Quality vs. Quantitative Testing For Hop Latent Viroids

For cannabis plants infected with the Hop Latent Viroid (HLVD), there are no effective cures at this time. It is possible to save contaminated plants via meristem tissue culture, but the procedure can take three to nine months and doesn't always produce plants that are viroid-free. Tissue culture remediation frequently has a cultivar-specific success rate. The time and effort required to preserve priceless heirloom cultivars will undoubtedly be expended by growers, but many others will discard contaminated plants and begin a new pheno quest to replace them.

The greatest method to defend your organization is undoubtedly through prevention. Cultivators can employ a variety of HLVD testing kits to confirm a suspected infection or spot asymptomatic plants. Regularly screening mother plants would help to guarantee that only clean plants enter production, especially before taking a round of cuttings. Additionally, cultivators must introduce new DNA from another facility with the utmost care. Before sharing a space with existing plants, new clones should be isolated and tested for HLVD and other infections.

A Case For Testing HLVd Using qPCR

With our youPCR Gender and THC/CBD testing, Verne Bioanalytics was the first firm to introduce Eiken Chemical Co, Ltd.'s revolutionary Loop-Mediated Isothermal Amplification (LAMP)-based testing to the cannabis industry. We gained a lot of knowledge from this venture and have some reasons to offer for why we have not yet used this patented technique for initial Hop Latent Viroid (HLVd) testing. First, binary inquiries like sex testing or the presence or absence of a specific allele are best suited for the LAMP technology. For this, LAMP is quick, affordable, and suitable for usage at the point of growth. LAMP is not the ideal technology, though, to address emerging biological issues like HLVd testing.

Here Are Some Things To Think About:

Internal Measures

Typically, LAMP is a single-plex response. Internal controls are essentially impossible because you can only amplify one target. Without internal controls, it is impossible to determine whether the plant was properly sampled. Negative results may be due to improper preparation, or they may actually come from a clean plant. Are you prepared to stake everything on that coin toss?

This issue doesn't exist with qPCR. In order to confirm that the DNA and RNA extraction was effective, we can utilize an internal cannabis control since we can employ different wavelengths to detect different targets. You run the danger of receiving many false negatives if internal controls aren't in place. With RNA preps that are used for virus and viroid identification, this is even more of an issue.

One study by Dadouh et al. shows that there are 1,000–10,000 fold variations when RNA viruses are detected in patient noses. This study cautions against using a single viral Ct value in qPCR to estimate viral load since two Ct values are required to accurately quantify a viral load (host and pathogen). You can easily exceed your limit of detection (LOD) and remain unaware of it if the sampling can vary by 1,000–10,000 fold. For +/- tests that don't even offer a single Ct score, this is considerably worse.

Risk Of Contamination

Opening a tube containing amplified DNA or RNA runs the risk of bringing a PCR product into your facility and causing false positive results. Because LAMP amplification generates 10 times as much DNA as qPCR, it is 10 times more prone to contamination. This means that in your facility, a LAMP amplification tube should never be opened. Because of this, we never create qPCR or LAMP assays that call for the user to open the tube following amplification.

The Capacity To Calculate

The viroid Ct score and the host Ct score are the two pieces of information that you require in order to create a legitimate viroid load, in accordance with MIQE guidelines. Without both, it is impossible to determine if a plant is mildly sick, a false positive, or both. But why measure viral load? Why not simply demolish every plant that tests positive? Maybe, but if you don't know how much viroid is an issue, it's challenging to remove an heirloom mother plant based on a single LAMP test. Do you have ten copies or ten million copies? The 10 copies can include LAMP contamination or be false positives. More of an issue is the 10 Million copies. Given how little is known about HLVD Testing, we think the best place to start is with quantitative tools so growers can determine whether their practices are reducing viroid load or making things worse. Since no viroid load is delivered by the test, binary, LAMP-based tests are unable to accomplish this. To mimic the results of a fully quantitative test, one has to resort to hyper sampling 10–50 locations on the plant to observe if the percent positive increases noticeably.

Scalability

Several hundred tests, in our opinion, are insufficient to fully assess the level of HLVD Testing contamination in a facility. The test should be run in duplicate to triplicate on numerous plants at various times. Before you know it, you'll be in 96 well plates, therefore you need a platform that can handle the data torrent. Platforms for qPCR are excellent for this. For SARS CoV2 testing, many LAMP-based methods were created fast, but few were used in high volume manufacturing because the CT score was too important to ignore for determining who was contagious and who was merely carrying dead virus bits.