Extraction-free clinical detection of SARS-CoV-2 virus from saline gargle samples using Hamilton STARlet liquid handler.

As part of the COVID-19 pandemic, clinical laboratories have been faced with massive increases in testing, resulting in sample collection systems, reagent, and staff shortages. We utilized self-collected saline gargle samples to optimize high throughput SARS-CoV-2 multiplex polymerase chain reaction (PCR) testing in order to minimize cost and technologist time. This was achieved through elimination of nucleic acid extraction and automation of sample handling on a widely available robotic liquid handler, Hamilton STARlet.

Evaluation of observed and unobserved self-collection of saline gargle samples for the detection of SARS-CoV-2 in outpatients.

The diagnostic sensitivity of observed and unobserved self-collected saline gargle samples for the molecular detection of SARS-CoV-2 in adults and school-aged children was evaluated against a reference standard of health care worker collected nasopharyngeal flocked swab. A total of 46 participants had a positive nasopharyngeal swab sample; of these, 10 were in the observed phase and 36 were in the unobserved phase. Only one matching saline gargle sample tested negative and this was in the unobserved phase, giving an overall sensitivity of 98%.

Development and validation of a new triplex real-time quantitative reverse Transcriptase-PCR assay for the clinical detection of SARS-CoV-2.

To increase the repertoire of PCR based laboratory developed tests (LDTs) for the detection of SARS-CoV-2, we describe a new multiplex assay (SORP), targeting the SARS-CoV-2's, Spike and ORF8 genes. The widely used human RNaseP internal control was modified to specifically co-amplify the RNaseP mRNA. The SORP triplex assay was tested on a cohort (n = 372; POS = 144/NEG = 228) of nasopharyngeal flocked swab (NPFS) specimens, previously tested for the presence of SARS-CoV-2 using a PCR assay targeting E and RdRp genes.

Self-Collected Saline Gargle Samples as an Alternative to Health Care Worker-Collected Nasopharyngeal Swabs for COVID-19 Diagnosis in Outpatients.

We assessed the performance, stability, and user acceptability of swab-independent self-collected saliva and saline mouth rinse/gargle sample types for the molecular detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in adults and school-aged children. Outpatients who had recently been diagnosed with COVID-19 or were presenting with suspected COVID-19 were asked to have a nasopharyngeal (NP) swab collected and provide at least one self-collected sample type. Participants were also asked about sample acceptability using a five-point Likert scale.

Asymptomatic Pharyngeal Carriage of Kingella kingae Among Young Children in Vancouver, British Columbia, Canada.

Background: Kingella kingae has emerged as a significant cause of osteoarticular infections in young children. Pharyngeal colonization is considered a prerequisite for invasive K. kingae infection.

Interaction between 2,4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing Pseudomonas brassicacearum LBUM300 and Clavibacter michiganensis subsp. michiganensis in the Tomato Rhizosphere.

We have previously demonstrated that inoculation of tomato plants with 2,4-diacetylphloroglucinol (DAPG)- and hydrogen cyanide (HCN)-producing LBUM300 could significantly reduce bacterial canker symptoms caused by subsp. In this study, in order to better characterize the population dynamics of LBUM300 in the rhizosphere of tomato plants, we characterized the role played by DAPG and HCN production by LBUM300 on rhizosphere colonization of healthy and subsp. -infected tomato plants.

Complete Genome Sequence of Pseudomonas fluorescens LBUM636, a Strain with Biocontrol Capabilities against Late Blight of Potato.

Herein provided is the full-genome sequence of Pseudomonas fluorescens LBUM636. This strain is a plant growth-promoting rhizobacterium (PGPR) which produces phenazine-1-carboxylic acid, an antibiotic involved in the biocontrol of numerous plant pathogens, including late blight of potato caused by the plant pathogen Phytophthora infestans.

Complete Genome Sequence of Pseudomonas brassicacearum LBUM300, a Disease-Suppressive Bacterium with Antagonistic Activity toward Fungal, Oomycete, and Bacterial Plant Pathogens.

