ERRATUM NOTICE
Important: There has been an erratum issued for this article. Read more …
Summary
En este vídeo, se demuestra un método alternativo para la detección y titulación de virus utilizando una técnica de detección de antígenos enzimática conocida como prueba de inmunoperoxidasa. Aquí le mostraremos cómo recoger las muestras virales, preparar las células para la prueba, y finalmente la prueba de inmunoperoxidasa utilizando diluciones seriadas para determinar el título viral.
Abstract
Cálculo de los títulos virales infecciosas representa un enfoque experimental básico y esencial para los virólogos. Ensayos clásica placa no se puede utilizar para detectar virus que no causan importantes efectos citopáticos, que es el caso de las cepas 229E y OC43 de coronavirus humano (HCoV). Un ensayo alternativo inmunoperoxidasa indirecta (IPA) es aquí descritos para la detección y valoración de estos virus. Células susceptibles son inoculadas con diluciones seriadas logarítmicas de las muestras en una placa de 96 pocillos. Después de un crecimiento viral, detección de virus por el IPA se obtiene el título de virus infecciosos, expresado como "la dosis infecciosa cultivo de tejidos" (TCID50). Esto representa la dilución de una muestra que contiene el virus en la que la mitad de una serie de pozos de laboratorio contienen replicación del virus. Esta técnica es un método fiable para la valoración de HCoV en muestras biológicas (células, tejidos o fluidos).
Protocol
El protocolo de texto completo de este enfoque experimental está disponible en los Protocolos Springer .
Tags
Microbiología Número 14 Springer Protocolos coronavirus humanos HCoV-229E HCoV-OC43 y muestra de tejido celular la titulación inmunoperoxidasa TCID50Erratum
Formal Correction: Erratum: Titration of Human Coronaviruses Using an Immunoperoxidase Assay
Posted by JoVE Editors on 04/01/2012.
Citeable Link.
A correction was made to: Titration of Human Coronaviruses Using an Immunoperoxidase Assay. A revised abstract was republished due to a publisher error.
Revised Abstract:
Determination of infectious viral titers is a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for prototype strains 229E and OC43 of human coronavirus (HCoV). Therefore, an alternative indirect immunoperoxidase assay (IPA) was developed for the detection and titration of these viruses and is described herein. Susceptible cells are inoculated with serial logarithmic dilutions of virus-containing samples in a 96-well plate format. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as 'Tissue Culture Infectious Dose 50 percent' (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain infectious replicating virus. This technique provides a reliable method for the titration of HCoV-229E and HCoV-OC43 in biological samples such as cells, tissues and fluids. This article is based on work first reported in Methods in Molecular Biology (2008) volume 454, pages 93-102.
Original Abstract:
Calculation of infectious viral titers represents a basic and essential experimental approach for virologists. Classical plaque assays cannot be used for viruses that do not cause significant cytopathic effects, which is the case for strains 229E and OC43 of human coronavirus (HCoV). An alternative indirect immunoperoxidase assay (IPA) is herein described for the detection and titration of these viruses. Susceptible cells are inoculated with serial logarithmic dilutions of samples in a 96-well plate. After viral growth, viral detection by IPA yields the infectious virus titer, expressed as "Tissue Culture Infectious Dose" (TCID50). This represents the dilution of a virus-containing sample at which half of a series of laboratory wells contain replicating virus. This technique is a reliable method for the titration of HCoV in biological samples (cells, tissues or fluids).
