Curtis Knox, global strategic marketing manager at Promega and Terri McDonnell, senior program manager at Promega, discuss the challenges and advantages of direct PCR amplification for infectious disease testing.
Testing for COVID-19 continues to be one of the most important tools in the global fight to slow and reduce the impact of the pandemic, particularly until vaccines are more widely available. Regular screening is likely to become the norm for many people around the world and demand for tests is expected to continue.
Most of the COVID-19 tests are molecular assays designed to detect viral ribonucleic acid (RNA) in patient samples. The general workflow for these COVID-19 assays can be broken down into a few steps:
- Collect and store the patient samples
- Ship samples to the testing lab
- Extract genetic material from samples (RNA)
- Amplify target
- Analyse and report the results.
A sample preparation method for direct polymerase chain reaction (PCR) amplification that simplifies and accelerates the time to results was needed. However, direct PCR amplification comes with both challenges and advantages.
Direct PCR amplification challenges
The first challenge is PCR inhibition. A robust and reliable PCR assay needs to have efficient DNA polymerisation and reverse transcription at detecting an RNA target. Primer and probe annealing should be specific to the intended target and the detection of the amplification signal should be above any background noise.
Sources of PCR inhibition include, but are not limited to:
- The sample itself: for example, saliva samples contain a high amount of protein which can be inhibitory, blood samples contain haemoglobin and faecal samples are known to contain a large variety of inhibitory substances.
- The sample collection device: for example, the swab or plastic used to collect the sample.
- The sample storage solution: these commonly contain high concentrations of guanidine, to inactivate pathogens. However, guanidine is also highly inhibitory to amplification reactions.
In a typical PCR workflow, the solution to minimising PCR inhibition is to purify the nucleic acid from the sample. If the level of nucleic acid in the sample is high enough, sample dilution can be used to minimise the number of inhibitors in a reaction. But this can impact the sensitivity of low copy targets. Master mix additives like bovine serum albumin (BSA), betane and trehalose are also commonly used as part of the strategy to overcome PCR inhibitors.
The second challenge to overcome in direct PCR amplification is assay sensitivity. Prior to the sample amplification and analysis by qPCR or RT-qPCR, sample extraction can serve as a concentrating step. In the example in Figure 1, a 200µl sample is concentrated to a 50µl eluent during the purification step. This results in a fourfold concentration increase of the sample before going into the amplification reaction. In direct amplification, there is no extraction step, so there is no concentration. Using the direct amplification protocol for Promega’s XpressAmp reagents, the sample is diluted 1:1 with lysis buffer, which results in a twofold dilution, or a 0.5X concentration of the original sample.
Figure 1: Comparison of amplification results of the same sample with a sample extraction workflow and a direct amplification workflow.
When comparing amplification results of the same sample, with the two example workflows, we would expect an eightfold difference in the amplification result. It is important to note that in qPCR, Cqs are on a log-scale, so a tenfold difference between samples would result in a 3.3 Cq difference, assuming 100% PCR efficiency.
Different sample extraction protocols vary in both sample input and elution volume, so it is important to set expectations when comparing a sample extraction workflow with a direct amplification workflow. Typically, most direct amplification workflows would be less sensitive compared to a sample extraction workflow, so the amplification assay may need additional optimisation when converting an assay from an extraction-based workflow to a direct amplification workflow.
Direct PCR amplification benefits
Despite these challenges, there are key advantages to direct amplification. In a typical testing workflow, the purification wash steps utilise many reagents and buffers, often supplied in configurations that are dependent upon plastic consumables and specific instrumentation. In a direct amplification workflow, you circumvent all those steps, reagents, and plastics, allowing you to avoid potential throughput limitations from different supply constraints.
Utilising a direct PCR workflow can also help you save time in the lab, though the amount of time saving will vary depending on your previous extraction method, and whether it was automated or manual. Direct amplification workflows are also high throughput automation friendly across multiple platforms.
As a leading supplier of sample extraction products and in direct response to the surging demand for COVID-19 testing, Promega has developed the XpressAmp direct amplification reagents.
The simple, direct amplification workflow involves three key steps:
- Collect and store sample – use common nasopharyngeal swabs in transport media.
- Lyse sample – mix sample and XpressAmp Lysis Buffer (1:1) and incubate for 10 minutes at room temperature.
- Amplify and analyse – add lysed sample to RT-qPCR containing XpressAmp Solution.
