Exploring Mitochondrial Localization of SARS-CoV-2 RNA by Padlock Assay: A Pilot Study in Human Placenta.

The ongoing COVID-19 pandemic dictated new priorities in biomedicine research. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, is a single-stranded positive-sense RNA virus. In this pilot study, we optimized our padlock assay to visualize genomic and subgenomic regions using formalin-fixed paraffin-embedded placental samples obtained from a confirmed case of COVID-19. SARS-CoV-2 RNA was localized in trophoblastic cells. We also checked the presence of the virion by immunolocalization of its glycoprotein spike. In addition, we imaged mitochondria of placental villi keeping in mind that the mitochondrion has been suggested as a potential residence of the SARS-CoV-2 genome. We observed a substantial overlapping of SARS-CoV-2 RNA and mitochondria in trophoblastic cells. This intriguing linkage correlated with an aberrant mitochondrial network. Overall, to the best of our knowledge, this is the first study that provides evidence of colocalization of the SARS-CoV-2 genome and mitochondria in SARS-CoV-2 infected tissue. These findings also support the notion that SARS-CoV-2 infection can reprogram mitochondrial activity in the highly specialized maternal-fetal interface.

[Rapid diagnostics of novel coronavirus infection by loop-mediated isothermal amplification].

This review presents the basic principles of application of the loop-mediated isothermal amplification (LAMP) reaction for the rapid diagnosis of coronavirus infection caused by SARS-CoV-2. The basic technical details of the method, and the most popular approaches of specific and non-specific detection of amplification products are briefly described. We also discuss the first published works on the use of the method for the detection of the nucleic acid of the SARS-CoV-2 virus, including those being developed in the Russian Federation. For commercially available and published LAMP-based assays, the main analytical characteristics of the tests are listed, which are often comparable to those based on the method of reverse transcription polymerase chain reaction (RT-PCR), and in some cases are even superior. The advantages and limitations of this promising methodology in comparison to other methods of molecular diagnostics, primarily RT-PCR, are discussed, as well as the prospects for the development of technology for the detection of other infectious agents.

Thermodynamic evaluation of the impact of DNA mismatches in PCR-type SARS-CoV-2 primers and probes.

BACKGROUND: DNA mismatches can affect the efficiency of PCR techniques if the intended target has mismatches in primer or probe regions. The accepted rule is that mismatches are detrimental as they reduce the hybridization temperatures, yet a more quantitative assessment is rarely performed. METHODS: We calculate the hybridization temperatures of primer/probe sets after aligning to SARS-CoV-2, SARS-CoV-1 and non-SARS genomes, considering all possible combinations of single, double and triple consecutive mismatches. We consider the mismatched hybridization temperature within a range of 5 ∘C to the fully matched reference temperature. RESULTS: We obtained the alignments of 19 PCR primers sets that were recently reported for the detection of SARS-CoV-2 and to 21665 SARS-CoV-2 genomes as well as 323 genomes of other viruses of the coronavirus family of which 10 are SARS-CoV-1. We find that many incompletely aligned primers become fully aligned to most of the SARS-CoV-2 when mismatches are considered. However, we also found that many cross-align to SARS-CoV-1 and non-SARS genomes. CONCLUSIONS: Some primer/probe sets only align substantially to most SARS-CoV-2 genomes if mismatches are taken into account. Unfortunately, by the same mechanism, almost 75% of these sets also align to some SARS-CoV-1 and non-SARS viruses. It is therefore recommended to consider mismatch hybridization for the design of primers whenever possible, especially to avoid undesired cross-reactivity.

High-coverage SARS-CoV-2 genome sequences acquired by target capture sequencing.

In this study, we designed a set of SARS-CoV-2 enrichment probes to increase the capacity for sequence-based virus detection and obtain the comprehensive genome sequence at the same time. This universal SARS-CoV-2 enrichment probe set contains 502 120 nt single-stranded DNA biotin-labeled probes designed based on all available SARS-CoV-2 viral sequences and it can be used to enrich for SARS-CoV-2 sequences without prior knowledge of type or subtype. Following the CDC health and safety guidelines, marked enrichment was demonstrated in a virus strain sample from cell culture, three nasopharyngeal swab samples (cycle threshold [Ct ] values: 32.36, 36.72, and 38.44) from patients diagnosed with COVID-19 (positive control) and four throat swab samples from patients without COVID-19 (negative controls), respectively. Moreover, based on these high-quality sequences, we discuss the heterozygosity and viral expression during coronavirus replication and its phylogenetic relationship with other selected high-quality samples from the Genome Variation Map. Therefore, this universal SARS-CoV-2 enrichment probe system can capture and enrich SARS-CoV-2 viral sequences selectively and effectively in different samples, especially clinical swab samples with a relatively low concentration of viral particles.

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.