Ae1 Ae3 Antibody Antibodies Assay Kits Bafilomycin A1 Bile Acid Assay Biology Cells Canine Albumin cDNA Choline Acetyltransferase Antibody Clia Kits Culture Cells Deoxycholic Acid Sodium Salt Devices DNA DNA Templates DNA Testing Enzymes Equipments Exosomes F Actin Antibody Fto Antibody Gels Glycodeoxycholic Acid Gsk3 Alpha Hama Antibodies Hdac Activity Assay Isotypes Laminin Alpha 5 Medium & Serums Mip 1B Mmp Activity Assay Mothers Against Decapentaplegic Mouse Albumin Elisa Panel Particles Peptides Phospho 4Ebp1 Primary Antibodies Reagents Recombinant Proteins Ria Kits Secreted Alkaline Phosphatase Tgf Alpha Elisa Trypsin Activity Assay Tubastatin A Valproic Acid Sodium Salt Vi Alpha
Clinical performance and accuracy of a qPCR-based SARS-CoV-2 mass-screening workflow for healthcare-worker surveillance using pooled self-sampled gargling solutions: a cross-sectional study
Introduction: The large number of asymptomatic SARS-CoV-2 infections necessitates general screening of employees. We evaluate the performance of a SARS-CoV-2 screening program in asymptomatic healthcare-workers (HCW), utilizing self-sampled gargling-solution and sample pooling for RT-qPCR.
Methods: We conducted a cross-sectional retrospective study to collect real-life data on the performance of a screening-workflow based on automated-pooling and high-throughput qPCR testing over a 3-month-period at the University Hospital Hamburg.
Results: Matrix validation reveals that lower limit of detection for SARS-CoV-2 RNA in gargling-solution was 180 copies/mL (5-sample-pool). A total of 55,122 self-collected gargle samples (=7,513 HCWs) was analyzed. The median time to result was 8.5 hours (IQR 7.2-10.8). Of 11,192 pools analyzed, 11,041 (98.7%) were negative, 69 (0.6%) were positive and 82 (0.7%) were invalid. Individual testing of pool participants revealed 57 SARS-CoV-2 previously unrecognized infections. All 57 HCWs were either pre-symptomatic or asymptomatic (prevalence 0.76%,CI95%0.58-0.98%). Accuracy based on HCWs with gargle-solution and NP-swab available within 3-day-interval (N=521) was 99.5% (CI95%98.3-99.9%), sensitivity 88.9% (CI95%65.3-98.6%) while specificity 99.8% (CI95%98.9-99.9).
Conclusion: This workflow was highly effective in identifying SARS-CoV-2 positive HCWs, thereby lowering the potential of inter-HCW and HCW-patient transmissions. Automated-sample-pooling helped to conserve qPCR reagents and represents a promising alternative strategy to antigen testing in mass-screening programs.
Keywords: SARS Coronavirus 2 RT-PCR Testing; mass Screening; pooling gargle solution.
RT-qPCR for Detection and Expression Monitoring of Core Genes Involved in the RNA Interference Pathways of Western Corn Rootworm (Diabrotica virgifera virgifera)
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays are a highly accurate and precise method for measuring transcript expression levels. A major drawback of RT-qPCR is the extensive optimization and validation necessary to produce high-quality assays, as described in the guidelines “Minimum Information for Publication of Quantitative Real-Time PCR Experiments.” This chapter describes use of designed and optimized RT-qPCR assays that accurately detect expression of eight genes predicted to be centrally involved in the RNA interference (RNAi) pathways of western corn rootworm (WCR), and appropriate accompanying parameters. Assay gene targets include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2, and detection has been validated at nine different points in the WCR life cycle. These assays can be used with this procedure to assess expression of any one of these core RNAi pathway genes in up to 96 samples per 384-well qPCR plate.
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%.
Multiple TaqMan qPCR and droplet digital PCR (ddPCR) diagnostics for pesticide resistance monitoring and management, in the major agricultural pest Tetranychus urticae
Background: Decisions on which pesticide to use in agriculture are expected to become more difficult, as the number of available chemicals is decreasing. For Tetranychus urticae, a major pest for which a number of candidate markers for pesticide resistance are in place, molecular diagnostics could support decision-making for the rational use of acaricides.
Results: A suite of twelve of TaqMan qPCR assays [G314D (GluCl1), G326E and I321T (GluCl3), G119S, F331W (Ace-1), H92R (PSST), L1024V, F1538I (VGSC), I1017F (CHS1)], G126S, S141F, P262T (cytb)], were validated against Sanger-sequencing, and subsequently adapted for use with the ddPCR technology. The concordance correlation coefficient between the actual and ddPCR measured mutant allelic frequencies was 0.995 (95% CI = 0.991 – 0.998), and no systematic, proportional or random differences detected.
The achieved Limit of Detection (LoD) was 0.1% (detection of a 1 mutant in a background of 999 wild type mites). The ddPCR assay panel was then assessed in terms of agreement with phenotypic resistance, through a pilot application in field populations from Crete, with strong correlation and thus predictive and diagnostic value of the molecular assays in some cases, e.g., etoxazole and abamectin resistance. Molecular diagnostics were able to capture incipient resistance that was missed by phenotypic bioassays in other cases. The molecular and phenotypic resistance screening of T. urticae field populations from Crete, revealed both multi-resistant and susceptible populations.
Conclusion: The highly sensitive T. urticae molecular diagnostic platforms developed in this study could prove a valuable tool for pesticide resistance management. This article is protected by copyright. All rights reserved.
Selection and validation of reference genes for RT-qPCR analysis in Desmodium styracifolium Merr
Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function. To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression.
The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP + UFM1, CCRP4 + BRM and NFD6 + NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively.
The most stable reference gene combination for all tissues was CCRP4 + CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A + SMUP1 for SA treatment, NCLN1 + ERDJ2A for ABA treatment and SF3B + VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium.
