Managing a PCR Contamination Event in a Molecular Pathology Laboratory

Hirschhorn, Julie; Avery, Alexandria; Schandl, Cynthia A.

doi:10.1007/978-1-0716-2950-5_2

Cited by 6 publications

(2 citation statements)

References 1 publication

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“…This is likely due to sample contamination prior to or during library preparation and is a commonly encountered issue when preparing tiled-amplicon sequencing assays [ 52 ]. Thus, extra precautions should be taken to reduce sample contamination during RNA extraction and PCR setup [ 53 ].…”

Section: Resultsmentioning

confidence: 99%

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

Nash,

Ellmen,

Knapp

et al. 2024

Viruses

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Tomato Brown Rugose Fruit Virus (ToBRFV) is a plant pathogen that infects important Solanaceae crop species and can dramatically reduce tomato crop yields. The ToBRFV has rapidly spread around the globe due to its ability to escape detection by antiviral host genes which confer resistance to other tobamoviruses in tomato plants. The development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detected ToBRFV in municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution of ToBRFV in WWI throughout Ontario, Canada. To aid in global ToBRFV surveillance efforts, we developed a tiled amplicon approach to sequence and track the evolution of ToBRFV genomes in municipal WWI. Our assay recovers 95.7% of the 6393 bp ToBRFV RefSeq genome, omitting the terminal 5′ and 3′ ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recovering ToBRFV genomes. Our ToBRFV assay was developed using existing ARTIC Network resources, including primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance of ToBRFV clades in samples.

show abstract

Section: Resultsmentioning

confidence: 99%

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

Nash,

Ellmen,

Knapp

et al. 2024

Viruses

View full text Add to dashboard Cite

show abstract

“…This is likely due to sample contamination prior to or during library preparation and is a commonly encountered issue when preparing tiled-amplicon sequencing assays (Mackie et al, 2022). Thus, extra precautions should be taken to reduce sample contamination during RNA extraction and PCR set up (Hirschhorn et al, 2023). 523, & (RR-OT) 2.449, critical t-value ±2.776.…”

Section: Taxonomic Profile Of Wwi Samples Prepared By Rna Shotgun And...mentioning

confidence: 99%

A Novel Tiled-Amplicon Sequencing Assay Targeting theTomato Brown Rugose Fruit Virus(ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

Nash,

Ellmen,

Knapp

et al. 2023

Preprint

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Tomato Brown Rugose Fruit Virus(ToBRFV) is a plant pathogen that infects importantSolanaceaecrop species and can dramatically reduce tomato crop yields. TheToBRFVhas rapidly spread around the globe due to its ability to escape detection by antiviral host genes, most notablyTm-22, which are used to confer resistance to otherTobamovirusesin tomato plants. Development of robust and reproducible methods for detecting viruses in the environment aids in the tracking and reduction of pathogen transmission. We detectedToBRFVin municipal wastewater influent (WWI) samples, likely due to its presence in human waste, demonstrating a widespread distribution ofToBRFVin WWI throughout Ontario, Canada. To aid in globalToBRFVsurveillance efforts, we developed a tiled-amplicon approach to sequence and track the evolution ofToBRFVgenomes in municipal WWI. Our assay recovers 97.5% of the 6393 bpToBRFVRefSeq genome, omitting the terminal 5’ and 3’ ends. We demonstrate that our sequencing assay is a robust, sensitive, and highly specific method for recoveringToBRFVgenomes. OurToBRFVassay was developed using existing ARTIC Network resources, which includes genome specific primer design, sequencing library prep, and read analysis. Additionally, we adapted our lineage abundance estimation tool, Alcov, to estimate the abundance ofToBRFVclades in samples.

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The contamination monitoring toolbox: Best practices for molecular microbiology testing

Starolis

2024

Clinical Microbiology Newsletter

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Managing a PCR Contamination Event in a Molecular Pathology Laboratory

Cited by 6 publications

References 1 publication

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

A Novel Tiled Amplicon Sequencing Assay Targeting the Tomato Brown Rugose Fruit Virus (ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

A Novel Tiled-Amplicon Sequencing Assay Targeting theTomato Brown Rugose Fruit Virus(ToBRFV) Genome Reveals Widespread Distribution in Municipal Wastewater Treatment Systems in the Province of Ontario, Canada

The contamination monitoring toolbox: Best practices for molecular microbiology testing

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