Development of a rapid, sensitive, and quantitative method to detect infective hepatitis, a virus in water
Microorganisms are responsible for more than 90% of the reported waterborne disease outbreaks in the United States; enteric viruses, such as hepatitis A virus, are identified as causing almost 10% of these. However, in 50% of the outbreaks, no causative agent is identified due to limitations in our ability to isolate and detect viruses in water samples. Historically, consumption of contaminated ground water has been the source of one-half of the reported outbreaks; in recent years, that fraction has risen to more than two-thirds. The most frequently reported source of contamination in these outbreaks is domestic sewage from septic tanks, leaking sewer lines, cesspools, etc.
As a result of the continuing waterborne disease outbreaks, and the growing fraction of them associated with consumption of ground water, the USEPA is finalizing a regulation, the Ground Water Rule, to minimize the risk of acquiring a microbial illness from ground water. This regulation will require all public water systems that use ground water as a source to assess the potential for fecal contamination of the water. One potential source of fecal contamination is the reuse of recycled water. Even tertiary treated wastewater may contain contaminants, including disease-causing microorganisms such as viruses, bacteria and parasites. Information about the numbers and types of some of these microorganisms, especially viruses, present in tertiary treated wastewater is scarce due to the limitations in the methods that are needed to detect these microorganisms in water. Current methods to detect viruses in water samples, for example, require highly specialized analytical facilities and at least two to four weeks to obtain results. In addition, the standard methods only detect one group of viruses, the enteroviruses (which includes polioviruses). From records on waterborne disease outbreaks in the U.S., it is known that the enteroviruses are not the major cause of reported viral waterborne disease; other viruses such as hepatitis A virus and caliciviruses are the public health concern. The lack of information about the presence of hepatitis A virus in water and its long survival times in water caused the USEPA to add it to the list of contaminants to be considered for regulation in drinking water.
When recycled water is used to artificially recharge groundwater, there is the potential for some microorganisms, especially viruses due to their small size, to contaminate the underlying groundwater. Although most of the microorganisms may be removed during recharge, if even a few are transported to the ground water, that is of concern, as the USEPA has determined that one virus in 10,000 liters of drinking water constitutes a public health concern.
Molecular beacons (MB) are oligonucleotide probes that become fluorescent upon hybridization and are ideal in providing real-time monitoring of target amplicons during the PCR reaction. In this project, a real-time PCR assay based on molecular beacons was developed for the rapid and specific detection of hepatitis A virus. Using the MB-based RT-PCR assay, a detection limit of 1 PFU per PCR reaction was obtained. The specificity of the MB-based PCR assay was evaluated using a variety of other enteric organisms, and only hepatitis A virus was positively identified. The method developed in this study should improve our ability to provide rapid, sensitive, and specific results for the detection and quantitation of viruses in samples from environmental waters.