One-step nested RT-PCR for COVID-19 detection: A flexible, locally developed test for SARS-CoV2 nucleic acid detection

Introduction: Due to the coronavirus pandemic, identifying the infected individuals has become key to limiting its spread. Virus nucleic acid real-time RT-PCR testing has become the current standard diagnostic method but high demand could lead to shortages. Therefore, we propose a detection strategy using a one-step nested RT-PCR. Methodology: The nucleotide region in the ORF1ab gene that has the greatest differences between the human coronavirus and the bat coronavirus was selected. Primers were designed after that sequence. All diagnostic primers are species-specific since the 3´ end of the sequence differs from that of other species. A primer set also creates a synthetic positive control. Amplified products were seen in a 2.5% agarose gel, as well as in an SYBR Green-Based Real-Time RT-PCR. Results: Amplification was achieved for the positive control and specific regions in both techniques. Conclusions: This new technique is flexible and easy to implement. It does not require a real-time thermocycler and can be interpreted in agarose gels, as well as adapted to quantify the viral genome. It has the advantage that if the coronavirus mutates in one of the key amplification nucleotides, at least one pair can still amplify, thanks to the four diagnostic primers.

Breve aproximación a la naturaleza genómica de Moniliophthora roreri CPMRT01 aislado de cacao en Tabasco, México//Brief approach to the genomic nature of Moniliophthora roreri CPMRT01 isolated from cocoa in Tabasco, Mexico

Moniliophthora roreri es un hongo de suma importancia agroeconómica principalmente en el continente americano, ya que es el agente causal de la moniliasis de al menos cuatro especies de cacao. El genoma de este hongo consta de aproximadamente 52,3 Mpb, cuyos genes de interés se reagrupan dependiendo de su naturaleza (e.g. hemibiotróficos, biotróficos y fitopatogénicos). Por otro lado, Moniliophthora es capaz de metabolizar proteínas involucradas en procesos de infección, regulación metabólica y mecanismos de defensa. El objetivo principal de este trabajo fue caracterizar un fragmento del genoma de una cepa de M. roreri, previamente aislada de cacao en el Estado de Tabasco, México. Para ello, se implementó una estrategia experimental que involucró la generación de bibliotecas genómicas así como una secuenciación de regiones nucleotídicas consenso, predicción heurística-funcional e identificación de hipotéticos dominios conservados, respectivamente. El estudio permitió explorar alrededor del 16 % del genoma de la cepa. Entre los resultados y conclusiones obtenidas más interesantes, se generaron reconstrucciones filogenéticas de máxima identidad respecto a un genoma de referencia y, cuyos transcritos se encontrarían involucrados en procesos de fitopatogenicidad y metabolismo secundario, identificándose aparentes dominios proteicos y sitios catalíticos activos en algunos de los cóntigos estudiados.ABSTRACTMoniliophthora roreri is a fungus of extreme agroeconomic importance mainly in the American continent, since it is the causal agent of moniliasis of at least four cocoa species. The genome of this fungus consists of approximately 52,3 Mbp, whose genes of interest may be regrouped depending on their nature (e.g. hemibiotrophics, biotrophics and phytopathogenics). On the other hand, Moniliophthora is liable to metabolize proteins involved in infection processes, metabolic regulation and defense mechanisms. The aim of this work was to partially characterize a fragment of the genome of a M. roreri strain previously isolated from cocoa in the State of Tabasco, Mexico. To reach this goal, an experimental strategy was implemented that involved the generation of genomic libraries as well as a consensus nucleotide region sequencing, functional-heuristic prediction and identification of hypothetical conserved domains, respectively. The study allowed to explore about 16 % of the strain´s genome. Among the most interesting results and conclusions obtained, phylogenetic reconstructions of maximum identity respect to the reference genome were reached, whose transcripts would be involved in phytopathogenic processes and secondary metabolism, identifying apparent protein domains and active catalytic sites in some of the contigs under study.

Fluid flow-induced left-right asymmetric decay of Dand5 mRNA in the mouse embryo requires Bicc1-Ccr4 RNA degradation complex

SUMMARYMolecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the 3’ untranslated region (3’-UTR) of Dand5 mRNA is necessary and sufficient for the left-sided decay and is responsive to the flow direction, loss of the cation channel Pkd2, and Ca2+. The 200-nucleotide proximal-most portion of the 3’-UTR, which is conserved among mammals, is essential for the asymmetric mRNA decay and binds Bicc1, an RNA binding protein specifically expressed at the node. Bicc1 preferentially recognizes GAC and GACR sequences in RNA, and these motifs are enriched in the 200-nucleotide region of the Dand5 3’-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. Our results thus suggest that leftward fluid flow induces binding of Bicc1 to the 3’-UTR of Dand5 mRNA in crown cells on the left side of the node, and that consequent recruitment of Ccr4-Not mediates mRNA degradation.

RNA Aptamers for a tRNA-Binding Protein from Aeropyrum pernix with Homologous Counterparts Distributed Throughout Evolution

In the present in vitro selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon Aeropyrum pernix. Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for A. pernix Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3′-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3′ terminus of tRNA in the interaction with Trbp-like structures.

