Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR

Abstract

Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

“…While our previous work demonstrated the ability to detect a SARS-CoV-2 positive sample within a 34-sample pool ( 26 ), identifying specific Va-SARS-CoV-2 sequences among SARS-CoV-2 positive samples proves to be more complex. This complexity arises because, in the former case, we were seeking a specific sequence within a vast array of DNA without similar sequences.…”

SARS‐CoV‐2 in digital era: Diagnostic techniques and importance of nucleic acid quantification with digital PCRs

“…95 Although studies relating to multiplexed variant screening are scarce, 120,121 utilization of such assays without a drop in efficiency can be a major utility in the diagnosis of COVID-19 as well as for pooled sample analysis in high-risk countries. 119 Further, developing and validating internationally approved simplex and multiplex dPCR assays can be utilitarian in detecting novel variants with increased pathogenicity and virulence.…”

“…have also evaluated the developed ddPCR assays on pooled sample analysis. A recent study from Heckel et al 119 . has also provided elaborate and practical considerations for developing a ddPCR assay for SARS‐CoV‐2 detection from pooled samples.…”

“…With the current increase in different variants of SARS‐CoV2, using dPCRs has become a primary requisite for the development of reference materials, validation of new kits and assays, pooled sample analysis and development of multiplex RT‐dPCR assays, which are of high importance for simultaneous detection of different variants. Although studies relating to multiplexed variant screening are scarce, 120,121 utilization of such assays without a drop in efficiency can be a major utility in the diagnosis of COVID‐19 as well as for pooled sample analysis in high‐risk countries 119 . Further, developing and validating internationally approved simplex and multiplex dPCR assays can be utilitarian in detecting novel variants with increased pathogenicity and virulence.…”

“…Studies like Kim et al108 and Milosevic et al118 have also evaluated the developed ddPCR assays on pooled sample analysis. A recent study from Heckel et al119 has also provided elaborate and practical considerations for developing a ddPCR assay for SARS-CoV-2 detection from pooled samples. The study has utilised the mentioned considerations in their developed ddPCR assay, where the assay could detect a single positive sample with a very low viral load in a pool of 34 samples.…”