Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples

Abstract

Human enteric viruses can be present in untreated and inadequately treated drinking water. Molecular methods, such as the reverse transcriptase PCR (RT-PCR), can detect viral genomes in a few hours, but they cannot distinguish between infectious and noninfectious viruses. Since only infectious viruses are a public health concern, methods that not only are rapid but also provide information on the infectivity of viruses are of interest. The intercalating dye propidium monoazide (PMA) has been used for distinguishing between viable and nonviable bacteria with DNA genomes, but it has not been used to distinguish between infectious and noninfectious enteric viruses with RNA genomes. In this study, PMA in conjunction with RT-PCR (PMA-RT-PCR) was used to determine the infectivity of enteric RNA viruses in water. Coxsackievirus, poliovirus, echovirus, and Norwalk virus were rendered noninfectious or inactivated by treatment with heat (72°C, 37°C, and 19°C) or hypochlorite. Infectious or native and noninfectious or inactivated viruses were treated with PMA. This was followed by RNA extraction and RT-PCR or quantitative RT-PCR (qRT-PCR) analysis. The PMA-RT-PCR results indicated that PMA treatment did not interfere with detection of infectious or native viruses but prevented detection of noninfectious or inactivated viruses that were rendered noninfectious or inactivatedby treatment at 72°C and 37°C and by hypochlorite treatment. However, PMA-RT-PCR was unable to prevent detection of enteroviruses that were rendered noninfectious by treatment at 19°C. After PMA treatment poliovirus that was rendered noninfectious by treatment at 37°C was undetectable by qRT-PCR, but PMA treatment did not affect detection of Norwalk virus. PMA-RT-PCR was also shown to be effective for detecting infectious poliovirus in the presence of noninfectious virus and in an environmental matrix. We concluded that PMA can be used to differentiate between potentially infectious and noninfectious viruses under the conditions defined above.

Optimization of Propidium Monoazide Quantitative PCR for Evaluating Performances of Bioaerosol Samplers for Sampling AirborneStaphylococcus aureus

“…The optimal light exposure time for the PMAqPCR assay varied from those reported in previously published studies. In general, 2-10 min of light exposure has typically been used to detect viable bacteria or viruses (Nocker et al 2006(Nocker et al , 2007Kobayashi et al 2009b;Parshionikar et al 2010;Liu and Mustapha 2014). Therefore, the optimal light exposure time for DNA amplification may vary and depend on the light source and bacterial species used (Liu and Mustapha 2014).…”

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

“…For culturable viruses this problem can be overcome using PCR in combination with culture 26,27 . The problem has also been addressed for some viruses through use of nucleic acid cross-linking agents [28][29][30] . This latter approach is more effective for viruses inactivated by hypochlorite and not effective for those inactivated by UV.…”

Amoeba-Related Health Risk in Drinking Water Systems: Could Monitoring of Amoebae Be a Complementary Approach to Current Quality Control Strategies?

“…PMA is a DNA-intercalating dye with the capacity to only penetrate membrane-compromised cells, blocking the amplification cycle. With this approach, cell viability is based on membrane integrity [52][53][54][55][56][57][58][59].…”