Immunoaffinity concentration and purification of waterborne enteric viruses for detection by reverse transcriptase PCR

Abstract

To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with 1MDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, ProCipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 l. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by the AbCap method; 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results for enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.

Detection of Viral Pathogens by Reverse Transcriptase PCR and of Microbial Indicators by Standard Methods in the Canals of the Florida Keys

“…The published sensitivity levels of the primer sets used in this study demonstrated that the detection limits vary. The sensitivities of the panenterovirus and hepatitis A virus primer sets varied from 10 3 to 0.01 poliovirus and hepatitis A virus PFU as determined with various virus recovery techniques (30,32). The Norwalk primer required at least 10 5 amplifiable units (approximate number of virions) before amplicon was detected (19).…”

PCR detection of pathogenic viruses in southern California urban rivers

“…Although primers and probes used in this study have been previously tested by several investigators (i.e. Tsai et al 1993;Schwab et al 1996;Griffin et al 1999) and periodically re-checked by submitting sequences to GenBank (NCBI) for matching with the most up-to-date sequence database to verify the specificity to human viruses, it is still difficult to completely rule out the possibility of amplifying nonhuman viral sequences because the vast majority of organisms in the environment have not been sequenced. For example, the primers used for detection of enteroviruses targeting at 5¢-nontranslated region that is highly conserved across enteroviruses including animal enteroviruses.…”

Diagnosis of Noncultivatable Gastroenteritis Viruses, the Human Caliciviruses

“…Antibody capture has been used for virus purification prior to amplification (74,219). Virus-specific antibodies are bound in a 96-well plate or to paramagnetic beads before exposure to a virus-containing sample.…”

“…The principal reason this method has not been explored further is the lack of high-titered antisera that react with a broad range of NLVs. Nevertheless, Schwab et al (219) were able to use human immunoglobulin preparations as a source of antibody to detect NLVs from water samples in which viruses had been concentrated by filtration and PEG precipitation. Polyclonal hyperimmune animal sera have also been coupled to paramagnetic beads and used to purify NV from fecal specimens prior to RT-PCR amplification (74).…”