Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples.

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

Facile method of curing toxicity in large viral genomes by high-throughput identification and removal of cryptic promoters.

Infectious plant virus clones are challenging to construct and manipulate due to the presence of cryptic promoter sequences that induce toxicity in bacteria. Common methods to overcome toxicity include intron insertion to interrupt toxic open reading frames and the use of Rhizobium or yeast species that do not recognize the same cryptic promoters. Unfortunately, intron insertion must be attempted on a trial and error basis within full-length clones and may change the infection characteristics of the virus. We have developed a facile method that can detect multiple cryptic bacterial promoters within large virus genomes. These promoters can then be silenced to obtain infectious clones that can be manipulated in E. coli. Our strategy relies on the generation of a viral library which is cloned upstream of either an eGFP open reading frame for low-throughput analysis or chloramphenicol for next generation sequencing. Pokeweed mosaic virus (PkMV), a 9.5 Kb ssRNA potyvirus, was used as a proof of concept. We found 16 putative promoter regions within 150-250 bp library fragments throughout the PkMV genome. 5'RACE allowed identification of the promoter sequence within each fragment, and subsequent silencing produced infectious clones. Our results indicate that cryptic promoters are ubiquitous within large viral genomes and that promoter screening is a desirable first step when constructing a viral clone. Our method can be applied to large plant and animal viruses as well as any DNA sequence for which low level of background transcriptional activity is required.

Prediction and Characterization of miRNA/Target Pairs in Non-Model Plants Using RNA-seq.

Plant microRNAs (miRNAs) are ∼20- to 24-nucleotide small RNAs that post-transcriptionally regulate gene expression of mRNA targets. Here, we present a workflow to characterize the miRNA transcriptome of a non-model plant, focusing on miRNAs and targets that are differentially expressed under one experimental treatment. We cover RNA-seq experimental design to create paired small RNA and mRNA libraries and perform quality control of raw data, de novo mRNA transcriptome assembly and annotation, miRNA prediction, differential expression, target identification, and functional enrichment analysis. Additionally, we include validation of differential expression and miRNA-induced target cleavage using qRT-PCR and modified RNA ligase-mediated 5' rapid amplification of cDNA ends, respectively. Our procedure relies on freely available software and web resources. It is intended for users that lack programming skills but can navigate a command-line interface. To enable an understanding of formatting requirements and anticipated results, we provide sample RNA-seq data and key input/output files for each stage. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Integration of the Pokeweed miRNA and mRNA Transcriptomes Reveals Targeting of Jasmonic Acid-Responsive Genes.

The American pokeweed plant, Phytolacca americana, displays broad-spectrum resistance to plant viruses and is a heavy metal hyperaccumulator. However, little is known about the regulation of biotic and abiotic stress responses in this non-model plant. To investigate the control of miRNAs in gene expression, we sequenced the small RNA transcriptome of pokeweed treated with jasmonic acid (JA), a hormone that mediates pathogen defense and stress tolerance. We predicted 145 miRNAs responsive to JA, most of which were unique to pokeweed. These miRNAs were low in abundance and condition-specific, with discrete expression change. Integration of paired mRNA-Seq expression data enabled us to identify correlated, novel JA-responsive targets that mediate hormone biosynthesis, signal transduction, and pathogen defense. The expression of approximately half the pairs was positively correlated, an uncommon finding that we functionally validated by mRNA cleavage. Importantly, we report that a pokeweed-specific miRNA targets the transcript of OPR3, novel evidence that a miRNA regulates a JA biosynthesis enzyme. This first large-scale small RNA study of a Phytolaccaceae family member shows that miRNA-mediated control is a significant component of the JA response, associated with widespread changes in expression of genes required for stress adaptation.

The Pokeweed Leaf mRNA Transcriptome and Its Regulation by Jasmonic Acid.

The American pokeweed plant, Phytolacca americana, is recognized for synthesizing pokeweed antiviral protein (PAP), a ribosome inactivating protein (RIP) that inhibits the replication of several plant and animal viruses. The plant is also a heavy metal accumulator with applications in soil remediation. However, little is known about pokeweed stress responses, as large-scale sequencing projects have not been performed for this species. Here, we sequenced the mRNA transcriptome of pokeweed in the presence and absence of jasmonic acid (JA), a hormone mediating plant defense. Trinity-based de novo assembly of mRNA from leaf tissue and BLASTx homology searches against public sequence databases resulted in the annotation of 59 096 transcripts. Differential expression analysis identified JA-responsive genes that may be involved in defense against pathogen infection and herbivory. We confirmed the existence of several PAP isoforms and cloned a potentially novel isoform of PAP. Expression analysis indicated that PAP isoforms are differentially responsive to JA, perhaps indicating specialized roles within the plant. Finally, we identified 52 305 natural antisense transcript pairs, four of which comprised PAP isoforms, suggesting a novel form of RIP gene regulation. This transcriptome-wide study of a Phytolaccaceae family member provides a source of new genes that may be involved in stress tolerance in this plant. The sequences generated in our study have been deposited in the SRA database under project # SRP069141.

A small RNA targets pokeweed antiviral protein transcript.

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.