scholarly journals Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

Genes ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 90
Author(s):  
Gustavo Barcelos Barra ◽  
Ticiane Henriques Santa Rita ◽  
Pedro Góes Mesquita ◽  
Rafael Henriques Jácomo ◽  
Lídia Freire Abdalla Nery
Keyword(s):  
Room Temperature ◽  
Limit Of Detection ◽  
Guanidine Hydrochloride ◽  
Significant Loss ◽  
Clinical Samples ◽  
Short Supply ◽  
Qualitative Detection ◽  
Probe Concentration ◽  
Assay Performance ◽  
The Stability

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Stability of Donepezil in an Extemporaneously Prepared Oral Liquid

2006 ◽  
Vol 19 (5) ◽  
pp. 282-285 ◽  
Author(s):  
Weeranuj Yamreudeewong ◽  
Eric Kurt Dolence ◽  
Deborah Pahl
Keyword(s):  
High Performance ◽  
Room Temperature ◽  
Storage Time ◽  
Microbial Growth ◽  
Storage Period ◽  
Significant Loss ◽  
Ambient Room Temperature ◽  
Oral Liquid ◽  
Liquid Preparation ◽  
The Stability

The stability of donepezil in an extemporaneously prepared oral liquid was studied. An aqueous liquid formulation of donepezil was prepared by reconstituting the powder from triturated 5-mg tablets with equal amounts of deionized water and 70% sorbitol solution with an expected donepezil concentration of 1 mg/mL. Polyethylene terephthalate plastic bottles containing donepezil liquid preparation were stored at ambient room temperature (22° C-26° C) and in the refrigerator (4° C-8° C). After a storage time of 1, 2, 3, and 4 weeks, donepezil liquid samples were analyzed in triplicate for donepezil concentrations by high-performance liquid chromatography. The concentrations of donepezil were found to be within the acceptable limit (± 10% of the initial concentration) in all test samples, which indicated that donepezil liquid preparation was stable at room temperature and in the refrigerator for up to 4 weeks. In addition, our study findings indicated that there was no microbial growth in the extemporaneously prepared donepezil liquid preparation after a storage period of 4 weeks in the refrigerator. In summary, the results of our study revealed that donepezil is stable (no significant loss of donepezil concentration and no microbial growth) in an extemporaneously prepared oral liquid when stored in the refrigerator for up to 4 weeks.

Stability of Aflatoxins in Solution

2012 ◽  
Vol 95 (4) ◽  
pp. 1084-1088 ◽  
Author(s):  
Gonzalo J Diaz ◽  
Sandra M Cepeda ◽  
Perry A Martos
Keyword(s):  
Nitric Acid ◽  
Organic Solvent ◽  
Aqueous Solutions ◽  
Chemical Etching ◽  
Room Temperature ◽  
Organic Content ◽  
Significant Loss ◽  
Type I ◽  
The Stability

Abstract The stability of aflatoxins B1, B2, G1, and G2 was studied in solutions containing different concentrations of water, acetonitrile, and/or methanol, and in autosampler vials treated with nitric acid or silanized. When stored at room temperature (20°C) for 24 h, aflatoxins G1 and G2 were stable only in solutions containing 100% organic solvent, whereas aflatoxins B1 and B2 were stable in solutions of methanol–water and acetonitrile–water at greater than 60 and 40% organic content, respectively. At 5°C, aflatoxins G1 and G2 showed a significant decrease in concentration only when kept in less than 20% aqueous organic solvent. Significant loss of aflatoxins was realized in standard, commercially available amber type I borosilicate autosampler vials, but chemical etching of the vials with nitric acid or with silanization prevented aflatoxin degradation. These results indicate that aflatoxins are unstable in aqueous solutions and that this instability can be counteracted by the presence of at least 20% organic solvent and keeping the solutions at 5°C or by the use of treated vials.

