scholarly journals Rapid Detection of Orthopoxviruses

2021 ◽  
pp. 106-113
Author(s):  
A. G. Poltavchenko ◽  
A. V. Ersh ◽  
P. V. Filatov ◽  
N. D. Ushkalenko ◽  
S. N. Yakubitsky ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Polyclonal Antibodies ◽  
Point Of Care ◽  
Rapid Test ◽  
Clinical Samples ◽  
Protein Arrays ◽  
One Stage ◽  
Immunochemical Test ◽  
The One ◽  
Dot Immunoassay

The aim of the study was to develop a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the “point of care” format.Materials and methods. The analyses were performed in cultured crude and purifed preparations of vaccinia virus, cowpoxvirus, rabbitpoxvirus and ectromelia virus, as well as in the blood and tissue suspensions of infected mice and rabbits. OPXV-antigen was detected by one-stage and two-stage protocols of dot-immunoassay based on flat protein arrays using rabbit polyclonal antibodies as capture and detection reagents.Results and discussion. The results show that the detection limit of OPXV is inversely related to the degree of their purifcation. The one-stage (rapid) protocol is specifc and allows detecting OPXV in crude culture samples of the virus and in clinical samples in the range of 104–103 PFU/ml within 39 minutes. Rapid dot-immunoassay can be applied to detect or exclude the presence of a viral threat in samples and can be useful in various aspects of biosafety provision. The simplicity of the one-stage protocol, the possibility to visually account the results and easy interpretation of the results allow the rapid test to be used in the “point of care” format.

scholarly journals Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

scholarly journals Performance of a Point-of-Care Test for the Rapid Detection of SARS-CoV-2 Antigen

2020 ◽  
Vol 9 (1) ◽  
pp. 58
Author(s):  
Annabelle Strömer ◽  
Ruben Rose ◽  
Miriam Schäfer ◽  
Frieda Schön ◽  
Anna Vollersen ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Point Of Care ◽  
Rapid Test ◽  
Vero Cells ◽  
Upper Respiratory Tract ◽  
Rt Pcr ◽  
Nucleoprotein Gene ◽  
Point Of Care Test ◽  
Polymerase Chain ◽  
Point Of Care Tests

The rapid detection of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary in the ongoing pandemic. Antigen-specific point-of-care tests (POCT) may be useful for this purpose. Here, such a POCT (SARS-CoV-2 NADAL® COVID-19 Ag) was compared to a laboratory-developed triplex real-time polymerase chain reaction (RT-PCR) designed for the detection of viral nucleoprotein gene and two control targets. This RT-PCR served as a reference to investigate POCT sensitivity by re-testing upper respiratory tract (URT) samples (n = 124) exhibiting different SARS-CoV-2 loads in terms of RT-PCR threshold cycle (Ct) values. The optical intensities of the antigen bands were compared to the Ct values of the RT-PCR. The infectivity of various virus loads was estimated by inoculating Vero cells with URT samples (n = 64, Ct 17-34). POCT sensitivity varied from 100% (Ct < 25) to 73.1% (Ct ≤ 30); higher SARS-CoV-2 loads correlated with higher band intensities. All samples with a Ct > 30 were negative; among SARS-CoV-2 free samples (n = 10) no false-positives were detected. A head-to-head comparison with another POCT (Abbott, Panbio™ COVID-19 Ag Rapid Test) yielded similar results. Isolation of SARS-CoV-2 in cell-culture was successful up to a Ct value of 29. The POCT reliably detects high SARS-CoV-2 loads and rapidly identifies infectious individuals.

scholarly journals Isothermal Recombinase Polymerase Amplification-lateral Flow Detection of SARS-CoV-2, the Etiological Agent of COVID-19

2020 ◽  
Author(s):  
Thomas R Shelite ◽  
Ashanti C Uscanga-Palomeque ◽  
Alejandro Castellanos ◽  
Peter C Melby ◽  
Bruno L Travi
Keyword(s):  
Rapid Detection ◽  
Limit Of Detection ◽  
Point Of Care ◽  
The United States ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
N Gene ◽  
Clinical Samples ◽  
Reference Test ◽  
Delay In Diagnosis

Abstract The rapid detection of novel pathogens necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. In December of 2019, novel coronavirus, SARS-CoV-2 (2019-nCoV), was isolated from a cluster of pneumonia patients in the Chinese city of Wuhan. The virus rapidly spread throughout the world and the first fatal cases of COVID-19 in the United States occurred in late February. The lack of testing and delay in diagnosis has facilitated the spread of this novel virus. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 clinical samples using CDC’s N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100% concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

scholarly journals Development of immunochromatographic lateral flow test for rapid detection of Clostridium perfringens α, β and ε toxins in clinical samples

2021 ◽  
Vol 24 (4) ◽  
pp. 497-507
Author(s):  
R Soliman ◽  
M. M. Magdy ◽  
A. Samir ◽  
Y. A. Abdalla ◽  
R. H. Sayed
Keyword(s):  
Guinea Pigs ◽  
Rapid Detection ◽  
Polyclonal Antibodies ◽  
Lateral Flow ◽  
Clinical Samples ◽  
Immunochromatographic Test ◽  
Flow Test ◽  
Faecal Samples ◽  
Lateral Flow Test ◽  
Sensitivity Specificity

