One-Step Quantitative RT-PCR Assay with Armored RNA Controls for Detection of SARS-CoV-2
COVID-19 has develop to be pandemic since March, 11, 2020. Thus, development and integration in clinics of fast and delicate diagnostic devices is essential. The aim of the analysis was a development and evaluation of a one-step RT-qPCR assay (COVID-19 Amp) for SARS-CoV-2 detection with an armored optimistic administration and inside controls constructed from synthetic MS2-phage-based RNA particles.
The COVID-19 Amp assay prohibit of detection was 103 copies/ml, the analytical specificity was 100%. 109 natural samples have been examined using COVID-19 Amp and WHO-based assay.
Discordance in 9 samples was observed (unfavourable by the WHO-based assay) and discordant samples have been retested as optimistic based mostly on the outcomes obtained from the Vector-PCRrv-2019-nCoV-RG assay.
The developed COVID-19 Amp assay has extreme sensitivity and specificity, incorporates virus particles-based controls, provides the direct definition of SARS-CoV-2 RdRp gene partial sequence, and is acceptable for any hospital and laboratory equipped for RT-qPCR. This textual content is protected by copyright. All rights reserved.
Results of graphene oxide on PCR amplification for microbial group survey
Background: Graphene oxide (GO) has been advised as an environment friendly assistant additive to eradicate non-specific amplification of the polymerase chain response (PCR). Though many research have centered on exploring its molecular mechanism, the apply of GO on the quantitation of microbial group has not been carried out but. On this examine, GO was added in PCR system to discover the adjustments on eradicating typical amplification errors, reminiscent of chimera and mismatches on two sorts of mock communities (an evenly blended and a staggered mock communities) and environmental samples
Outcomes: Excessive-throughput sequencing of bacterial and fungal communities, based mostly on 16S rRNA genes and inside transcribed spacers (ITS) respectively, confirmed that GO may considerably enhance giant segmental error (chimeric sequence) in PCR process whereas had no particular impact on level error (mismatched sequence). Moreover, GO decreased the α-diversity of group, and altered the composition of fungal group extra clearly than bacterial group.
Conclusions: Our examine offers the primary quantitative knowledge on microbial group degree to show the unfavourable impact of GO, and likewise signifies that there could also be a extra complicated interplay between GO and complete DNA fragments in PCR course of.
Description: Rat Primary Vein Fibroblasts from Gentaur are isolated from tissue of adult Sprague-Dawley Rats. Rat Primary Vein Fibroblasts are grown in T75 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Cells are negative for bacteria, yeast, fungi, and mycoplasma. Rat Primary Vein Fibroblasts are tested for expression of marker using the antibody of anti-FSP1/S100A4 by immunofluorescence staining and can be expanded by 2-4 passages at a split ratio of 1:2 under the cell culture conditions specified by Gentaur. Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with fibroblast cultures include the assays of cell to cell interaction, PCR, Western blotting, immunoprecipitation, immunofluorescent staining, immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Recombinant Gallid herpesvirus 2 Portal protein UL6 homolog (MDV018), partial
Description: Rat Vein Endothelial Cells from Gentaur are isolated from tissue of adult Sprague-Dawley Rats. Rat Vein Endothelial Cells are grown in T75 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and are delivered frozen.Rat Vein Endothelial Cells can be used in assays of cell to cell adhesion, migration, vascular tube formation, or transendothelial resistance (TER). Standard biochemical procedures performed with endothelial cell cultures include PCR, Western blotting, immunoprecipitation, immunofluorescent staining or immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Tissue cDNA, First Strand, Human Diseased (Adult), Hypertension, Vein, BioGenomics
Description: Rat Primary Pulmonary Vein Fibroblasts from Gentaur are isolated from tissue of neonatal Sprague-Dawley Rats. Rat Primary Pulmonary Vein Fibroblasts are grown in T75 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Cells are negative for bacteria, yeast, fungi, and mycoplasma. Rat Primary Pulmonary Vein Fibroblasts are tested for expression of marker using the antibody of anti-FSP1/S100A4 by immunofluorescence staining and can be expanded by 2-4 passages at a split ratio of 1:2 under the cell culture conditions specified by Gentaur. Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with fibroblast cultures include the assays of cell to cell interaction, PCR, Western blotting, immunoprecipitation, immunofluorescent staining, immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Description: Rat Vein Smooth Muscle Cells from Gentaur are isolated from tissue of Sprague-Dawley Rats. Rat Vein Smooth Muscle Cells are grown in T75 tissue culture flasks pre-coated with gelatin-based coating solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Cells are negative for bacteria, yeast, fungi, and mycoplasma and are characterized by immunofluorescent staining with antibodies to α-smooth muscle actin. Cells can be expanded on a multiwell culture plate ready for experiments under the cell culture conditions specified by Gentaur.Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with smooth muscle cell cultures include the assays of cell to cell interaction, PCR, Western blotting, immunoprecipitation, immunofluorescent staining, immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Description: Lewis Rat Vein Endothelial Cells are isolated from inferior vena cava tissue of 6-8 week old Lewis rat. Lewis Rat Vein Endothelial Cells are cryopreserved at passage 3 and delivered frozen.
