broward primer method
using PCR for virus identification
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The advent of the PCR has greatly enhanced our ability to detect human enteric viral pathogens in the environment, including water, municipal wastes, sewage, food, air, and fomites (2, 3, 59, 69, 79). This is especially true for those viruses which do not grow in cell culture. Despite great sensitivity, PCR methods do have some serious limitations for environmental viral analysis, including small sample volumes, the presence of PCR-inhibitory substances, and an inability to differentiate between infective and noninfective viruses (66). The ability of PCR to assess infectivity would greatly enhance its application for the monitoring of water and food quality and for treatment processes (e.g., disinfection). This review focuses on approaches to overcome these limitations.
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DETERMINATION OF VIRAL INFECTIVITY
Viral infectivity can be described as the capacity of viruses to enter the host cell and use cell resources to ultimately produce infectious viral particles (virions) (10). The virion of most enteric viruses is composed of two major components, the capsid and the genome (83). The protein capsid is involved in the interaction of the virus with the host cell surface and contains antigens specific to cell receptors used to gain entry into the cell. The capsid also has the function of protecting the viral genome from degradation by nucleases and abiotic stresses, such as humidity, pH, UV radiation, and temperature. Thus, an undamaged viral capsule is critical for the initiation of a successful infection.
In addition to the viral capsule, the replication and translation of the viral genome to viral proteins and enzymes are also important for the successful production of new viral particles (83). The properties of the genome vary among the different groups of enteric viruses, which include positive-stranded RNA viruses, double-stranded RNA viruses, and double-stranded DNA viruses. Therefore, each viral group has its own mechanism for translation and replication of genetic information. Only positive-stranded viruses can initiate an infection by means of intact naked viral RNA without the viral capsid. However, this is very difficult and inefficient; in the case of poliovirus only 1 naked positive strand of RNA in 10,000 can initiate an infection (78).
Standard methods for the detection of infectious viruses in water require the use of susceptible cell lines within which the viruses can propagate and produce cytopathic effects (CPE) observable under a light microscope (17). It is important to emphasize that even with cell culture the detection of infectious viruses in environmental samples is difficult. Each virus has different capabilities to propagate in any given cell line. For example, not all enteroviruses can propagate effectively in any one cell line (15); therefore, the use of multiple cell lines is required to detect all the enteroviruses that may be present in a sample (72). In addition, detection of infectious viruses in a sample will greatly depend on the assay conditions, i.e., duration of exposure to host cells, volume of inocula, age of the cells, and the presence of inhibitory or toxic substances.