In the present work a lateral flow immunochromatographic test (LFT) for rapid detection of Clostri­dium perfringens toxins types, alpha (α), beta (β) and epsilon (ε) in clinical samples was developed. C. perfringens toxins were prepared, purified and inactivated with 0.2% formalin. Polyclonal antibodies specific to C. perfringens toxins types α, β and ε toxoids were prepared in rabbits and guinea pigs. The toxoid specific polyclonal antibodies prepared in rabbits were labelled with gold chloride nanoparticles. The prepared toxin specific rabbit and guinea pigs antibodies and goat anti-rabbit antibodies were utilised in development of a lateral flow immunochromatographic test and the latter - evaluated for detection of C. perfringens α, β and ε toxins in clinical samples. The sensitivity and specificity and accuracy of the developed LFT were determined by comparison with a commercially available ELISA used for detection of these toxins. The prepared LFT was capable to detect C. perfringens α, β and ε toxins in quantities of 2 μg/ml, 250 ng/ml and 60 ng/ml, respectively. One hundred poultry suspected faecal samples was examined both with the prepared LFT and commercial ELISA to test the validity of developed LFT. The sensitivity, specificity and accuracy of the LFT for detection of C. perfringens toxins were 81%, 95.2% and 90%, respectively, for α toxin, 76.6%, 98.5% and 72%, respectively, for β toxin and 66.6%, 98.8% and 95%, respectively, for ε toxin.

scholarly journals Comparison of seven commercial SARS-CoV-2 rapid Point-of-Care Antigen tests

2020 ◽  
Author(s):  
Victor M. Corman ◽  
Verena Claudia Haage ◽  
Tobias Bleicker ◽  
Marie Luisa Schmidt ◽  
Barbara Mühlemann ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Point Of Care ◽  
Rapid Test ◽  
Infectious Period ◽  
Clinical Samples ◽  
Virus Concentration ◽  
Viral Loads ◽  
And Performance ◽  
Antigen Tests ◽  
Point Of Care Tests

AbstractBackgroundAntigen point of care tests (AgPOCT) can accelerate SARS-CoV-2 testing. As first AgPOCT are becoming available, there is a growing interest in their utility and performance.MethodsHere we compare AgPOCT products by seven suppliers: the Abbott Panbio™ COVID-19 Ag Rapid Test; the RapiGEN BIOCREDIT COVID-19 Ag; the Healgen® Coronavirus Ag Rapid Test Cassette (Swab); the Coris Bioconcept Covid.19 Ag Respi-Strip; the R-Biopharm RIDA®QUICK SARS-CoV-2 Antigen; the NAL von minden NADAL COVID19-Ag Test; and the Roche/SD Biosensor SARS-CoV Rapid Antigen Test. Tests were evaluated on recombinant nucleoprotein, cultured endemic and emerging coronaviruses, stored clinical samples with known SARS-CoV-2 viral loads (n=138), stored samples from patients with respiratory agents other than SARS-CoV-2 (n=100), as well as self-sampled swabs from healthy volunteers (n=35).FindingsLimits of detection in six of seven tested products ranged between 2.08 × 106 and 2.88 × 107 copies per swab, the outlier at 1.58 × 1010 copies per swab. Specificities ranged between 98.53% and 100% in five products, with two outliers at 94.85% and 88.24%. False positive results were not associated with any specific respiratory agent. As some of the tested AgPOCT were early production lots, the observed issues with specificity are unlikely to persist.InterpretationThe sensitivity range of most AgPOCT overlaps with viral load figures typically observed during the first week of symptoms, which marks the infectious period in the majority patients. AgPOCTs with a limit of detection that approximates the virus concentration above which patients are infectious may enable shortcuts in decision-making in various areas of healthcare and public health.

Performances of an Artificial Reagent for the One-stage Factor IX Assay

1975 ◽  
Vol 33 (03) ◽  
pp. 547-552 ◽  
Author(s):  
L Meunier ◽  
J. P Allain ◽  
D Frommel
Keyword(s):  
Human Plasma ◽  
Factor Ix ◽  
Severe Haemophilia ◽  
Normal Human Plasma ◽  
Haemophilia B ◽  
One Stage ◽  
Normal Human ◽  
The One

SummaryA mixture of adsorbed normal human plasma and chicken plasma was prepared as reagent for factor IX measurement using a one-stage method. The substrate was found to be specific for factor IX. Its performances tested on samples displaying factor IX activity ranging from <l%–2,500% compared favorably with those obtained when using the plasma of severe haemophilia B patients as substrate.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

The Formation of Intermediate Product I in a Purified System The Role of Factor IX or of its Precursor and of a Hageman Factor-PTA Fraction

1961 ◽  
Vol 6 (02) ◽  
pp. 224-234 ◽  
Author(s):  
E. T Yin ◽  
F Duckert
Keyword(s):  
Intermediate Product ◽  
Factor Ix ◽  
Assay Method ◽  
Lag Phase ◽  
Factor X ◽  
Haemophilia B ◽  
Hageman Factor ◽  
One Stage ◽  
The One

Summary1. The role of two clot promoting fractions isolated from either plasma or serum is studied in a purified system for the generation of intermediate product I in which the serum is replaced by factor X and the investigated fractions.2. Optimal generation of intermediate product I is possible in the purified system utilizing fractions devoid of factor IX one-stage activity. Prothrombin and thrombin are not necessary in this system.3. The fraction containing factor IX or its precursor, no measurable activity by the one-stage assay method, controls the yield of intermediate product I. No similar fraction can be isolated from haemophilia B plasma or serum.4. The Hageman factor — PTA fraction shortens the lag phase of intermediate product I formation and has no influence on the yield. This fraction can also be prepared from haemophilia B plasma or serum.

The Preparation of an Artificial Reagent for the One-Stage Factor VIII Assay

1970 ◽  
Vol 23 (02) ◽  
pp. 306-312 ◽  
Author(s):  
D Nyman
Keyword(s):  
Factor Viii ◽  
Artificial Substrate ◽  
One Stage ◽  
The One

SummaryA method for preparation of an artificial substrate for the one-stage factor VIII assay is described. Standardization of the test is given. The method is specific and reproducible.

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