Description: Rat Pulmonary Vein Endothelial Cells from Gentaur are isolated from tissue of adult Sprague-Dawley Rats. Rat Pulmonary Vein Endothelial Cells are grown in T75 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and are delivered frozen.Rat Pulmonary Vein Endothelial Cells can be used in assays of cell to cell adhesion, migration, vascular tube formation, or transendothelial resistance (TER). Standard biochemical procedures performed with endothelial cell cultures include PCR, Western blotting, immunoprecipitation, immunofluorescent staining or immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Description: Human vein tissue lysate was prepared by homogenization using a proprietary technique. The tissue was frozen in liquid nitrogen immediately after excision and then stored at -70°C. The human vein tissue total protein is provided in a buffer including HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium deoxycholate, NP-40, and a cocktail of protease inhibitors. For quality control purposes, the vein tissue pattern on SDS-PAGE gel is shown to be consistent for each lot by visualization with coomassie blue staining. The vein tissue is then Western analyzed by either GAPDH or β-actin antibody, and the expression level is consistent with each lot.
Description: Rat Pulmonary Vein Smooth Muscle Cells from Gentaur are isolated from tissue of Sprague-Dawley Rats. Rat Pulmonary Vein Smooth Muscle Cells are grown in T75 tissue culture flasks pre-coated with gelatin-based coating solution for 0.5 hour and incubated in Gentaur's Cell Culture Medium for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Cells are negative for bacteria, yeast, fungi, and mycoplasma and are characterized by immunofluorescent staining with antibodies to α-smooth muscle actin. Cells can be expanded on a multiwell culture plate ready for experiments under the cell culture conditions specified by Gentaur.Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with smooth muscle cell cultures include the assays of cell to cell interaction, PCR, Western blotting, immunoprecipitation, immunofluorescent staining, immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Description: Porcine Vein Endothelial Cells are derived from healthly porcinine vein tissue. Porcine Vein Endothelial Cells are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and are delivered frozen.Porcine Vein Endothelial Cells can be used in assays of cell to cell adhesion, migration, vascular tube formation, or transendothelial resistance (TER). Standard biochemical procedures performed with endothelial cell cultures include RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining or immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Myc-DDK-tagged ORF clone of viral ORF for capsid portal protein [Human herpesvirus 1], codon optimized for human cell expression, NP_044607
Description: Porcine Pulmonary Vein Endothelial Cells are derived from healthly porcinine pulmonary vein tissue. Porcine Pulmonary Vein Endothelial Cells are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur's Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and are delivered frozen.Porcine Pulmonary Vein Endothelial Cells can be used in assays of cell to cell adhesion, migration, vascular tube formation, or transendothelial resistance (TER). Standard biochemical procedures performed with endothelial cell cultures include RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining or immunofluorescent flow cytometry or generating cell derivatives for desired research applications.
