Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation

Ho, Johannes; Seidel, Michael; Nießner, Reinhard; Eggers, Jutta; Tiehm, Andreas

doi:10.1016/j.watres.2016.07.032

Cited by 41 publications

(18 citation statements)

References 43 publications

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“…Also, the qPCR method is based on the amplification of DNA fragments of 100−200 bp, which contains less UV-reactive sites. 60,61 Thus, the qPCR results of the shorter opr gene may not truly reflect the damage to iDNA of P. aeruginosa induced by UV irradiation, further resulting in an underestimation of the inactivation of P. aeruginosa by UV. 62 Short opr gene fragments decreased by 18 and 30%, respectively, after 30 min chlorine and UV/chlorine treatments, whereas long opr gene fragments decreased by 22 and 72%, respectively.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…Unexpectedly, the numbers of copies of the opr gene after UV irradiation by qPCR and PCR did not decrease significantly, which was consistent with previous studies. ,, This is likely due to the percentage of adjacent thymine (TT) in total adjacent pyrimidines (TT + CC + TC + CT) being as low as 12.4% (Table S4), which was relatively low and resulted in less formation of thymine dimers. Also, the qPCR method is based on the amplification of DNA fragments of 100–200 bp, which contains less UV-reactive sites. , Thus, the qPCR results of the shorter opr gene may not truly reflect the damage to iDNA of P. aeruginosa induced by UV irradiation, further resulting in an underestimation of the inactivation of P.…”

Section: Results and Discussionmentioning

confidence: 99%

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Assessment of the UV/Chlorine Process in the Disinfection of Pseudomonas aeruginosa: Efficiency and Mechanism

Wang

Guo

et al. 2021

Environ. Sci. Technol.

142

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UV irradiation and chlorination have been widely used for water disinfection. However, there are some limitations, such as the risk of generating viable but nonculturable bacteria and bacteria reactivation when using UV irradiation or chlorination alone. This study comprehensively evaluated the feasibility of the UV/chlorine process in drinking water disinfection, and Pseudomonas aeruginosa was selected as the target microorganism. The number of culturable cells was effectively reduced by more than 5 orders of magnitude (5-log 10 ) after UV, chlorine, and UV/chlorine treatments. However, intact and VBNC cells were detected at 10 3 to 10 4 cells/mL after UV and chlorine treatments, whereas they were undetectable after UV/chlorine treatment due to the primary contribution of reactive chlorine species (Cl • , Cl 2•− , and ClO • ). After UV/chlorine treatment, the metabolic activity determined using single cell Raman spectroscopy was much lower than that after UV. The level of toxic opr gene in P. aeruginosa decreased by more than 99% after UV/chlorine treatment. Importantly, bacterial dark reactivation was completely suppressed by UV/chlorine treatment but not UV or chlorination. This study suggests that the UV/chlorine treatment can completely damage bacteria and is promising for pathogen inactivation to overcome the limitations of UV and chlorine treatments alone.

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Section: ■ Results and Discussionmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Assessment of the UV/Chlorine Process in the Disinfection of Pseudomonas aeruginosa: Efficiency and Mechanism

Wang

Guo

et al. 2021

Environ. Sci. Technol.

142

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“…In recent decades, PCR-based methods have also been applied for measuring genome integrity after disinfection (24). While several of these studies have drawn correlations between, for example, reductions of infectious virus and qPCR detection of the whole genome (18) or genome segments (25)(26)(27), this success depends on several factors. First, different disinfectants vary in the type and severity of damage caused to the genome (e.g., RNA structural modification, phosphate backbone scission, etc.…”

