Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: ( a , b ) qPCR and ( c , d ) LAMP experiments targeting E. coli 23S rRNA gene, which shows increased impact of reaction inhibition at low NA concentrations. ( a ) qPCR and ( c ) LAMP spiked with 4-fold dilution series of E. coli 23S rRNA gene copies and comparing with and without Zymo Viral DNA/RNA Buffer. Each bar represents the average of technical qPCR or LAMP triplicates (black circles). Numbers above a bar indicate the number of samples which amplified if not all triplicates were detected. Dashed boxes indicate axes for zoomed-in graphs of ( b ) qPCR and ( d ) LAMP. Numbers above each pair of bars indicate the difference in either C q or TTP between the control and the reaction with added lysis buffer. Samples marked N.D. were not detected within either 60 cycles or 40 min.

Article Snippet: For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 10 4 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration.

Techniques: Inhibition, Amplification, Lysis

Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: Identifying the most effective TPW in ( a ) qPCR and ( b ) LAMP reactions and subsequent validation of 1-undecanol as a candidate TPW with ( c ) qPCR and ( d ) LAMP at low eluent dilutions. TPW candidates for ( a ) qPCR and ( b ) LAMP reactions were spiked with 5 × 10 4 copies λ phage DNA and primers, made to 10 µL, and 1 µL of each wash candidate was added to yield 11 µL total. The number 2 next to the 1-octanol bar indicates that only two of the three replicates amplified. The dashed lines show the C q or TTP of the uninhibited 10 µL “No Additive” control. ( c ) qPCR with 2.2x diluted eluent and ( d ) LAMP with 2x diluted eluent on a λ phage DNA sample extracted with a Zymo Quick-Viral DNA/RNA kit. Protocol was performed according to manufacturer instructions as provided or with an additional TPW (+1-undecanol) between the ethanol wash and elution steps. Each bar represents the average of technical triplicates (black circles). We ran 6 extractions (3 silica columns x 2 conditions) and used the same eluent for both the qPCR and LAMP analyses. Samples marked N.D. were not detected within either 40 cycles or 40 min. NTC, no-template control. ( a , b ) We asked whether TPW candidates fell within the 99% CI of the “No Additive” control (qPCR: 20.01-20.17, LAMP: 6.25-6.83) with outliers indicated with a *. ( d ) We asked whether the average TTP was statistically different between the manufacturer protocol and the +1-undecanol condition using a t -test.

Article Snippet: For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 10 4 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration.

Techniques: Amplification

Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: Comparing the performance of different TPWs with eluent at 2.2x dilution in qPCR ( a , d ), 2x dilution in LAMP ( b , e ), and 100x dilution in digital PCR (dPCR) ( c , f ). Samples were spiked with 2.5 × 10 6 copies λ phage DNA and extracted in 50 µL water with a Zymo Quick-Viral DNA/RNA kit. We compared each manufacturer’s protocol (Manuf. protocol) with the same protocol plus an additional TPW of either 1-undecanol, 1-octanol, 2-dodecanol, silicone oil, or FC-40. To observe inhibition, a low eluent dilution was used in qPCR and LAMP with λ phage primers. To get a highly accurate quantification of NAs (for comparing these results), we ran each sample using dPCR with a high dilution of eluent (100x), which eliminates the effects of inhibitors. Each bar represents the average of qPCR or LAMP technical triplicates (black circles) or single dPCR measurements. We ran 24 extractions (3 silica columns x 8 conditions) and the same eluent was used to run the qPCR, LAMP, and dPCR analyses. Where shown, numbers above a bar indicate the number of samples which amplified if not all triplicates were detected. Dashed lines (panels c and f) indicate the average NA recovery following manufacturer protocol. Samples marked N.D. were not detected within 40 cycles by qPCR or 40 min by LAMP. ( a – f ) For each of the five TPW candidates, we asked whether the mean value was statistically different from the manufacturer protocol by t -test. N.S. stands for not significant ( P > 0.05).

Article Snippet: For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 10 4 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration.

Techniques: Digital PCR, Inhibition, Amplification

Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: Evaluation of TPW for different silica-column NA extraction kit protocols on pure water samples using ( a – c ) qPCR and ( d – f ) LAMP. All reactions were spiked with 5 × 10 4 copies λ phage DNA and primers. By manufacturer protocol, the ( a , d ) Zymo Quick-DNA/RNA Viral Kit and ( b , e ) Zymo ZR Viral DNA/RNA Kit do not include the dry spin (+dry spin) whereas the ( c , f ) Qiagen QIAquick PCR Purification Kit does. The left of each graph shows high dilution and the right shows low dilution. Each bar represents the result from a single qPCR or LAMP measurement. We ran 27 silica-column extractions (3 silica columns × 3 conditions × 3 extraction protocols) and the kit extract was shared between high and low dilutions of both qPCR and LAMP. Dashed lines show the C q or TTP for a reaction without inhibitors (“No Extract”). Samples marked N.D. were not detected within either 40 cycles or 40 min. ( a – f ) We asked whether the manufacturer protocol replicates (“No Dry Spin for Zymo kits, “+dry spin” for Qiagen kit) fell within the 95% CI of the corresponding +1-undecanol condition for the low kit extract dilution case. The number of replicates that lie outside the 95% CI are indicated by the number of + (above) and - (below).

Article Snippet: For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 10 4 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration.

Techniques: Purification

Journal: Scientific Reports

Article Title: Two-phase wash to solve the ubiquitous contaminant-carryover problem in commercial nucleic-acid extraction kits

doi: 10.1038/s41598-020-58586-3

Figure Lengend Snippet: Evaluating TPW for compatibility with Zymo Quick-DNA/RNA MagBead extraction with ( a ) qPCR, ( b ) LAMP, and ( c ) dPCR. Extraction performed on 1 × 10 6 λ phage DNA copies with either a 10 min air dry (Manuf. protocol), no air dry, or with the air dry replaced by a TPW (+1-undecanol) step. The resulting eluent is spiked at either high dilution or low dilution into ( a ) qPCR and ( b ) LAMP or 100x dilution into ( c ) dPCR. For dPCR ( d ), the bars to the right of the solid black line show the results for an extraction protocol with a +1-undecanol wash using a high-yield protocol from a separate experiment (normalized to the no TPW control in that experiment). Bars represent single qPCR and LAMP or the merged result from a duplicate dPCR measurement. Dashed line in dPCR ( c ) indicates the average NA recovery following manufacturer protocol. We ran 9 extractions (3 magnetic-bead extractions x 3 conditions) and the eluent was shared among qPCR, LAMP, and dPCR analyses. Samples marked N.D. were not detected within either 40 cycles for qPCR or 40 min for LAMP. ( a , b ) We asked whether the manufacturer protocol replicates fell within the 95% CI of the corresponding +1-undecanol condition for the low eluent dilution case. The number of replicates that lie outside the 95% CI were indicated by the number of *s.

Article Snippet: For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 10 4 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration.

Techniques:

Journal: Cell

Article Title: Emergence and rapid transmission of SARS-CoV-2 B.1.1.7 in the United States

doi: 10.1016/j.cell.2021.03.052

Figure Lengend Snippet:

Article Snippet: Omega BioTek MagBind Viral DNA/RNA Kit , Omega Biotek , Cat#M6246-03.

Techniques: Isolation, Ligation, Sequencing, Purification, Multiplex Assay, Software, Variant Assay