Pseudomonas brassicacearum LBUM300, a plant rhizosphere-inhabiting bacterium, produces 2,4-diacetylphloroglucinol and hydrogen cyanide and has shown antagonistic activity against the plant pathogens Verticillium dahliae, Phytophthora cactorum, and Clavibacter michiganensis subsp. michiganensis. Here, we report the complete genome sequence of P.

Validation of endogenous reference genes in Buglossoides arvensis for normalizing RT-qPCR-based gene expression data.

Selection of a stably expressed reference gene (RG) is an important step for generating reliable and reproducible quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) gene expression data. We, in this study, have sought to validate RGs for Buglossoides arvensis, a high nutraceutical value plant whose refined seed oil is entering the market under the commercial trade name Ahiflower™. This weed plant has received attention for its natural ability to significantly accumulate the poly-unsaturated fatty acid (PUFA) stearidonic acid (SDA, C18:4n-3) in its seeds, which is uncommon for most plant species.

Fidelity and representativeness of two isothermal multiple displacement amplification systems to preamplify limiting amounts of total RNA.

In this study we investigated the fidelity and representativeness of two novel multiple displacement amplification (MDA) protocols leading to whole transcriptome amplification (WTA). WTA is used to amplify a limiting amount of experimental RNA, allowing its use in downstream applications. Using Phi29 and Bst DNA polymerase-based MDA, henceforth referred to as WTA-Phi and WTA-Bst, respectively, we successfully amplified very low amounts of linearly concatenated cDNA originating from 10 to 100 ng of starting RNA.

Quantitative Real-time Polymerase Chain Reaction for Tracking Microbial Gene Expression in Complex Environmental Matrices.

Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.

Development of a versatile TaqMan™ real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA.

Background: In bacterial systems, the sequence congruence of genomic DNA (gDNA) and cDNA obtained following reverse transcription of RNA, makes gDNA an automatic target for qPCR primers. This could lead to aberrant gene expression quantification. This is why a rigorous treatment of bacterial RNA with DNase I is usually required to remove any traces of carryover gDNA.

A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement amplification.

In various experimental systems, limiting available amounts of RNA may prevent a researcher from performing large-scale analyses of gene transcripts. One way to circumvent this is to 'pre-amplify' the starting RNA/cDNA, so that sufficient amounts are available for any downstream analysis. In the present study, we report the development of a novel protocol for constructing amplified cDNA libraries using the Phi29 DNA polymerase based multiple displacement amplification (MDA) system.

Production of DAPG and HCN by Pseudomonas sp. LBUM300 contributes to the biological control of bacterial canker of tomato.

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment.

A novel method to perform genomic walks using a combination of single strand DNA circularization and rolling circle amplification.

Characterization of regions flanking a known sequence within a genome, known as genome walking, is a cornerstone technique in modern genetic analysis. In the present work we have developed a new PCR-dependent, directional genome walking protocol based on the unique circularization property of a novel DNA ligase, CircLigase. In the first step, PCR based primer extension is performed using a phosphorylated primer, designed to extend from the boundary of the known sequence, into the flanking region.

Verticillium dahliae alters Pseudomonas spp. populations and HCN gene expression in the rhizosphere of strawberry.

The production of hydrogen cyanide (HCN) by beneficial root-associated bacteria is an important mechanism for the biological control of plant pathogens. However, little is known about the biotic factors affecting HCN gene expression in the rhizosphere of plants. In this study, real-time reverse transcription PCR (qRT-PCR) assays were developed to investigate the effect of the plant pathogen Verticillium dahliae on hcnC (encoding for HCN biosynthesis) gene expression in Pseudomonas sp.

Relative and absolute quantitative real-time PCR-based quantifications of hcnC and phlD gene transcripts in natural soil spiked with Pseudomonas sp. strain LBUM300.

Transcriptional analysis of microbial gene expression using relative quantitative real-time PCR (qRT-PCR) has been hampered by various technical problems. One such problem is the unavailability of an exogenous standard robust enough for use in a complex matrix like soil. To circumvent this technical issue, we made use of a recently developed artificial RNA (myIC) as an exogenous "spike-in" control.

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato.

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S.