Hop latent viroid detection: A review of cannabis testing methods

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Written By Tassa Saldi

A look at the strengths and weaknesses of different HLVd detection techniques and the role of third-party labs in securing cannabis harvests

Maintaining cannabis plant health is essential, and it starts by checking for hidden threats like hop latent viroid using sophisticated testing tools. This is especially important because some pathogens, like hop latent viroid, can be present without showing any initial symptoms. 

Detecting an active infection before it becomes widespread and highly contagious can significantly reduce the risk of spreading the disease and losing your crops. So, choosing the right test that gives accurate results is vital. However, understanding the different pathogen testing technologies and choosing a third-party lab can be challenging. In this blog, we'll discuss the most common cannabis testing methods for HLVd and address their strengths and weaknesses.


For more information on Hop Latent Viroid, read Everything you need to know about Hop Latent Viroid.

PCR (Polymerase Chain Reaction):

PCR is an amplification technology that finds specific genetic material (i.e. DNA/ genes) in a sample and amplifies (or increases the amount of) that identified material. For the ingredients of a PCR reaction to accomplish this task the reactions need to move through a certain pattern (or cycle) of specific temperatures. This precise temperature cycling requires expensive equipment (i.e. a PCR machine). 

There are two main types of PCR you might come across when searching for a pathogen testing lab: quantitative PCR (qPCR) and endpoint PCR. As the name suggests, quantitative PCR allows you to estimate the total amount of pathogen in each sample. This is because while amplification is occurring, the PCR equipment is measuring the amount of material as it is produced. 

A specialized type of qPCR, called TaqMan qPCR, can measure more than one type of material simultaneously. This pathogen testing method can identify an infection of interest (i.e. HLVd), and an internal control (like a plant gene) that should be detected as long as the sample isn’t degraded. By detecting an internal control, labs can confirm sample quality after having been exposed to varying temperatures during shipping. Both standard and TaqMan qPCR use specialized lasers and light filters inside the PCR machine to analyze the amount of amplified material.

qPCR vs TaqMan qPCR

In standard qPCR, amplification is measured indirectly by using a fluorescent dye that glows when bound to any amplified material, including artifacts, which can lead to false positive results. TaqMan qPCR detects results using multiple fluorescent colors that only glow if the amplified material matches the desired sequence (i.e. the amplified material is actually HLVd and not a non-specific signal). TaqMan qPCR assays can be designed to produce multiple signals from a signal target, dramatically improving specificity. Because of the target-specific detection method, TaqMan qPCR generally has increased accuracy over standard qPCR.    

In contrast, endpoint PCR looks for the presence or absence of the pathogen once the DNA amplification is finished. Endpoint PCR assays are not quantitative, meaning they cannot provide a pathogen load estimate. While it is possible to include an internal control in endpoint PCR, this is rarely done.

results for different PCR Methods

LAMP (Loop Mediated Isothermal Amplification) and RPA (Recombinant Polymerase Amplification)

LAMP and RPA tests are “isothermal” amplification technologies. These methods amplify DNA at a single temperature, as opposed to the various temperature cycles needed for PCR. Because these pathogen testing methods operate at one set temperature, they can be performed in a simple, inexpensive heat block instead of an expensive PCR machine. While both technologies can be quantitative with specialized equipment, most isothermal HLVd tests are “endpoint” tests, meaning the results are measured at the end of the amplification. The results from many isothermal endpoint tests can be judged by eye-enhancing ease of use, but these analysis methods can also involve key weaknesses.

While LAMP and RPA are isothermal (single temperature) tests, they do have some key differences. These tests use a different set of ingredients to amplify the HLVd material and are run at different set temperatures, 65°C vs. ~40°C, respectively. These distinctions make for key differences in test performance and reliability (reference table).

The simplicity of isothermal tests makes them ideal for at-home or on-site use. However, certain qualities make their use limited and less appropriate for a complex laboratory setting, including reduced sensitivity and accuracy compared to PCR.

What are the strengths and weaknesses of PCR and isothermal amplification technologies?

All three methods – PCR, LAMP, and RPA – work by finding HLVd in a sample and amplifying its DNA/RNA so it is easily detectable.  However, the differences in needed equipment, enzymes used, and detection method, mean the cost, sensitivity, and accuracy of these different technologies are dramatically different. 

  • PCR is the most sensitive, accurate, and quantitative of the three technologies. However, PCR is also more expensive, takes longer, and requires specialized scientific expertise.

  • LAMP is simple, and specific, and produces results that can be judged by the naked eye.  However, this method can be less sensitive, and less accurate (due to indirect detection methods) and the results can be arbitrary when determined visually. While an internal control is included when detecting the result with test strips, this method requires opening the amplified sample, risking contamination and subsequent false positives.  

  • RPA is the fastest technology of these three pathogen testing methods and can function well within a range of temperatures. However, recent studies using plant tissue show this technology may not be as sensitive as PCR or LAMP and is prone to user error and false positives due to non-specific amplification (Zou, 2020). 

A detailed summary of key strengths and weaknesses of common HLVd detection technologies is shown below:

What type of test is best for me?

The type of HLVd test you choose depends on your goals and expectations. If you are looking for a highly sensitive, accurate test to confirm the health of your prized mother stock population, PCR will provide the most reliable result. Among PCR types, TaqMan qPCR analysis provides a highly sensitive, specific and accurate result, while it also ensures the integrity of samples shipped through the mail. 

If you are looking for a quick screening test that can be done on-site, LAMP and RPA technologies can be very powerful. However, due to their decreased accuracy and flexibility, these methods are best for identifying visibly infected plants rather than low-level infections or non-infected plants. 


The best way to test can only be determined by you, though. Pick the test that meets your requirements and protect your plants. And don’t forget that sanitation and facility operations are also critical. Our S.T.O.P. Pathogens program guide offers valuable insights and resources to support your pathogen prevention efforts.

References and Further Reading

About TUMI Genomics

TUMI Genomics is advancing cannabis cultivation by bringing biological insights to help commercial growers thrive. Through comprehensive preventative solutions, superior pathogen diagnostics, and AI-driven insights, TUMI Genomics is helping cultivators start clean and stay clean. The unrivaled diagnostics, transparent validation and industry-leading pathogen detection innovations provided by TUMI Genomics are raising the standards of commercial cannabis cultivation. The team at TUMI Genomics is composed of deeply experienced Ph.D. molecular biologists, bioinformaticians, business strategists, and accomplished serial entrepreneurs.

Media Contact: Hailey Spencer, Marketing Director

hailey@tumigenomics.com


Research Inquiries: Tassa Saldi, Ph.D., Co-Founder and CSO

tassa@tumigenomics.com

 
Tassa Saldi
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