Quantification and discovery of PCR inhibitors found in food matrices commonly associated with foodborne viruses

Suther, Cassandra; Moore, Matthew D.

doi:10.1016/j.fshw.2019.09.002

Cited by 21 publications

(18 citation statements)

References 22 publications

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“…In animal species including humans, diagnostic methods, such as nucleic acid testing, allow for the direct detection of the microorganism causing the infection, typically after an amplification step using, preferentially but not exclusively, the polymerase chain reaction (PCR), whilst indirect methods, such as serological assays, allow for the detection of antibodies against microorganism antigens. Pathogen detection in plants that lack an immune system do rely on the proper detection of the pathogen, but with a couple of notable exceptions: the amplification of specific targets through PCR is often proven difficult due to the presence of inhibitors in plant cells ( Dorn et al, 1999 ; Schrader et al, 2012 ; Rački et al, 2014 ; Lacroix et al, 2016 ; Lardeux et al, 2016 ; Suther and Moore, 2019 ), whereas, the role of the detected pathogen component is rather obscure at least from the etiological point of view. In the case of viral plant pathogens, for instance, the capsid protein subunits or the virus particles containing the viral genome may be detected simultaneously ( Manoussopoulos and Tsagris, 2015 ), indicating the establishment of the virus in its host.…”

Section: Discussionmentioning

confidence: 99%

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples

et al. 2021

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Serological methods serve as a direct or indirect means of pathogen infection diagnosis in plant and animal species, including humans. Dot-ELISA (DE) is an inexpensive and sensitive, solid-state version of the microplate enzyme-linked immunosorbent assay, with a broad range of applications in epidemiology. Yet, its applicability is limited by uncertainties in the qualitative output of the assay due to overlapping dot colorations of positive and negative samples, stemming mainly from the inherent color discrimination thresholds of the human eye. Here, we report a novel approach for unambiguous DE output evaluation by applying machine learning-based pattern recognition of image pixels of the blot using an impartial predictive model rather than human judgment. Supervised machine learning was used to train a classifier algorithm through a built multivariate logistic regression model based on the RGB (“Red,” “Green,” “Blue”) pixel attributes of a scanned DE output of samples of known infection status to a model pathogen (Lettuce big-vein associated virus). Based on the trained and cross-validated algorithm, pixel probabilities of unknown samples could be predicted in scanned DE output images, which would then be reconstituted by pixels having probabilities above a cutoff. The cutoff may be selected at will to yield desirable false positive and false negative rates depending on the question at hand, thus allowing for proper dot classification of positive and negative samples and, hence, accurate diagnosis. Potential improvements and diagnostic applications of the proposed versatile method that translates unique pathogen antigens to the universal basic color language are discussed.

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Section: Discussionmentioning

confidence: 99%

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples

et al. 2021

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“… Reque et al (2014) also showed an increase in the activity of anthocyanins in frozen blueberries after 3 months of storage followed by a decrease during the subsequent 3 months. Lastly, the presence of pectin found in blueberries may also inhibit RT-qPCR ( Suther and Moore, 2019 ). These findings are consistent with the significant variations seen in our detections of HAV RNA at 60 and 90 days of storage.…”

Section: Discussionmentioning

confidence: 99%

Persistence of Hepatitis A Virus RNA in Water, on Non-porous Surfaces, and on Blueberries

