Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
Primers obtained from gene sequences coding for heat shock proteins (HSP) were used to specifically detect enteric protozoans of the genus Giardia. The HSP primers amplified Giardia DNA or the corresponding RNA sequences obtained from lysed cysts and gave a 163-bp product. Since the presence of the product did not indicate whether the cysts were viable, these amplifications are a presence/absence test only. In contrast, amplification of heat shock-induced mRNA utilizing the same HSP primers was indicative of viable Giardia cysts. The limit of sensitivity of the presence/absence test was 1 cyst, whereas for the viability test it was 10 cysts. Thus, viable Giardia cysts can be rapidly and specifically detected with great sensitivity through the use of PCR amplifications.
Information has not been previously available on the occurrence of enteric pathogenic viruses and protozoan parasites in composted municipal domestic solid waste. A potential source of these pathogens in domestic solid waste is disposal diapers. The occurrence of enteroviruses, Giardia cysts, Cryptosporidium oocysts and Salmonella were determined in municipal composted domestic solid waste, and solid waste to which extra diapers had been added to increase their concentration 2-4 fold (6.6-7.7% by weight) above that found normally in municipal solid waste before composting. The compost was tested at various periods of time during aging (101-203 days). No enteroviruses or protozoan cysts or oocysts were detected in any of the samples collected during this period. One sample out of 19 collected (after 175 days aging) was positive for Salmonella. These results suggest that enteric pathogens were destroyed during the composting process, or were present in numbers below the detection method (i.e. one organism per 40-50 g of compost) used in this study.
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