Contamination-pipetting: relative efficiency of filter tips compared to Microman® positive displacement pipette

Rouzic, Elise Le

doi:10.1038/nmeth887

Cited by 19 publications

(9 citation statements)

References 2 publications

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“…The total mass of the simulated carryover contamination for each reaction was approximately equivalent to a 0.124 μm-diameter aerosol droplet, which cannot be efficiently blocked by either high efficiency particulate air (HEPA) filters48 in the biosafety cabinets or fibrous pipette tip filters49. The detection limits of the rRT-LAMP reactions with and without UDG treatment before amplification were compared to confirm whether the studied UDG-rRT-LAMP assay can eliminate false positives.…”

Section: Methodsmentioning

confidence: 99%

“…To evaluate the ability of the developed UDG-rRT-LAMP method to reduce false-positive results due to carryover contaminants in detecting TMUV, we conducted both rRT-LAMP and UDG-rRT-LAMP reactions by adding 2 μL of diluted viral RNA templates (0.5 × 10 −1 − 0.5 × 10 4 copies/μL) and 2 μL of simulated carryover contamination of 0.5 × 10 −18 g/μL in the same reaction tube. The total mass of the simulated carryover contamination for each reaction was approximately equivalent to a 0.124 μm-diameter aerosol droplet, which cannot be efficiently blocked by either high efficiency particulate air (HEPA) filters 48 in the biosafety cabinets or fibrous pipette tip filters 49 . The detection limits of the rRT-LAMP reactions with and without UDG treatment before amplification were compared to confirm whether the studied UDG-rRT-LAMP assay can eliminate false positives.…”

Section: Methodsmentioning

confidence: 99%

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Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Tang

Chen

Diao

2016

Sci Rep

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Tembusu virus (TMUV) is a mosquito-borne flavivirus which threatens both poultry production and public health. In this study we developed a complete open reading frame alignment-based rRT-LAMP method for the universal detection of TUMV. To prevent false-positive results, the reaction was supplemented with uracil DNA glycosylase (UDG) to eliminate carryover contamination. The detection limit of the newly developed UDG-rRT-LAMP for TMUV was as low as 100 copies/reaction of viral RNA and 1 × 100.89 − 1 × 101.55 tissue culture infectious dose/100 μL of viruses. There were no cross-reactions with other viruses, and the reproducibility of the assay was confirmed by intra- and inter-assay tests with variability ranging from 0.22–3.33%. The new UDG-rRT-LAMP method for TMUV produced the same results as viral isolation combined with RT-PCR as the “gold standard” in 96.88% of cases for 81 clinical samples from subjects with suspected TMUV infection. The addition of UDG can eliminate as much as 1 × 10−16 g/reaction of contaminants, which can significantly reduce the likelihood of false-positive results during the rRT-LAMP reaction. Our result indicated that our UDG-rRT-LAMP is a rapid, sensitive, specific, and reliable method that can effectively prevent carryover contamination in the detection of TMUV.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Tang

Chen

Diao

2016

Sci Rep

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“…Second, improper handling during the DNA extraction or the DNA dispersal to PCR plates can occur ( Ballenghien et al, 2017 ). Thus, DNA from actual samples may be aerosolized ( Le Rouzic, 2006 ) and cross-contaminate adjacent wells, including low-biomass control blanks. For the CoDL dataset, testing for aerosolization in control samples is difficult, as most studies did not include controls for sequencing.…”

Section: Resultsmentioning

confidence: 99%

Identification and Removal of Contaminant Sequences From Ribosomal Gene Databases: Lessons From the Census of Deep Life