Section: Discussionmentioning

confidence: 99%

Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes

Torrey

Gunten

Kohn

2019

Appl Environ Microbiol

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Virus inactivation mechanisms can be elucidated by methods that measure the loss of specific virus functionality (e.g., host attachment, genome internalization, and genome replication). Genome functionality is frequently assessed by PCRbased methods, which are indirect and potentially inaccurate; genome damage that affects detection by high-fidelity PCR enzymes may not adversely affect the ability of actual cellular enzymes to produce functional virus. Therefore, we developed here a transfection-based assay to quantitatively determine viral genome functionality by inserting viral RNA into host cells directly to measure their ability to produce new functional viruses from damaged viral genomes. Echovirus 11 was treated with ozone, free chlorine (FC), UV light at 254 nm (UV 254 ), or heat, and then the reductions in genome functionality and infectivity were compared. Ozone reduced genome functionality proportionally to infectivity, indicating that genome damage is the main mechanism of virus inactivation. In contrast, FC caused little or no loss of genome functionality compared to infectivity, indicating a larger role for protein damage. For UV 254 , genome functionality loss accounted for approximately 60% of virus inactivation, with the remainder presumably due to protein damage. Heat treatment resulted in no reduction in genome functionality, in agreement with the understanding that heat inactivation results from capsid damage. Our results indicate that there is a fundamental difference between genome integrity reductions measured by PCR enzymes in previous studies and actual genome functionality (whether the genome can produce virus) after disinfection. Compared to PCR, quantitative transfection assays provide a more realistic picture of actual viral genome functionality and overall inactivation mechanisms during disinfection. IMPORTANCE This study provides a new tool for assessing virus inactivation mechanisms by directly measuring a viral genome's ability to produce new viruses after disinfection. In addition, we identify a potential pitfall of PCR for determining virus genome damage, which does not reflect whether a genome is truly functional. The results presented here using quantitative transfection corroborate previously suggested virus inactivation mechanisms for some virus inactivation methods (heat) while bringing additional insights for others (ozone, FC, and UV 254 ). The developed transfection method provides a more mechanistic approach for the assessment of actual virus inactivation by common water disinfectants.W aterborne infectious viruses (IVs) (e.g., norovirus, hepatitis A virus, rotavirus, etc.) have a great impact on human health, causing a range of diseases from acute self-limiting diarrheal/vomiting episodes to chronic hepatic infections with long-lasting

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“…Novel approaches like modifying the targeted gene sequence or the length of the PCR product, or amplifying less stable messenger RNA after reverse transcription to DNA (e.g. (Ho et al, 2016; Ko et al, 2003; Polston et al, 2014; Wu et al, 2019)) generally lack robustness and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – a systematic review

Leifels¹,

Cheng²,

Sozzi

et al. 2020

Preprint

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20Capsid-integrity quantitative PCR (qPCR), a molecular detection method for infectious viruses 21 combining azo-dye pretreatment with qPCR, has been widely used in recent years; however, 22 variations in pretreatment conditions for various virus types can limit the efficacy of specific 23 protocols. By identifying and critically synthesizing forty-two recent peer-reviewed studies 24 employing capsid-integrity qPCR for viruses in the last decade (2009 to 2019) in the fields of food 25 safety and environmental virology, we aimed to establish recommendations for the detection of 26 infectious viruses. Intercalating dyes are effective measures of viability in PCR assays provided 27 the viral capsid is damaged; viruses that have been inactivated by other causes, such as loss of 28 attachment or genomic damage, are less well detected using this approach. Although optimizing 29 specific protocols for each virus is recommended, we identify a framework for general assay 30 conditions. These include concentrations of ethidium monoazide, propidium monoazide or its 31 derivates between 10 and 200 µM; incubation on ice or at room temperature (20 -25ºC) for 5 to 32 120 min; and dye activation using LED or high light (500 -800 Watts) exposure for periods 33 ranging from 5 to 20 min. These simple steps can benefit the investigation of infectious virus 34 transmission in routine (water) monitoring settings and during viral outbreaks such as the current 35 COVID-19 pandemic or endemic diseases like dengue fever. 36 37 Graphical abstract 38 39 Keywords: (6) azo dye, EMA, PMA, microbial contamination, viability, water quality 40 41

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Long amplicon (LA)-qPCR for the discrimination of infectious and noninfectious phix174 bacteriophages after UV inactivation

Cited by 41 publications

References 43 publications

Assessment of the UV/Chlorine Process in the Disinfection of Pseudomonas aeruginosa: Efficiency and Mechanism

Assessment of the UV/Chlorine Process in the Disinfection of Pseudomonas aeruginosa: Efficiency and Mechanism

Differences in Viral Disinfection Mechanisms as Revealed by Quantitative Transfection of Echovirus 11 Genomes

Capsid integrity quantitative PCR to determine virus infectivity in environmental and food applications – a systematic review

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