The measures required for ensuring clarity and fidelity of qPCR testing results

Molecular methods such as quantitative polymer chain reaction (qPCR) have gained significant momentum for use in a variety of diagnostic settings. Within the realm of cannabis, qPCR methods have taken root and include a growing number of testing protocols and commercial kits for genomic characterization of other applications. A rapidly expanding area involves the use of qPCR-based testing for the detection and quantification of microbial pathogens in cannabis.

Importance of microbial testing in cannabis

Microbial contamination can enter the cannabis production cycle from a number of routes. Excess moisture during growth, harvesting, and storage can lead to fungal pathogens including Aspergillus spp.  Improper attention to clean hygiene and processing guidelines can lead to Salmonella, E coli STEC, and other bacterial contamination. These pathogens can have serious effects on cannabis consumers, particularly medicinal cannabis users and those who suffer from immunocompromised conditions.

Advantages of molecular methods for microbial testing

Molecular methods such as qPCR can offer significant advantages over traditional measures such as culture identification for microbial testing in cannabis. Culture methods depend on the successful outgrowth of potential pathogens on appropriate media. This necessitates materials and time for incubation, in addition to successful access to potential pathogens in cannabis samples. Variation in these and other parameters can lead to results which lack a definitive outcome and/or must be repeated for verification. The qPCR technique is positioned to save time and resources, provide a high level of reliability for definitive detection, and has the added ability to be adapted to high-throughput workflows.

Advantages and considerations for qPCR microbial testing in cannabis

There are distinct advantages to the use of qPCR for microbial testing. These include:

  • Specificity. The availability of genetic sequences for pathogenic and non-pathogenic species allows for the design of primers that can differentiate between these two types. An example includes the iQ-Check STEC (Shiga toxin-producing E. coli) kits from Bio-Rad, which use primers directed towards multiple virulence genes. The design of high-confidence primers can lend to higher extension efficiencies, which equate to better sensitivity and specificity of the qPCR reaction.
  • Time. Rather than requiring extended incubation periods for cell culture and colony detection, qPCR methods require only 60-90 minutes from the time DNA is prepped to the point results are returned.
  • Safety. Handling and incubating cultures can present a safety hazard, particularly for human pathogens. Methods used for qPCR can involve cannabis processing, DNA extraction, and qPCR analysis, without directly handling and coming contact with virulent cells or spores.

There are many other advantages of qPCR including the ability to multiplex and achieve high-throughput workflows for testing lab operations.

There are additional considerations that must be addressed, and solutions that must be built-in, to enable successful qPCR testing for microbial contamination. These include:

  • Selectivity. An issue to consider with any infectious disease diagnostic is determination of live versus dead cells in testing samples. Culture media methods avoid this potential pitfall by only examining live cells for virulence markers after successful incubation. On the other hand, samples taken directly from the source without an enrichment step can contain DNA (or RNA in the case of virus testing) from both live and dead cells.  For this reason, qPCR methods should be paired with an initial pre-enrichment incubation of samples in culture media. This involves growth in defined media to differentiate live versus dead cells, with the added bonus of generating increased amounts of DNA for analysis.  Another advantage of pre-enrichment is the dilution of potential interfering substances of PCR inhibitors (salts, enzymes) in the source sample.
  • Enrichment. Since there is a requirement for a pre-enrichment step, the time to result for qPCR testing is pushed back from minutes to hours. Enrichment culturing can take 10-15 hrs or more depending on the nature of the potential pathogen. The time and the culture materials and methods required must be taken into consideration, although the ability to multiplex still supports high-throughput operations if resources allow.
  • Accuracy.  Although less important in qPCR, inclusion of standards with known concentrations (internal standards) can ensure absolute quantitative measurements of target DNA are obtained.
  • Analytical Sensitivity and Specificity. Both positive and negative DNA standards should be included in the qPCR workflow.  A negative control protects against false positives by helping to determine whether the sample is contaminated with DNA or another substance which may confound readings and produce a positive result. A positive control, on the other hand, permits successful PCR results from target DNA, provided the conditions are right. This protects against false negatives that may arise from faulty PCR primers, reagents, or reaction conditions.
  • Significance. Depending on the nature of the samples, the performance of the qPCR test, and the evolving requirements from testing authorities, statical significance of the results may be a consideration.  The appropriate use of controls, sample repetitions, and multiplexing should help satisfy significance concerns. A well constructed qPCR method and workflow protocol will assist in this respect.

Outlook

There are a growing number of qPCR kits on the market designed to detect pathogens in cannabis, foods, and other consumables. These kits harness to power of PCR technology to address the shortcomings of traditional culture media methods, which many testing authorities currently adhere to as the gold standard adapted from the food industry. Molecular methods may eventually replace these protocols, and attention to the considerations for successful testing by qPCR will help to accelerate this process. Much progress has already been made, such as the recent validation of the iQ-Check Aspergillus test by the AOAC, with more developments certain to come in this important area of the cannabis testing industry.

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