A multiplex reverse transcription-PCR method for detection of human enteric viruses in groundwater

Abstract

Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for 1 year for enteroviruses, hepatitis A virus, Norwalk virus, reoviruses, and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure with which to remove environmental RT-PCR inhibitors from groundwater samples was developed. The procedure allowed an average of 71 liters of the original groundwater to be assayed per RT-PCR, with an average virus recovery rate of 74%, based on seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed, with reoviruses being the most frequently detected virus group.

FIG. 1.

Inhibitor removal from groundwater samples. Single-enterovirus primer RT-PCRs were performed on seven solvent-treated groundwater samples. Unseeded samples (lanes 3 to 8) or samples seeded with 500 particles of poliovirus (lanes 10 to 15) were analyzed. PBS and virus-seeded PBS were run as a negative (lane 2) and positive controls (lane 9). Products were analyzed on a 1.5% agarose gel. Lane 1 contains a 123-bp ladder. The arrow indicates the location of the 196-bp poliovirus RT-PCR fragment.

FIG. 2.

Multiplex RT-PCR. Standard multiplex RT-PCR mixture A (lanes 2 to 4) and B (lanes 5 to 7) reactions were performed and analyzed on a 3% NuSieve agarose gel. Mixture A viruses (poliovirus, reovirus, and rotavirus) were added to the reaction mixture shown in lane 3. A 10-fold dilution of mixture A was added to the reaction mixture shown in lane 4. Mixture B viruses (HAV and Norwalk virus) were added to the reaction mixture shown in lane 6 (RT-PCR mixture B contains two primer sets for HAV, generating two unique PCR fragments). A 10-fold dilution of mixture B was added to the reaction mixture shown in lane 7. Lanes 2 and 5 contained mixture A and B negative control samples, respectively. Lane 1 contains a 123-bp ladder.