2021

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Enteric viruses, such as human norovirus and hepatitis A virus (HAV), are the leading cause of transmissible foodborne illness. Fresh produce such as berries are often contaminated by infected food handlers, soiled water, or food contact surfaces. The gold-standard method for virus detection throughout the food chain is RT-qPCR, which detects portions of genomes including non-infectious viral particles and naked viral RNA. The aim of this study was to evaluate the persistence of heat-inactivated HAV in water, phosphate-buffered saline, on stainless steel and polyvinyl chloride, and on blueberries at −80°C, −20°C, 4°C, and room temperature. In water and phosphate-buffered saline, viral RNA could be detected for up to 90 days regardless of temperature when the initial load was 2.5 × 104 or 2.5 × 106 genome copies. It was detected on polyvinyl chloride and blueberries under most conditions. On stainless steel, the large initial load persisted for 90 days, while the medium-level load was detected only up to 16 days at room temperature or 60 days at 4°C. The detection of non-infectious viral RNA can confound investigations of gastroenteritis outbreaks. Pretreatments that discriminate between naked RNA, non-infectious virions and infectious virions need to be included in the RT-qPCR method in order to reduce the risk of positive results associated with non-infectious viral particles.

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“…Despite its advantages, saliva, like other biological samples, is prone to matrix-specific factors that influence diagnostics including intrinsic degrading enzymes, changes in salivary flow over time (e.g., circadian rhythms), or oral microbiome composition [8][9][10]12]. Moreover, various PCR inhibitors have been described in milk, vegetables, and foods high in protein and fat which interfere with robust diagnostic detection of pathogen nucleic acids [8,9]. Together with unique dietary habits of children, these factors pose challenges to molecular microbiology diagnostics and are essential to consider as we learn to exploit saliva for capturing infections with upper respiratory pathogens.…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…We [2] and others [3][4][5][6] have demonstrated the diagnostic utility of saliva for SARS-CoV-2 testing, and several have demonstrated comparable sensitivities for SARS-CoV-2 detection in saliva versus NP specimens [5,7]; but few have assessed performance in children [4,5] whose saliva can be impacted by dietary and oral hygiene behaviors distinct from adults. With routine self-collection, robust detection of viruses are vulnerable to inhibitors found in saliva secondary to foods, dental care, or native salivary environment [8][9][10].…”

Section: Introductionmentioning

confidence: 99%

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Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection

Hernandez

Riollano‐Cruz

Boyle

et al. 2021

Preprint

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BackgroundSaliva is an optimal specimen for detection of viruses that cause upper respiratory infections including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to its cost-effectiveness and non-invasive collection. However, together with intrinsic enzymes and oral microbiota, children’s unique dietary habits may introduce substances that interfere with diagnostic testing.MethodsTo determine whether children’s dietary choices impact SARS-CoV-2 detection in saliva, we performed a diagnostic study that simulates testing of real-life specimens provided from healthy children (n=5) who self-collected saliva at home before and at 0, 20, and 60 minutes after eating from 20 foods they selected. Each of seventy-two specimens was split into two volumes and spiked with SARS-CoV-2-negative or -positive standards prior to side-by-side testing by reverse-transcription polymerase chain reaction matrix-assisted laser desorption ionization time-of-flight (RT-PCR/MALDI-TOF) assay.ResultsDetection of internal extraction control and SARS-CoV-2 nucleic acids was reduced in replicates of saliva collected at 0 minutes after eating 11 of 20 foods. Interference resolved at 20 and 60 minutes after eating all foods except hot dog in one participant. This represented a significant improvement in detection of nucleic acids compared to saliva collected at 0 minutes after eating (P=0.0005).ConclusionsWe demonstrate successful detection of viral nucleic acids in saliva self-collected by children before and after eating a variety of foods. Fasting is not required before saliva collection for SARS-CoV-2 testing by RT-PCR/MALDI-TOF, but waiting 20 minutes after eating is sufficient for accurate testing. These findings should be considered for SARS-CoV-2 testing and broader viral diagnostics in saliva specimens.

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Quantification and discovery of PCR inhibitors found in food matrices commonly associated with foodborne viruses

Cited by 21 publications

References 22 publications

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples

Pixel-Based Machine Learning and Image Reconstitution for Dot-ELISA Pathogen Diagnosis in Biological Samples

Persistence of Hepatitis A Virus RNA in Water, on Non-porous Surfaces, and on Blueberries

Food for thought: Eating before saliva collection and interference with SARS-CoV-2 detection

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