Virus Assay Sampling Best Practices

Best practices for collecting samples for use with the PathoSEEK® Virus Multiplex Assay

The PathoSEEK® Cannabis Virus Multiplex Detection Assay is a reverse-transcriptase quantitative PCR (RT-qPCR or QRT-PCR) assay that screens for the presence of hops latent viroid, lettuce chlorosis virus, and cannabis cryptic virus. If these three viruses are found in cannabis and hemp they can impact yield. All three targets are tested in a single PCR reaction. Like all PathoSEEK® assays, cannabis DNA is also targeted and utilizes the HEX optical channel.

This product must be used with our Leaf Punch Lysis Buffer and our RT-qPCR Master Kit. There are three individual controls sold separately, one for each of the three genomic targets.

  • HLV Positive Control
  • LCV Positive Control
  • CCV Positive Control

This product requires the following optical channels on your qPCR instrument.

  • Hops Latent Viroid is on the FAM channel
  • Lettuce Chlorosis Virus is on the CY5 channel
  • Cannabis Cryptic Virus is on the ROX (Texas Red) channel
  • Cannabis Internal Control is on the HEX channel

Leaf sampling recommendations:

  • Samples should be taken from the midrib of the leaf.  Older plant material is more likely to contain viroid RNA.
  • One to multiple punches can be transferred into one Leaf Punch Lysis tube.  We have lysed from 1 to 10 punches at once.  The amount of plant material necessary is dependent on the level of infection. 
  • Determining number of leaf punches necessary will depend on:

    • Level of viral infection

    • Where the leaf is sampled (broad part of leaf vs midrib)
    • Age/thickness of leaf
  • Too much leaf material can result in overloading the assay, and a dilution of the lysed material may be necessary.


Leaves | Biology I

How to avoid RNases in work area 

RNases are very stable and robust enzymes that degrade RNA. Autoclaving solutions and glassware are not always sufficient to actively remove these enzymes. The first step when preparing to work with RNA is to create an RNase-free environment. The following precautions are recommended as your best defense against these enzymes.

  • The RNA area should be located away from microbiological work stations.
  • Clean, disposable gloves should be worn at all times when handling reagents, samples, pipettes, disposable tubes, etc. It is recommended that gloves are changed frequently to avoid contamination.
  • There should be designated solutions, filtered tips, tubes, lab coats, pipettes, etc. for RNA only.
  • All RNA solutions should be prepared using molecular biology grade nuclease-free water.
  • Clean all surfaces with commercially available RNase decontamination solutions.
  • When working with purified RNA samples, ensure that they remain on ice during downstream applications.