Stability of Ondansetron in Large-Volume Parenteral Solutions

1992 ◽  
Vol 26 (6) ◽  
pp. 768-771 ◽  
Author(s):  
C. Lynn Graham ◽  
George E. Dukes ◽  
Cheng-Fang Kao ◽  
Jeanne M. Bertch ◽  
Lawrence J. Hak
Keyword(s):  
Large Volume ◽  
Room Temperature ◽  
Design Method ◽  
Hplc Method ◽  
Significant Loss ◽  
Fluorescent Light ◽  
All Solutions ◽  
Ondansetron Hydrochloride ◽  
Clinically Significant ◽  
The Stability

OBJECTIVE: To determine the stability of ondansetron hydrochloride in large-volume parenteral solutions under four storage and time-period conditions. DESIGN/METHOD: Ondansetron was added to each of the following commercially available solutions to make final concentrations of approximately 24 and 96 μg/mL: NaCl 0.9%, D5W, and lactated Ringer's solution. SETTING: University analytical laboratory. MAIN OUTCOME MEASURES: Each solution was studied at both concentrations under the following conditions: (1) 1 day refrigerated, 2 days room temperature; (2) 7 days refrigerated, 2 days room temperature; (3) 14 days refrigerated, 2 days room temperature; and (4) 14 days room temperature. All solutions were exposed to fluorescent light when under room temperature conditions and were studied in triplicate. Ondansetron concentrations of samples were obtained periodically throughout each storage/time condition via a specific, stability-indicating HPLC method. RESULTS: A clinically significant loss of concentration was defined as >10 percent decrease from the initial concentration. In all solutions and at both concentrations studied, the mean ondansetron concentration was ≥90 percent under all storage and time conditions. CONCLUSIONS: Ondansetron can be stored and administered in these solutions without loss of potency.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

2021 ◽  
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Cytopathic Effect ◽  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
The Stability

AbstractBackgroundUpper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings.MethodsWe evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream RT-PCR testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C.ResultsSARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on vero-E6 cell lines was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions.ConclusioneNAT and similar guanidinium thiocyanate-based media may be of value for transport, preservation, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

THE STABILITY OF ENDOGENOUS AND EXOGENOUS CORTICOTROPHIN IN RAT PLASMA AS ESTABLISHED BY A BIOASSAY IN INTACT RATS

1968 ◽  
Vol 41 (4) ◽  
pp. 491-497 ◽  
Author(s):  
H. D. PURVES ◽  
NANCY E. SIRETT
Keyword(s):  
Low Temperature ◽  
Room Temperature ◽  
Rat Plasma ◽  
Significant Loss ◽  
Working Standard ◽  
The Stability ◽  
Saline Medium ◽  
Intact Rats

SUMMARY Corticotrophin (ACTH) activity in rat plasma, both endogenous (adrenalectomized rat plasma) and exogenous (International Working Standard, 1962), has been assayed in dexamethasone-treated rats and the stability studied under various conditions of storage. Exogenous ACTH added to rat plasma and assayed immediately had only 76% of the activity of the same ACTH dissolved in gelatine acid saline medium. This difference is ascribed to the effects of the medium and not to inactivation. When allowance was made for the effects of the medium on potency, added ACTH showed a similar stability on incubation or storage to endogenous ACTH. For endogenous ACTH the following loss of activity was found in vitro: after 45 min. at 37°, 64%; after 1 day at room temperature, more than 85%; after 1 day at 3°, 58%. No loss of potency was detected on storage at −17° for 18 months. It is concluded that plasma can be frozen, stored at low temperature, and thawed without significant loss of corticotrophic potency provided that the blood is chilled as soon as drawn and the subsequent operations performed expeditiously.

scholarly journals Saliva-Based Molecular Testing for SARS-CoV-2 that Bypasses RNA Extraction

2020 ◽  
Author(s):  
Diana Rose E. Ranoa ◽  
Robin L. Holland ◽  
Fadi G. Alnaji ◽  
Kelsie J. Green ◽  
Leyi Wang ◽  
...  
Keyword(s):  
Large Scale ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Rna Isolation ◽  
Clinical Samples ◽  
Molecular Testing ◽  
Short Supply ◽  
Isolation And Purification ◽  
Repeat Testing ◽  
Large Populations

