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1.
Sci Rep ; 12(1): 505, 2022 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-35017592

RESUMO

Current methods used for diagnosis of acute infection of pathogens rely on detection of nucleic acids, antigens, or certain classes of antibodies such as IgM. Here we report a virus enzyme assay as an alternative to these methods for detection of acute viral infection. In this method, we used a luciferin derivative as the substrate for detection of the enzyme activity of influenza viral neuraminidase as a means for diagnosis of influenza. The resulting commercial test, the qFLU Dx Test, uses a different supply chain that does not compete with those for the current tests. The assay reagents were formulated as a master mix that accommodated both the neuraminidase and luciferase reactions, thereby enabling rapid and prolonged production of stable light signal in the presence of influenza virus in the sample. The assay was evaluated using depository throat swab specimens. As expected, the assay exhibited similar detection rates for all influenza types and subtypes except for A(H7N9), which exhibited lower detection rate due to lower viral titer in the specimens. When throat swab specimens were diluted with the sample buffer of the test kit and tested with the qFLU Dx Test. The sensitivity and specificity were 82.41% (95% confidence interval: 79.66-85.84%) and 95.39% (95% confidence interval: 94.32-96.46%), respectively, for these diluted specimens in comparison to a real-time polymerase chain reaction assay. The uniqueness of the qFLU Dx Test as an enzymatic assay makes it highly complementary with currently available methods.


Assuntos
Testes Diagnósticos de Rotina/métodos , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Influenza Humana/diagnóstico , Neuraminidase/análise , Proteínas Virais/análise , Testes Diagnósticos de Rotina/instrumentação , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Subtipo H7N9 do Vírus da Influenza A/isolamento & purificação , Influenza Humana/virologia , Neuraminidase/genética , Neuraminidase/metabolismo , Faringe/virologia , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Clin Exp Med ; 22(4): 577-581, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-35001207

RESUMO

A biochemiluminescent assay of influenza diagnosis is presented. The assay diagnoses influenza based on detection of the influenza viral neuraminidase activity. An instrument designed for the assay is also reported. This assay solves the problem that current influenza virus diagnosis assays are susceptible to virus mutation. A luciferase-based complex is synthesized as biochemiluminescent substrate. The substrate is cleaved to free luciferin with presence of influenza neuraminidase in specimen. Luciferase is oxidized to oxyluciferin with luciferin as catalyzer resulting in luminescence, which is proportional to the neuraminidase activity and measured by instrument. The instrument uses a photomultiplier tube as sensor, with 24 test channels. Fine optical arrangements enable the instrument with high sensitivity and accuracy. A total of 389 clinical specimens were collected to evaluate the performance of the assay in clinical settings. This assay had a sensitivity and specificity of 95.92% (95% confidence interval 91.38-98.12%) and 97.93% (95% confidence interval 95.26-99.11%), respectively, compared to the colloidal gold assay. As a biochemiluminscence assay, this assay is advantageous in sensitivity and specificity. It does not require any washing or separation steps, which makes the instrument simple in design and easy to operate or maintenance. The assay is suitable for the rapid diagnosis of influenza virus in point-of-care settings.


Assuntos
Influenza Humana , Humanos , Influenza Humana/diagnóstico , Neuraminidase/genética , Sensibilidade e Especificidade , Coloide de Ouro
3.
Sci Rep ; 9(1): 20024, 2019 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-31882933

RESUMO

Bacterial vaginosis (BV) is a common condition among women of reproductive age. A sensitive, quantitative and rapid assay is needed for the diagnosis of and, particularly, therapy monitoring for BV. Bacterial sialidase appears to play an important role in bacterial biofilms on vaginal epithelium, a condition closely associated with BV. Here, we report a biochemiluminescent sialidase assay that uses a substrate derivatized with firefly luciferin. In the presence of sialidase in the reaction, the substrate is cleaved to release luciferin, which is subsequently oxidized by firefly luciferase to generate a light signal. Thus, the light signal intensity can be used to detect and measure the relative concentration of sialidase in a vaginal sample as a means of BV diagnosis. All reagents are present in a reagent bead and sample buffer, enabling essentially a one-step assay. The assay is highly sensitive and quantitative, with a sensitivity and specificity of 95.40% and 94.94%, respectively, compared to the Amsel method. Interestingly, only 27.6% of those with BV had high levels of sialidase activity with a signal to cutoff ratio of 10 or more. The assay may be used for diagnosis of BV, risk assessment of BV patients in terms of sialidase activity levels, and monitoring antibiotic therapy.


Assuntos
Neuraminidase/metabolismo , Vaginose Bacteriana/diagnóstico , Adulto , Feminino , Humanos , Luminescência , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Especificidade por Substrato , Vaginose Bacteriana/microbiologia
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