Rat Saphenous Vein Smooth Muscle Cells Complete Medium
Description: Gentaur's normal Human Umbilical Vein Endothelial Cells (HUVEC), when grown in Gentaur's LIVas Medium, provides an ideal low serum culture model, with or without human VEGF, for the study of angiogenesis, atherosclerosis or vascular biology. Gentaur's HUVEC are cryopreserved as primary cells to ensure the highest viability and plating efficiency. Our HUVEC are quality tested in LIVas 2% serum medium to ensure optimal reduced-serum growth over a period of at least 15 population doublings at rates equal to or greater than serum-supplemented medium. Cell Features:HUVEC are isolated from human umbilical cords, cultured on plastic and cryopreserved as primary cells.HAoEC and HUVEC 10-Donor Pool are isolated from human aorta or umbilical cord, cultured on plastic and cryopreserved as secondary cells.HPAEC are isolated from human pulmonary artery, cultured on plastic and cryopreserved as secondary cells.HCAEC are isolated from human coronary arteries, cultured on plastic and cryopreserved as tertiary cells.Human endothelial cells can be grown in low serum (2%) medium without phenol red or antimicrobials when cultured in VascuLife medium.Culture human endothelial cells with or without VEGF.Gentaur's human endothelial cells are extensively tested to meet quality standards and exhibit optimal performance.Gentaur guarantees performance and quality.
Description: BALB/c Mouse Primary Vein Fibroblasts from Gentaur are isolated from tissue of pathogen-free laboratory mice. Mouse Primary Vein Fibroblasts are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Mouse Primary Vein Fibroblasts are negative for bacteria, yeast, fungi, and mycoplasma. Cells are tested for expression of marker using the antibody of anti-FSP1/S100A4 by immunofluorescence staining. Cells can be expanded for 2-4 passages at a split ratio of 1:2 under the cell culture conditions specified by Gentaur. Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with cell cultures include the assay of cell to cell interaction, RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining, flow cytometry or generating cell derivatives for desired research applications.
Description: C57BL/6 Mouse Primary Vein Fibroblasts from Gentaur are isolated from tissue of pathogen-free laboratory mice. Mouse Primary Vein Fibroblasts are grown in T25 tissue culture flasks pre-coated with gelatin-based solution for 0.5 hour and incubated in Gentaur Culture Complete Growth Medium generally for 3-7 days. Cultures are then expanded. Prior to shipping, cells are detached from flasks and immediately cryo-preserved in vials. Each vial contains at least 1x10^6 cells per ml and is delivered frozen.Mouse Primary Vein Fibroblasts are negative for bacteria, yeast, fungi, and mycoplasma. Cells are tested for expression of marker using the antibody of anti-FSP1/S100A4 by immunofluorescence staining. Cells can be expanded for 2-4 passages at a split ratio of 1:2 under the cell culture conditions specified by Gentaur. Repeated freezing and thawing of cells is not recommended.Standard biochemical procedures performed with cell cultures include the assay of cell to cell interaction, RT-PCR, Western blotting, immunoprecipitation, immunofluorescent staining, flow cytometry or generating cell derivatives for desired research applications.
Description: Gentaur endothelial cells from large vessels are available from different locations, e.g. umbilical vein, umbilical artery, aorta, coronary artery, pulmonary artery, and saphenous vein. In addition, Gentaur offers HUVEC isolated in standard Endothelial Cell Growth Medium and also in Endothelial Cell Growth Medium 2. The cells are supplied either from single donors*or from pooled donors (from up to four different umbilical cords). Furthermore, HUVEC are available which are prescreened for VEGF (vascular endothelial cell growth factor) response. Shortly after isolation, Gentaur Human Endothelial Cells from large vessels are cryopreserved at passage 1 or passage 2 (see page 5) using Gentaur's proprietary, serum-free freezing medium, Cryo-SFM. Each cryo vial contains more than 500,000 viable cells after thawing.Proliferating cell cultures are made from 500,000 cryopreserved cells that have been thawed and cultured for three days at Gentaur.