Localization of genetic determinants for pathogenicity of Maize dwarf mosaic virus and Bermudagrass southern mosaic virus

Maize dwarf mosaic virus (MDMV) and Bermuda grass southern mosaic virus (BgSMV) are economically important potyviruses of cereals. BgSMV is very similar in genomic sequence to MDMV, but cannot infect Johnsongrass and is not transmitted by Rhopalosiphum maidis. Comparison of their genomes showed an additional stretch of 90 nucleotides in BgSMV coat protein but not in MDMV. Since the 90-nucleotide region is located in the N-terminal of BgSMV coat protein, it seems to have a role in biological properties such as vector transmission and pathogenicity. Recombinant virus constructs were made with and without the 90 nucleotides using SOEing PCR (MDMV (+90) and BgSMV (−90). Johnsongrass plants inoculated with the wild-type MDMV and recombinant BgSMV (−90) showed mosaic symptoms after 16 and 23 days, respectively, whereas plants inoculated with the wild-type BgSMV and recombinant MDMV (+90) didn’t show any symptoms until three months after inoculation. The qRT-PCR results detected significantly higher levels of BgSMV (−90) and MDMV compared to BgSMV and MDMV (+90), respectively. Also, R. maidis was able to transfer only the wild type MDMV and BgSMV (−90) from infected to healthy plants. These results confirmed that the insertion of the 90-nt region into the coat protein of MDMV affects the pathogenicity of the virus.

Grapevine red blotch-associated virus is Present in Free-Living Vitis spp. Proximal to Cultivated Grapevines

Red blotch is an emerging disease of grapevine associated with grapevine red blotch-associated virus (GRBaV). The virus spreads with infected planting stocks but no vector of epidemiological significance has been conclusively identified. A vineyard block of red-blotch-affected Vitis vinifera ‘Cabernet franc’ clone 214 was observed in California, with a clustering of infected, symptomatic vines focused along one edge of the field proximal to a riparian habitat with free-living Vitis spp. No genetic heterogeneity was observed in a 587-nucleotide region of the GRBaV genome in a population of 44 Cabernet franc clone 214 isolates. By contrast, genetic differences were observed in isolates from other cultivars and clones growing in adjacent blocks. GRBaV was confirmed infecting four free-living vines, two of which were shown to be V. californica × V. vinifera hybrids. The genomes of three free-living GRBaV vine isolates and seven from V. vinifera cultivars were compared; free-living vine isolates were shown to be more similar to each other and a ‘Merlot’ isolate than to the other cultivated vine isolates. The finding that GRBaV is present in free-living Vitis spp. indicates the virus can be spread by natural (nonhuman-mediated) means, and we hypothesize that in-field spread of GRBaV is occurring.

High infection rate of bank voles (Myodes glareolus) with Puumala virus is associated with a winter outbreak of haemorrhagic fever with renal syndrome in Croatia

SUMMARYAn outbreak of haemorrhagic fever with renal syndrome (HFRS) started on Medvednica mountain near Zagreb in January 2012. In order to detect the aetiological agent of the disease in small rodents and to make the link with the human outbreak, rodents were trapped at four different altitudes. Using nested RT–PCR, Puumala virus (PUUV) RNA was detected in 41/53 (77·4%) bank voles (Myodes glareolus) and Dobrava virus (DOBV) RNA was found in 6/61 (9·8%) yellow-necked mice (Apodemus flavicollis). Sequence analysis of a 341-nucleotide region of the PUUV S segment, obtained from all infected bank voles and five HFRS patients, showed 98·8–100% sequence similarity, indicating that the patients were probably exposed to PUUV on Medvednica mountain. A very large bank-vole population combined with an extremely high infection rate of PUUV was responsible for this unusual winter outbreak of HFRS in Croatia.

A stem–loop structure in the 5′ untranslated region of bean pod mottle virus RNA2 is specifically required for RNA2 accumulation

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family Secoviridae. Its RNA1 encodes all proteins needed for genome replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought to identify RNA elements in RNA2 required for its replication. The exchange of 5′ untranslated regions (UTRs) between genome segments revealed an RNA2-specific element in its 5′ UTR. Further mapping localized a 66 nucleotide region that was predicted to fold into an RNA stem–loop structure, designated SLC. Additional functional analysis indicated the importance of the middle portion of the stem and an adjacent two-base mismatch. This is the first report of a cis-acting RNA element in RNA2 of a bipartite secovirus.

Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs

ABSTRACT RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome.

Spatially Restricted Translation of the xCR1 mRNA in Xenopus Embryos

ABSTRACT The xCR1 protein is a maternal determinant and cofactor for nodal signaling in vertebrate embryos. The xCR1 protein accumulates specifically in the animal cells of Xenopus embryos, but maternal xCR1 mRNA is distributed equally throughout all embryonic cells. Here, we show that vegetal cell-specific translational repression of xCR1 mRNA contributes to this spatially restricted accumulation of the xCR1 protein in Xenopus embryos. xCR1 mRNA was associated with polyribosomes in animal cells but not vegetal cells. A 351-nucleotide region of xCR1 mRNA's 3′ untranslated region was sufficient to confer a spatially restricted pattern of translation to a luciferase reporter mRNA by repressing translation in vegetal cells. Repression depended upon the mRNA's 5′ cap but not its 3′ poly(A) tail. Furthermore, the region of xCR1 mRNA sufficient to confer vegetal cell-specific repression contained both Pumilio binding elements (PBEs) and binding sites for the CUG-BP1 protein. The PBEs and the CUG-BP1 sites were necessary but not sufficient for translation repression. Our studies of xCR1 mRNA document the first example of spatially regulated translation in controlling the asymmetric distribution of a maternal determinant in vertebrates.