et al. 2018

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Earth’s subsurface environment is one of the largest, yet least studied, biomes on Earth, and many questions remain regarding what microorganisms are indigenous to the subsurface. Through the activity of the Census of Deep Life (CoDL) and the Deep Carbon Observatory, an open access 16S ribosomal RNA gene sequence database from diverse subsurface environments has been compiled. However, due to low quantities of biomass in the deep subsurface, the potential for incorporation of contaminants from reagents used during sample collection, processing, and/or sequencing is high. Thus, to understand the ecology of subsurface microorganisms (i.e., the distribution, richness, or survival), it is necessary to minimize, identify, and remove contaminant sequences that will skew the relative abundances of all taxa in the sample. In this meta-analysis, we identify putative contaminants associated with the CoDL dataset, recommend best practices for removing contaminants from samples, and propose a series of best practices for subsurface microbiology sampling. The most abundant putative contaminant genera observed, independent of evenness across samples, were Propionibacterium, Aquabacterium, Ralstonia, and Acinetobacter. While the top five most frequently observed genera were Pseudomonas, Propionibacterium, Acinetobacter, Ralstonia, and Sphingomonas. The majority of the most frequently observed genera (high evenness) were associated with reagent or potential human contamination. Additionally, in DNA extraction blanks, we observed potential archaeal contaminants, including methanogens, which have not been discussed in previous contamination studies. Such contaminants would directly affect the interpretation of subsurface molecular studies, as methanogenesis is an important subsurface biogeochemical process. Utilizing previously identified contaminant genera, we found that ∼27% of the total dataset were identified as contaminant sequences that likely originate from DNA extraction and DNA cleanup methods. Thus, controls must be taken at every step of the collection and processing procedure when working with low biomass environments such as, but not limited to, portions of Earth’s deep subsurface. Taken together, we stress that the CoDL dataset is an incredible resource for the broader research community interested in subsurface life, and steps to remove contamination derived sequences must be taken prior to using this dataset.

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“…Total mass of simulated carryover contaminants (1 × 10 -18 g) for each reaction is approximately equivalent to a 0.2-μm-diameter aerosol droplet. In the clinical and basic laboratories, the aerosol droplet could not be completely eliminated by either high efficiency particulate air filters or fibrous pipette tip filters in the biosafety cabinets ( Le Rouzic, 2006 ; Barhate and Ramakrishna, 2007 ; Wang et al, 2017a ). The LoD of m-MCDA assay with AUDG and without AUDG digestion before amplification was compared to validate whether the AUDG enzyme has the ability to remove false-positive results in the m-MCDA methods.…”

Section: Methodsmentioning

confidence: 99%

Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus

Wang

Yan²,

et al. 2018

Front. Microbiol.

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Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), is one of the most important human pathogens, which is responsible for bacteremia, soft-tissue infections, and food poisoning. Hence, multiple cross displacement amplification (MCDA) is employed to detect all S. aureus strains, and differentiates MRSA from methicillin-sensitive S. aureus. Multiplex MCDA (m-MCDA), which targets the nuc gene (S. aureus-specific gene) and mecA gene (encoding penicillin-binding protein-2′), could detect S. aureus strains and identify MRSA within 85 min. Detection of the m-MCDA products is achieved using disposable lateral flow biosensors. A total of 58 strains, including various species of Gram-positive and Gram-negative strains, are used for evaluating and optimizing m-MCDA assays. The optimal amplification condition is found to be 63°C for 40 min, with detection limits at 100 fg DNA/reaction for nuc and mecA genes in the pure cultures, and 10 CFU/tube for nuc and mecA genes in the blood samples. The analytical specificity of m-MCDA assay is of 100%, and no cross-reactions to non-S. aureus strains are produced according to the specificity testing. Particularly, two additional components, including AUDG enzyme and dUTP, are added into the m-MCDA amplification mixtures, which are used for eliminating the unwanted results arising from carryover contamination. Thus, the m-MCDA technique appears to be a simple, rapid, sensitive, and reliable assay to detect all S. aureus strains, and identify MRSA infection for appropriate antibiotic therapy.

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Contamination-pipetting: relative efficiency of filter tips compared to Microman® positive displacement pipette

Cited by 19 publications

References 2 publications

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus

Identification and Removal of Contaminant Sequences From Ribosomal Gene Databases: Lessons From the Census of Deep Life

Multiple Cross Displacement Amplification Coupled With Nanoparticles-Based Lateral Flow Biosensor for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus

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