Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva

Abstract

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Ionic liquid-caged nucleic acids enable active folding-based molecular recognition with hydrolysis resistance

“…When the MnSOD MB and its target RNA were incubated together in the nuclease cocktail, both strands were fully digested within 30 min, eventually releasing the Cy3 fluorophore that induced a false-positive detection error (Figure 5A , solid black circle). Moreover, due to thermostable and high RNase activities ( Supplementary Figure S14 ) ( 67 ), both 50% non- and heat-treated saliva elicited predominance of RNA digestion, thereby causing a true-negative error in MB-based mRNA detection (Figure 5A , empty black circle and solid blue diamond). Similarly, both the aptameric target recognition and the ribozymatic product formation suffered from the multifaceted hydrolysis problems in the samples with various nucleases (Figure 5B , C, left).…”

Colorimetric Detection of SARS-CoV-2 Using Plasmonic Biosensors and Smartphones

“…Challenges are often found in processing and avoiding degradation in saliva, which has hampered diagnostic tools . Furthermore, effective detection of SARS-CoV-2 in saliva requires high sensitivity because of its enzymes that may destabilize nucleic acid and inhibit proteases. , For SARS-CoV-2, most infectious saliva and cough specimens have virus loads near 10 6 PFU mL –1 (PFU stands for plaque-forming units), indicating that 10–100 μL droplets could deposit 10 4 –10 5 PFU of infectious material. − The minimal contagious dose in humans ranges from 1 to 5 PFU …”