contained in the plant is what carries the hop latent viroid, and this moisture will transfer to other plants on clippers, contaminating further samples and rendering your data inaccurate.
Label and number each sample, and be sure to keep track of these throughout the testing process. Once your test tubes are placed, you’ll be able to record this information in the Amplifire testing machine. For now, set your samples aside, and retrieve your Sample Extract Pouches.
Prepare the Sample Extract Pouches
For eight samples, you will need to retrieve eight GEB-filled mesh extraction bags. These bags have a textured lining and are filled with a GEB extraction buffer we want blended with the plant’s hormones. One at a time, cut an opening in each bag, and place your first cannabis leaf sample inside. From here, you will need to emulsify the leaf inside the solution. Using a blunt object, pen, or marker, you can grind the leaves from outside the bag, using the mesh lining inside to macerate the sample. It takes some time before the leaves are adequately dissolved in the solution, and you should see a sort of green foam appearing inside. This is how you know the sample is ready. You may set each extraction pouch to the side until all pouches are complete.
Prepare the Test Tubes
Preparing the test tubes for HLV testing is a multi-step process. First, retrieve a PD1-filled tube for each of your samples (these are the colored, not clear, tubes), and label each to match its corresponding sample number. Check to make sure all liquid contained in the tubes is settled at the bottom, then open the cap on each tube.
Next, you’ll need your sample extract pouches containing the mixtures of leaf samples and GEB extraction buffer, and a pipette. Using the pipette, retrieve 5 µL of your first sample extract and place it in a PD1-filled tube. Repeat this process for your remaining samples, and close the lids on each tube. With the caps securely in place, shake the tubes to thoroughly mix the sample and PD1 diluent (you may do this one at a time, if you separated your tubes, or altogether if the tubes are connected). Your test tubes are now complete.
Prepare the Reaction Pellets
From here, retrieve one reaction pellet for each sample (these look like clear test tubes), which should still be stored in a cool, dark place. Reaction pellets are light sensitive and must be kept hidden away as long as possible. These come packaged in a white foil pouch, which is labeled with a barcode you will need to keep on hand. Open each pellet, as well as your test tubes, and one sample at a time, transfer exactly 25 µL of sample mixture (from the colored test tubes) into a fresh reaction pellet (clear).
Once all samples have been added to a reaction pellet, close all the pellets, and mix thoroughly. The best method is to use a centrifuge to mix and settle the liquid inside. However, without a centrifuge, you can simply flick and manually shake the pellets until the liquid inside is mixed and settled completely at the bottom. Your reaction pellets are now complete and ready for transfer to the testing machine.
Bring Out the AmpliFire
Power on the AmpliFire, and press “Execute Reaction.” From here, you’ll be able to follow prompts on-screen, which guide you through the process of entering product codes, naming reactions, and configuring individual sample IDs. Although it isn’t necessary to individually ID each sample, we recommend doing so. Not only does this help keep your samples organized, you’ll be able to easily track detection by strain on-screen during the testing process.
Once configured, the machine is closed, and testing begins. After incubating for five minutes, the samples must be removed, shaken thoroughly, and gently returned to the machine. Another twenty minutes of incubation, and your results are ready!
During the 25 minutes of testing, results display in real time on the Amplifire screen. Some samples will appear to register pretty quickly, but results will continue to change or update continuously. When a sample hits around 600fg, the Amplifire marks the sample with a red icon (+), indicating a positive HLV infection. Samples that test below 600fg are considered HLV-negative, with a green dash (-) indication. The third result is “invalid” (!), indicating an error in the testing process, ultimately meaning you will need to re-test the sample.
Use Your Results to Take Action
Once you have the results from your HLV testing, you’ll have two options moving forward. For samples that come back negative for the hop latent viroid, there’s nothing to do! Your plant is safe to be around other plants, no harm done. In the unfortunate case a sample comes back negative, there are some definite steps you need to take to prevent a viral spread.
Isolating the sample’s mom and all her clones is the only way to ensure HLV won’t reach your healthy plants. Ideally, you can locate, remove, and quarantine these plants to a separate room or space, where members of your team know the infection is present. Dedicated clippers and other tools for pruning and harvest should be used, and must always be disinfected before use on another plant, especially a healthy one. As you repeat the testing process on your remaining plants, continue this quarantine, slowly moving all infected plants to a single area.
Thankfully, HLV doesn’t completely kill or ruin the plants it infects, it just never goes away. You’ll see lower yields and potency on these infected plants, but they can still be utilized in different ways. So don’t fret! One low-yield harvest isn’t ideal, but if you can eliminate HLV altogether, and maintain a sterile space in the future, you’ll start seeing the proliferative harvests you expect.
Do you have any questions about the Hop Latent Viroid or the testing process? Drop us a line! And stay tuned for more posts, as we dive deeper into ways to make the most of HLV-infected plants and what we're learning from the testing process.