Use of ethidium monoazide and propidium monoazide to determine viral infectivity upon inactivation by heat, UV- exposure and chlorine

Leifels, Mats; Jurzik, Lars; Wilhelm, Michael; Hamza, Ibrahim Ahmed

doi:10.1016/j.ijheh.2015.02.003

Cited by 86 publications

(87 citation statements)

References 53 publications

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Section: Discussioncontrasting

confidence: 79%

“…In those studies, PMA was less effective (0.14 to 1.38 log 10 reduction) in discriminating between non-infectious and infectious murine norovirus [13,25] than shown by our results using PEMAX TM (~2.0 log 10 reduction). This difference may be explained by the use of a 90 • C virus inactivation temperature in our study, much higher than the 65 • C and 72 • C temperatures used elsewhere [13,25]. As Millard and colleagues reported that complete inactivation of another enteric virus, hepatitis A virus, was only achieved when heated to 90 • C and maintained for 90 s [27], therefore, 90 • C for 3 min was chosen to ensure that complete capsid damage was achieved, with virus capsid would be likely permeable to the PEMAX TM dye [26].…”

Section: Discussioncontrasting

confidence: 79%

“…The photoactivatable dyes PMA and EMA have been used individually for the selective detection of infectious murine norovirus from heat-inactivated samples [8,13,25,26]. In those studies, PMA was less effective (0.14 to 1.38 log 10 reduction) in discriminating between non-infectious and infectious murine norovirus [13,25] than shown by our results using PEMAX TM (~2.0 log 10 reduction).…”

Section: Discussioncontrasting

confidence: 57%

“…Photoactivatable dyes such as propidium monoazide (PMA) and ethidium monoazide (EMA) (Biotium, Inc., Fremont, CA, USA) have been used with RT-qPCR with some success for the selective detection of infectious norovirus using RT-qPCR [8][9][10][11][12][13]. Theoretically, these dyes penetrate a damaged capsid and, in the presence of light, a covalent bond forms with the nucleic acid, resulting in a reduction in nucleic acid extraction efficiency and in the RT-qPCR signal [14].…”

Section: Introductionmentioning

confidence: 99%

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Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

Gyawali

Hewitt

2018

Water

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Rapid detection of infectious noroviruses from environmental samples is essential to minimize the risk of norovirus outbreaks associated with environmental transmission. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) methods are rapid and sensitive, but cannot differentiate between infectious and non-infectious noroviruses. In this study, a PEMAX TM treatment followed by RT-qPCR (PEMAX TM -RT-qPCR) method was developed for murine norovirus and norovirus GI/GII, and evaluated for the selective detection of infectious viruses following heat inactivation. The norovirus PEMAX TM -RT-qPCR method was then evaluated for the selective detection of infectious viruses from environmental samples. Following heat-treatment (90 • C for 3 min), the murine norovirus PEMAX TM -RT-qPCR showed at least a 2.04 log 10 reduction in detectable virus, compared to a 0.43 log 10 reduction for RT-qPCR alone. Under the same conditions, the norovirus PEMAX TM -RT-qPCR showed a 0.34 to 0.98 log 10 (GI.3) and 0.63 to 2.06 log 10 (GII.4) reduction in detectable viruses, compared to 0.05 to 0.18 log 10 (GI.3) and 0.06 to 0.25 log 10 (GII.4) for RT-qPCR alone. Evaluation of the norovirus PEMAX TM -RT-qPCR on norovirus-contaminated influent and effluent wastewater, and seawater indicated a high proportion of non-infectious norovirus GI and GII (i.e., 56 to 100% in seawater, 32 to 76% in effluent, and 11 to 79% in influent) was present in samples. While potentially overestimating the amount of infectious noroviruses, this approach has potential to provide better information on viral infectivity than RT-qPCR alone.

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Section: Discussioncontrasting

confidence: 79%

Section: Discussioncontrasting

confidence: 79%

Section: Discussioncontrasting

confidence: 57%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

Gyawali

Hewitt

2018

Water

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“…Thus, PMA‐qPCR could be used to discriminate VBNC from antibiotics‐inactivated bacteria cells when antibiotic pressure induces the VBNC state in P. aeruginosa PAO1, which is known to be an opportunistic pathogen‐ and antibiotic‐resistant bacterium. However, PMA‐qPCR based on cell membrane integrity might be incapable of discriminating VBNC cells from other conditions such as UV irradiation (Leifels et al ., 2015; Weigel et al ., 2017) because the cell membrane of the VBNC state might be still intact meaning the cell will be identified as live. This will result in an overestimation of VBNC cells.…”

Section: Discussionmentioning

confidence: 99%

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Lee

Bae

2017

Microbial Biotechnology

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SummaryNucleic acid amplification‐based methods are limited by their inability to discriminate between viable and dead cells. To overcome this drawback, propidium monoazide (PMA) combined with qPCR has been used to differentiate viable from nonviable cells in environmental samples. However, assessing bacterial physiology using PMA‐qPCR remains a challenge due to its incapability of detecting metabolic activities, leading to overestimation of the viable bacteria population under an inactivation condition (e.g. antibiotic treatments). A recent advanced technique to amplify ribosomal RNA precursors (pre‐rRNA) has been shown to detect viable cells because pre‐rRNAs are intermediates in rRNA synthesis. This study investigated the effect of different types of antibiotics on the bacterial viability or viable but non‐culturable (VBNC) state using both PMA‐qPCR and pre‐rRNA analyses with Pseudomonas aeruginosa. This study demonstrated that P. aeruginosa was more sensitive to colistin than it was to carbenicillin, gentamicin and levofloxacin. We could discriminate VBNC P. aeruginosa cells using PMA‐qPCR when antibiotic pressure induced the VBNC state. Also, pre‐rRNA was able to distinguish viable cells from colistin‐inactivated bacteria cells, and it could detect the presence of VBNC and persister cells. Our results showed that these two molecular methods could successfully eliminate false‐positive signals derived from antibiotics‐inactivated cells.

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Progress in the study of virus detection methods: The possibility of alternative methods to validate virus inactivation

2019

Biotech & Bioengineering

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Virus inactivation validation studies have been widely applied in the risk assessment of biogenic material-based medical products, such as biological products, animal tissue-derived biomaterials, and allogeneic biomaterials, to decrease the risk of virus transmission. Traditional virus detection methods in an inactivation validation study utilize cell culture as a tool to quantify the infectious virus by observing cytopathic effects (CPEs) after virus inactivation. However, this is susceptible to subjective factors because CPEs must be observed by experts under a microscope during virus titration. In addition, this method is costly and time-and labor-consuming. Molecular biological technologies such as quantitative polymerase chain reaction (qPCR) have been widely used for virus detection but cannot distinguish infectious and noninfectious viruses. Therefore, qPCR cannot be directly applied to virus inactivation validation studies. In this paper, methods to detect viruses and progress in the challenge of differentiating infectious and noninfectious viruses with the combination of pretreatment and qPCR techniques such as the integrated cell culture-qPCR (ICC-qPCR) method are reviewed. In addition, the advantages and disadvantages of each new method, as well as its prospect in virus inactivation validation studies, are discussed.

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Use of ethidium monoazide and propidium monoazide to determine viral infectivity upon inactivation by heat, UV- exposure and chlorine

Cited by 86 publications

References 53 publications

Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

Detection of Infectious Noroviruses from Wastewater and Seawater Using PEMAXTM Treatment Combined with RT-qPCR

Molecular viability testing of viable but non‐culturable bacteria induced by antibiotic exposure

Progress in the study of virus detection methods: The possibility of alternative methods to validate virus inactivation

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