SARS-CoV-2 RT-LAMP in saliva: enhancing the results via a combination of cooling and specimen dilution procedure.

Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a potential and relatively simple rapid diagnostics method for COVID-19 detection. This study aims to evaluate and optimize the RT-LAMP performance on saliva specimens based on a commercially available kit.Modifications on an established protocol (Protocol A) were used, including Proteinase K supplementation (Protocol B); pre-treatment using nuclease-free water and proteinase K (Protocol C); Saliva cooling (Protocol D); saliva dilution after pre-treatment (Protocol E); lastly a combination of saliva cooling and dilution (Protocol F). Protocol performances were evaluated by comparing success rates (SR), diagnostic accuracy (DA), sensitivity, specificity, and predictive values. Additionally, a correlation between the Ct value by RT-qPCR and RT-LAMP performance was analyzed.. A total of 106 specimens were used in this study. Protocols B and C showed 100% unreadable results, therefore were paused. Protocol F showed the highest SR (87.65%) compared to other protocols, with a slight compromise to DA (81.69%), sensitivity (57.14%), specificity (97.67%), PPV (94.12%), and NPV (77.78%). In the sub-analysis of the low Ct value group (Ct < 30), Protocol F demonstrated a higher success rate (86.57%) compared to protocol A (64.18%); increased 3.08% sensitivity and 2.42% NPV; comparable DA; minor reduction in specificity (A = 100%; F = 97.67%) and PPV (A = 100%; F = 92.31%). A combination of saliva cooling-dilution substantially increased the tested kit's success rate, despite a slight decrease in specificity and PPV. Findings confirmed the saliva cooling-dilution procedure was beneficial to the test's SR, sensitivity, and NPV in the low Ct value group. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00870-1.

RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

Accelerating the Laboratory Testing Capacity through Saliva Pooling Prior to Direct RT-qPCR for SARS-CoV-2 Detection.

The testing capacity of the laboratory is paramount for better control of the pandemic caused by SARS-CoV-2. The pooling method is promising to increase testing capacity, and the use of direct NAAT-based detection of SARS-CoV-2 on a non-invasive specimen such as saliva will ultimately accelerate the testing capacity. This study aims to validate the pooling-of-four method to quadruple the testing capacity using RNA-extraction-free saliva specimens. In addition, we intend to investigate the preferable stage of pooling, including pre- or post-heating. The compatibility of this approach was also tested on five commercial kits. Saliva specimens stored at -80 °C for several months were proven viable and were used for various tests in this study. Our findings revealed that pooling-of-four specimens had an overall agreement rate of 98.18% with their individual testing. Moreover, we proved that the pooling procedure could be conducted either pre- or post-heating, with no discordance and no significant difference in Ct values generated. When compared to other commercial detection kits, it demonstrated an overall agreement greater than 85%, which exhibits broad compatibility and ensures easy adaptability in clinical settings. This method has been proven reliable and increases the testing capacity up to fourfold.

Wide Application of Minimally Processed Saliva on Multiple RT-qPCR Kits for SARS-CoV-2 Detection in Indonesia.

Saliva as a sample matrix has been an attractive alternative for the detection of SARS-CoV-2. However, due to potential variability in collection and processing steps, evaluating a proposed workflow amongst the local population is recommended. Here, we aim to validate the collection and treatment of human saliva as a direct specimen for RT-qPCR-based detection of SARS-CoV-2 in Indonesia. We demonstrated that SARS-CoV-2 target genes were detected in saliva specimens and remained stable for five days either refrigerated or stored at room temperature. The method of processing saliva specimens described in this report bypasses the need for an RNA-extraction process, thereby reducing the cost, time, and manpower required for processing samples. The developed method was tested across nine commercial kits, including the benchmark, to demonstrate its wide applicability on multiple existing workflows. Our developed method achieved an 86% overall agreement rate compared to paired nasopharyngeal and oropharyngeal swab specimens (NPOP). With the assistance of a saliva sampling device, the collection was found to be more convenient for individuals and improved the overall agreement rate to 97%.