A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples

Coudray-Meunier, Coralie; Fraisse, Audrey; Martin‐Latil, Sandra; Guillier, Laurent; Delannoy, Sabine; Fach, Patrick; Perelle, Sylvie

doi:10.1016/j.ijfoodmicro.2015.02.006

Cited by 89 publications

(53 citation statements)

References 36 publications

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“…Photoactivatable intercalating dyes have begun to show promise in being able to selectively detect infectious HAV (Sanchez et al, 2012; Coudray-Meunier et al, 2015; Moreno et al, 2015; Fuster et al, 2016; Randazzo et al, 2018b) and human NoV (Parshionikar et al, 2010; Randazzo et al, 2016, 2018a; Jeong et al, 2017). Recently, Fraisse et al (2018) proposed PtCl 4 as a successful viability marker for human NoV.…”

Section: Resultsmentioning

confidence: 99%

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

et al. 2018

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The hepatitis E virus (HEV) is an emerging pathogen showing a considerable increase in the number of reported cases in Europe mainly related to the ingestion of contaminated food. As with other relevant viral foodborne pathogens, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) is the gold standard for HEV detection in clinical, food, and environmental samples, but these procedures cannot discriminate between inactivated and potentially infectious viruses. Thus, the aim of this study was to develop a viability PCR method to discriminate between native, heat-, and high-pressure processing (HPP)-treated HEV using the hepatitis A virus (HAV) as a cultivable surrogate. To this end, different concentrations of viability markers (PMAxx and platinum chloride, PtCl4) were screened firstly on purified viral RNA using different RT-qPCR assays. Reductions of HEV RNA signals of >17.5, >15.0, and >15.5 quantification cycles (Cq) were reported for PtCl4 and 1.6, 2.9, and 8.4 Cq for PMAxx, clearly indicating a better performance of PtCl4 than PMAxx irrespective of the RT-qPCR assay used. The most efficient viability pretreatment (500 μM PtCl4 incubated at 5°C for 30 min) was then assessed on native, heat-, and HPP-treated HEV suspension. The optimized viability RT-qPCR discriminated successfully between native, heat-, and HPP-treated HEV, to different extents depending on the experimental conditions. In particular, approximately 2-log10 reduction was reported by PtCl4-RT-qPCR at both 72 and 95°C compared to the control. Additionally, both viability pretreatments were tested for HPP-treated HAV without success, while PtCl4-RT-qPCR completely eliminated (>5.6-log10 reduction) the RT-qPCR signals of HPP-treated HEV. Although this viability procedure may still overestimate infectivity, the PtCl4 pretreatment represents progress to better interpreting the quantification of intact HEV, and it could be included in molecular procedures used to quantify enteric viruses in food and environmental samples.

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Section: Resultsmentioning

confidence: 99%

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

et al. 2018

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“…dPCR could be used on undiluted samples, whereas qPCR could only be performed on diluted stool samples because of the high concentrations of PCRinterfering substances that affect the sensitivity of the qPCR reaction. Similarly, better tolerability of reverse transcription (RT)-dPCR to PCR inhibition was shown to detect viral hepatitis A and norovirus RNA in lettuce and bottled water [24]. The higher tolerance to PCR inhibition may also allow direct quantification of viruses from samples without prior extraction, as shown by Pavšič et al on CMV viruses, yielding a higher concentration of detectable DNA copies compared with extracted DNA [21].…”

Section: Pcr Inhibitory Substancesmentioning

confidence: 95%

“…Alternatively, dPCR has been frequently explored for testing low-levels of b Due to the presence of a large amount of rain (grey), the distinction between negative (red) and positive partitions (green) is not clear, and setting an inappropriate threshold may result in a large number of false positives or false negatives. dPCR digital polymerase chain reaction viral contaminants in environmental samples [22][23][24]. These studies indicate that dPCR may indeed form a promising tool in standard diagnostic virology; however, extensive validation of this platform will still be required in order to move to a clinical setting.…”

Section: Applications Of Digital Polymerase Chain Reaction (Dpcr) In mentioning

confidence: 99%

The Future of Digital Polymerase Chain Reaction in Virology

et al. 2016

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Driven by its potential benefits over currently available methods, and the recent development of commercial platforms, digital polymerase chain reaction (dPCR) has received increasing attention in virology research and diagnostics as a tool for the quantification of nucleic acids. The current technologies are more precise and accurate, but may not be much more sensitive, compared with quantitative PCR (qPCR) applications. The most promising applications with the current technology are the analysis of mutated sequences, such as emerging drug-resistant mutations. Guided by the recent literature, this review focuses on three aspects that demonstrate the potential of dPCR for virology researchers and clinicians: the applications of dPCR within both virology research and clinical virology, the benefits of the technique over the currently used real-time qPCR, and the importance and availability of specific data analysis approaches for dPCR. Comments are provided on current drawbacks and often overlooked pitfalls that need further attention to allow widespread implementation of dPCR as an accurate and precise tool within the field of virology.

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“…This improvement is achieved by partitioning sample amplification reactions into tens to thousands of picoliter-scale compartments on microfluidic chips or microdroplets so that each mini-reaction contains zero or one copy of the target nucleic acid molecule. A comparative study of digital RT-PCR (RT-dPCR) and RT-qPCR for the detection and quantification of HAV and NoVs in lettuce and water samples was recently reported [86]. This RT-dPCR assay showed that the sensitivity was either comparable or slightly (around 1 log 10 ) decreased to that of RT-qPCR for detecting viral RNA and cDNA of HAV and NoV, but the viral recoveries were found to be significantly higher than that of RTqPCR for NoV GI and HAV in water, and for NoV GII and HAV in lettuce.…”

Section: Digital Pcrmentioning

confidence: 99%

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

Chen¹

2016

Nucleic Acids - From Basic Aspects to Laboratory Tools

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Human noroviruses (hNoVs) and hepatitis A virus (HAV) are the leading cause of foodborne viral illness, and they exact a considerable economic and health toll worldwide. The detection of viral contamination in foods requires highly sensitive and accurate methods, due to the intrinsically low amounts of viruses and the complexity of the sample matrices. In recent years, a wide variety of nucleic acid-based molecular methods have been developed for the detection of hNoVs and HAV, displaying superior sensitivity, specificity, and speed. This chapter aims to provide a summary of the application of the molecular methods for the detection of the two important foodborne viruses.

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A comparative study of digital RT-PCR and RT-qPCR for quantification of Hepatitis A virus and Norovirus in lettuce and water samples

Cited by 89 publications

References 36 publications

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

Viability RT-qPCR to Distinguish Between HEV and HAV With Intact and Altered Capsids

The Future of Digital Polymerase Chain Reaction in Virology

Nucleic Acid Detection of Major Foodborne Viral Pathogens Human: Noroviruses and Hepatitis A Virus

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