Description: HUVEC are isolated from the vein of the umbilical cord and are commonly used for physiological and pharmacological investigations, such as macromolecule transport, blood coagulation, angiogenesis, and fibrinolysis. Human umbilical vein endothelial cells have played a major role as a model system for the study of the regulation of endothelial cell function and the role of the endothelium in the response of the blood vessel wall to stretch, shear forces, and the development of atherosclerotic plaques.Each human umbilical cord vein is individually processed to isolate endothelial cells through collagenase digestion and culture. Frozen HUVEC products are cryopreserved at the end of the primary culture. HUVEC are guaranteed to reach 15 population doublings when cultured with Gentaur's recommended medium.Samples from each donor are tested via PCR to confirm non-reactivity.
Routine blood evaluation drastically reduces the false-negative price of RT-PCR testing for Covid-19
Background: The COVID-19 outbreak is now a pandemic illness reaching as a lot as 210 international locations worldwide with greater than 2.5 million contaminated folks and almost 200.000 deaths. Amplification of viral RNA by RT-PCR represents the gold commonplace for affirmation of an infection, but it confirmed false-negative charges as giant as 15-20% which could jeopardize the impact of the restrictive measures taken by governments. We beforehand confirmed that a number of hematological parameters had been considerably totally different between COVID-19 constructive and unfavourable sufferers. Amongst them aspartate aminotransferase and lactate dehydrogenase had predictive values as giant as 90%. Thus a mix of RT-PCR and blood assessments may cut back the false-negative price of the genetic check.
Strategies: We retrospectively analyzed 24 sufferers displaying a number of and inconsistent RT-PCR, check throughout their first hospitalization interval, and in contrast the genetic assessments outcomes with their AST and LDH ranges.
Outcomes: We confirmed that when contemplating the hematological parameters, the RT-PCR false-negative charges had been decreased by virtually 4-fold.
Conclusions: The examine represents a preliminary work aiming on the improvement of methods that, by combining RT-PCR assessments with routine blood assessments, will decrease and even abolish the speed of RT-PCR false-negative outcomes and thus will establish, with excessive accuracy, sufferers contaminated by COVID-19.
Multiplex detection of ctDNA mutations in plasma of colorectal most cancers sufferers by PCR/SERS assay
Molecular diagnostic testing of KRAS and BRAF mutations has develop into important within the administration of colorectal most cancers (CRC) sufferers. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), however sluggish evaluation for DNA sequencing strategies has restricted fast diagnostics.
Different strategies reminiscent of quantitative PCR and extra lately, droplet digital PCR (ddPCR), have limitations in multiplexed capability and the necessity for costly specialised tools. Therefore, a strong, fast and facile technique is required for detecting a number of ctDNA mutations to enhance the administration of CRC sufferers. To handle this important downside, herein, we suggest a brand new software of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a quick and non-invasive method to diagnose and stratify sufferers for efficient remedy.
Strategies: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers had been designed for the amplification of three clinically essential DNA level mutations in CRC together with KRAS G12V, KRAS G13D and BRAF V600E. Floor-enhanced Raman scattering (SERS) nanotags had been labelled with a brief and particular sequence of oligonucleotide, which may hybridize with the corresponding PCR amplicons. The PCR/SERS assay was carried out by firstly amplifying the a number of mutations, adopted by binding with multicolor SERS nanotags particular to every mutation, and subsequent enrichment with magnetic beads. The mutation standing was evaluated utilizing a transportable Raman spectrometer the place the fingerprint spectral peaks of the corresponding SERS nanotags point out the presence of the mutant targets. The tactic was then utilized to detect ctDNA from CRC sufferers beneath a blinded check, the outcomes had been additional validated by ddPCR.
Outcomes: The PCR/SERS technique confirmed excessive specificity and sensitivity for genotyping CRC cell strains and plasma ctDNA, the place as few as 0.1% mutant alleles may very well be detected from a background of plentiful wild-type cfDNA. The blinded check utilizing 9 samples from superior CRC sufferers by PCR/SERS assay was validated with ddPCR and confirmed good consistency with pathology testing outcomes.
Conclusions: With ddPCR-like sensitivity but on the comfort of ordinary PCR, the proposed assay reveals nice potential in delicate detection of a number of ctDNA mutations for scientific decision-making.
