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

Introduction: The declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance. Methods: Using 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples. Results: The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes. Discussion: The employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method's robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method's independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

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

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.

CNBP controls transcription by unfolding DNA G-quadruplex structures.

Guanine-rich DNA strands can fold into non-canonical four-stranded secondary structures named G-quadruplexes (G4). Experimental evidences suggest that G4-DNA surrounding transcription start sites act as cis-regulatory elements by either stimulating or inhibiting gene transcription. Therefore, proteins able to target and regulate specific G4 formation/unfolding are crucial for G4-mediated transcriptional control. Here we present data revealing that CNBP acts in vitro as a G4-unfolding protein over a tetramolecular G4 formed by the TG4T oligonucleotide, as well as over the G4 folded in the promoters of several oncogenes. CNBP depletion in cellulo led to a reduction in the transcription of endogenous KRAS, suggesting a regulatory role of CNBP in relieving the transcriptional abrogation due to G4 formation. CNBP activity was also assayed over the evolutionary conserved G4 enhancing the transcription of NOGGIN (NOG) developmental gene. CNBP unfolded in vitro NOG G4 and experiments performed in cellulo and in vivo in developing zebrafish showed a repressive role of CNBP on the transcription of this gene by G4 unwinding. Our results shed light on the mechanisms underlying CNBP way of action, as well as reinforce the notion about the existence and function of G4s in whole living organisms.