Saliva Testing is a Robust Non-Invasive Method for SARS-CoV-2 RNA Detection.

PURPOSE: The precise diagnostic testing is of high importance in fighting the coronavirus pandemic. While nasopharyngeal (NP) swab testing is currently the gold standard, the SARS-CoV-2 virus could be also detected in some other body fluids. In this study, we aimed to compare the SARS-CoV-2 RNA detection results, obtained using saliva samples and NP swab samples, collected from infected patients and healthy volunteers. PATIENTS AND METHODS: A total of 111 individuals were enrolled in this study: 53 healthy volunteers, participating in routine testing and 58 COVID-19 patients. Diagnosis for both groups was confirmed using a set of diagnostic CE-IVD labeled RT-qPCR kits. Most of the saliva samples were collected within 48 hours after the NP swabs were taken. RNA was purified from saliva samples and analyzed using a laboratory-developed kit (Diagnolita). Detection results for both sample types were compared and analyzed in terms of result agreement, Ct variation, and quantity of internal control, as well as population analysis. RESULTS: We found a good concordance between the NP swab and saliva samples. The positive percent agreement was 98.28% (CI 90.76-99.96%) and negative percent agreement was 98.11% (CI 89.93-99.95%). Additionally, we observed a statistically significant (p<0.05) and moderately strong (R = 0.53) correlation between Ct values in saliva and NP swab samples. The saliva collection method is more robust since the Ct variation of internal control ribonuclease P mRNA detection is lower in saliva samples. CONCLUSION: Saliva sample testing is a robust and reliable non-invasive alternative to the NP swab method for SARS-CoV-2 RNA detection, as well as a promising tool for COVID-19 screening.

Compartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: a novel tool for the directed evolution of enzymes.

We have developed an in vitro evolution method for the selection for catalytic activity under the conditions of free intermolecular interaction between the enzyme and a substrate. The destabilized ternary enzyme-mRNA-ribosome complexes generated by a ribosome display of the mutant library are compartmentalized in vitro by forming a water-in-oil emulsion in such a way, that every droplet would on average contain no more than a single complex. After the complex dissociates within the droplet, the released enzyme molecule is free to interact with a substrate under the selection pressure on all its enzymatic properties (substrate binding, product formation, rate acceleration and turnover) simultaneously-an opportunity for the most efficient selection for catalytic activity. By using the M-MuLV reverse transcriptase as a model, we demonstrated the high efficiency of the method selecting for mutants synthesizing cDNA at increased temperature. A slightly modified compartmentalized ribosome display (CRD) could be used for the selection of other enzymes activities (e.g. DNA polymerase, RNA or DNA ligase terminal nucleotidyl transferase activity). Employment of microfluidics technique could broaden the scope of CRD technique furthermore providing an opportunity to select almost any enzyme at single molecule level under desired conditions.

Generation and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variants.

In vitro synthesis of cDNA is one of the most important techniques in present molecular biology. Faithful synthesis of long cDNA on highly structured RNA templates requires thermostable and processive reverse transcriptases. In a recent attempt to increase the thermostability of the wt Moloney Murine leukemia virus reverse transcriptase (M-MuLV RT), we have employed the compartmentalized ribosome display (CRD) evolution in vitro technique and identified a large set of previously unknown mutations that enabled cDNA synthesis at elevated temperatures. In this study, we have characterized a group of the M-MuLV RT variants (28 novel amino acid positions, 84 point mutants) carrying the individual mutations. The performance of point mutants (thermal inactivation rate, substrate-binding affinity and processivity) correlated remarkably well with the mutation selection frequency in the CRD experiment. By combining the best-performing mutations D200N, L603W, T330P, L139P and E607K, we have generated highly processive and thermostable multiply-mutated M-MuLV RT variants. The processivity of the best-performing multiple mutant increased to 1500 nt (65-fold improvement in comparison to the wt enzyme), and the maximum temperature of the full-length 7.5-kb cDNA synthesis was raised to 62°C (17° higher in comparison with the wt enzyme).