Real-time polymerase chain reaction reverse transcription (RT-qPCR) has been described as the gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection. While the process was not originally intended for diagnostic purposes, the necessity of the situation led to optimization and has resulted in several very reliable procedures to detect infection.
Study: Direct lysis RT-qPCR of SARS-CoV-2 in cell culture supernatant allows for fast and accurate quantification of virus, opening a vast array of applications. Image Credit: Corona Borealis Studio/Shutterstock
Researchers from the University of Edinburgh have been exploring ways to optimize this process further, allowing RT-qPCR to work more efficiently by eliminating one of the key steps.
A preprint version of the study is available on the
bioRxiv* server while the article undergoes peer review. The study
The researchers began using 1-Step-RTqPCR with fluorescent dye detection, which is currently the most cost-effective method. Primer pairs widely used by the Centers for Disease Control (CDC) and German Center for Infection Research (DZIF) to detect SARS-CoV-2 were also selected for optimization.
First, the manufacturer's standard protocol was tested, following basic procedures and recommendations. Every primer pair tested showed around a 5-fold increase in sensitivity, with similar increases in efficiency. Unfortunately, using the standard conditions with CDC N1, DZIF N, and RdRp consistently led to the formation of primer dimers. Increasing the concentrations of N2 reduced the formation of primer dimers but did not eliminate the formation of these dimers completely.
The DZIF N primers also formed larger products than expected, and sequencing revealed circularization or multiplication of a section of the product. An increase in annealing temperature did not reduce this. The DZIF N primer's efficiency did not rise above 78.24%, the DZIF RdRp above 89.61%. The CDC N3 primer performed the best at 93.14%.
Heat lysis was first tested to release viral RNA from the virus production medium (viral culture supernatant, or VPM). Unfortunately, the release of vRNA was limited, roughly corresponding to a 1000-fold loss in sensitivity.
Following this, the researchers attempted to use lysis buffers instead, basing all buffers on a 150mM NaCl, 10mM Tris-Hcl (7.5pH) solution supplemented with lysis detergents including Triton X-100, IGEPAL-620, or Tween-20. All of these were also supplemented with RNasin – an RNA inhibitor. Proteinase K treatment was also added to assess whether it could improve vRNA release.
All the lysis buffers showed similar sensitivities, but the Tween and Triton buffers impaired the qPCR reaction. When the detergent lysis buffers were tested with heat and proteinase K for vRNA, an increase by 1.22-1.37Cts was observed. This increase was higher for 10% Triton X-100, where proteinase K digest led to a loss of 5.86Ct. The researchers decided to continue with the IGEPAL-630 buffer for further optimization, as it has similar sensitivity levels to the other buffers at lower concentrations.
Trying different concentrations of the IGEPAL-630 buffer showed efficiencies decrease at higher concentrations, but the lowest concentration, 2.5%, showed complete inactivation in cellular supernatants. No significant decrease in sensitivity was observed for the RT-PCR reaction at this concentration, but efficiency was impacted.
The researchers performed further experiments to investigate why direct lysis RPM RT-qPCR sensitivities were lower than those in extracted vRNA. They prepared 10-fold dilution series of RNA templates. Only a slight loss of efficiency could be observed across the dilution curve, and Cts were increased by an average of 8.75 cycles across all concentrations in samples involving media. Incubation in IGEPAL-630 buffer or media also increases Ct, rising if samples were incubated in both.
Addition of RNasin rescued the decrease in sensitivity this caused. Further investigation revealed the inhibition of the PCR only occurred in samples incubated in 'spent' media, not fresh media. Betaine is known to improve PCR amplification by relaxing secondary structures, but when added, it decreased the efficiency significantly or decreased the signal at lower concentrations. Sensitivity was not impacted. Increased primer concentration also failed to improve sensitivity.
Finally, the scientists tested the effect of increased amounts of lyase on the reaction to find that this significantly decreased efficiency and sensitivity. In response, they tried diluting the lysate in the reaction with NF-H2O. Lowering the concentration of lysate from VPM significantly improved sensitivity and accuracy compared to vRNA at similar amounts showed no significant difference.
The authors highlight that the direct lysis protocol they have developed makes a one-step RT-qPCR from cell culture supernatant possible, achieving similar results to those seen using more traditional methods that require vRNA purification. This works well with SARS-CoV-2 and could help both testing and research laboratories worldwide produce results quicker, helping to end the pandemic that has lasted over two years.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information