AbstractConvenient, repeatable, large-scale molecular testing for SARS-CoV-2 would be a key weapon to help control the COVID-19 pandemic. Unfortunately, standard SARS-CoV-2 testing protocols are invasive and rely on numerous items that can be subject to supply chain bottlenecks, and as such are not suitable for frequent repeat testing. Specifically, personal protective equipment (PPE), nasopharyngeal (NP) swabs, the associated viral transport media (VTM), and kits for RNA isolation and purification have all been in short supply at various times during the COVID-19 pandemic. Moreover, SARS-CoV-2 is spread through droplets and aerosols transmitted through person-to-person contact, and thus saliva may be a relevant medium for diagnosing SARS-CoV-2 infection status. Here we describe a saliva-based testing method that bypasses the need for RNA isolation/purification. In experiments with inactivated SARS-CoV-2 virus spiked into saliva, this method has a limit of detection of 500-1000 viral particles per mL, rivalling the standard NP swab method, and initial studies also show excellent performance with 100 clinical samples. This saliva-based process is operationally simple, utilizes readily available materials, and can be easily implemented by existing testing sites, thus allowing for high-throughput, rapid, and repeat testing of large populations.Graphical Abstract

Methods for the Concentration of Bovine Ac-Globulin (Factor V)

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Room Temperature ◽  
Barium Carbonate ◽  
Deae Cellulose ◽  
Reducing Agents ◽  
Factor V ◽  
Isoelectric Precipitation ◽  
Stabilizing Agent ◽  
Bovine Plasma ◽  
The Stability

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

Survival of mouse blastocysts after low-temperature preservation under high pressure

2004 ◽  
Vol 52 (4) ◽  
pp. 479-487 ◽  
Author(s):  
Cs. Pribenszky ◽  
M. Molnár ◽  
S. Cseh ◽  
L. Solti
Keyword(s):  
Phase Transition ◽  
Hydrostatic Pressure ◽  
Low Temperature ◽  
High Hydrostatic Pressure ◽  
Room Temperature ◽  
Freezing Point ◽  
Significant Loss ◽  
Embryo Cryopreservation ◽  
Subzero Temperatures ◽  
Survival Capacity

Cryoinjuries are almost inevitable during the freezing of embryos. The present study examines the possibility of using high hydrostatic pressure to reduce substantially the freezing point of the embryo-holding solution, in order to preserve embryos at subzero temperatures, thus avoiding all the disadvantages of freezing. The pressure of 210 MPa lowers the phase transition temperature of water to -21°C. According to the results of this study, embryos can survive in high hydrostatic pressure environment at room temperature; the time embryos spend under pressure without significant loss in their survival could be lengthened by gradual decompression. Pressurisation at 0°C significantly reduced the survival capacity of the embryos; gradual decompression had no beneficial effect on survival at that stage. Based on the findings, the use of the phenomena is not applicable in this form, since pressure and low temperature together proved to be lethal to the embryos in these experiments. The application of hydrostatic pressure in embryo cryopreservation requires more detailed research, although the experience gained in this study can be applied usefully in different circumstances.

Tyrosine-Selective Bioconjugation with Iminoxyl Radicals

2020 ◽  
Author(s):  
Katsuya Maruyama ◽  
Takashi Ishiyama ◽  
Yohei Seki ◽  
Kounosuke Oisaki ◽  
Motomu Kanai
Keyword(s):  
Reaction Time ◽  
Steric Hindrance ◽  
Room Temperature ◽  
Water Soluble ◽  
Light Irradiation ◽  
Sodium Ascorbate ◽  
Iminoxyl Radical ◽  
Short Reaction Time ◽  
Modified Peptides ◽  
The Stability

A novel Tyr-selective protein bioconjugation using the water-soluble persistent iminoxyl radical is described. The conjugation proceeded with high Tyr-selectivity and short reaction time under biocompatible conditions (room temperature in buffered media under air). The stability of the conjugates was tunable depending on the steric hindrance of iminoxyl. The presence of sodium ascorbate and/or light irradiation promoted traceless deconjugation, restoring the native Tyr structure. The method is applied to the synthesis of a protein-dye conjugate and further derivatization to azobenzene-modified peptides.

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