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Virus Titer Test Services for Adenoviruses
Syd Labs provides comprehensive virus titer test services: Multiplicity of Infection (MOI) assay, 50% Tissue Culture Infective Dose (TCID50), Replication-Competent Adenovirus (RCA) assays with PCR and A549 cells, and plaque assay with Plaque Forming Units (PFU).
Multiplicity of Infection (MOI) = Plaque Forming Units (PFU) of virus used for infection / number of cells.
50% Tissue Culture Infective Dose (TCID50): The endpoint dilution assay quantifies the number of virus required to kill 50% of infected host cells or to produce a cytopathic effect in 50% of inoculated tissue culture cells.
Replication-Competent Adenovirus (RCA) assay in A549 cells: The standard method to determine the presence of RCA is to inoculate A549 cells with a sample of the viral stock. Since A549 cells are very readily infected by adenovirus but do not express the E1 gene, no plaque will form unless viral particles that have acquired the E1 gene are present. The sensitivity of this method is limited by the cytotoxic effects of the replication-deficient virus on the A549 cells, which limits the number of particles that can be added to the cultured cells. Approximately 1 RCA per 10^6 viral particles (determined spectrophotometrically) can be detected by this method.
Replication-Competent Adenovirus (RCA) assay by PCR: The DNA purified from viral particles is used as a template for a PCR procedure in which E1 gene specific primers are used to detect the presence of the E1 gene. The sensitivity of this method is limited by the amount of template DNA that can be added. Greater sensitivity (10-fold) can be achieved by doing 10 replicate PCR reactions. The PCR procedure to detect the presence of RCA is faster and more sensitive than the assay that employs A549 cells and is recommended before amplification of a viral stock.
Plaque assay with Plaque Forming Units (PFU): The abundance of adenoviral particles can be determined in a variety of ways. Two of the most useful are the spectrophotometric method, which is fast and highly reproducible, and the plaque assay, which estimates the number of infectious particles that are present. The plaque assay is performed by treating host cells in which the virus can replicate with serial dilutions of the virus preparation for several days. During the incubation, infected cells grow new viral particles that infect neighboring cells, which die and release more adenovirus until a plaque becomes visible. The plaque assay is slower and less reproducible than the spectrophotometric assay, but it provides a result that may be a more realistic estimate of the number of viral particles needed to evoke a biological response. A normal ratio between the spectrophotometric and plaque assay results indicates that a purified viral product is not contaminated with UV absorbent material.