Journal: BMC Genomics

Article Title: Digital PCR provides sensitive and absolute calibration for high throughput sequencing

doi: 10.1186/1471-2164-10-116

Figure Lengend Snippet: A Schematic of the universal template (UT) PCR assay . The forward primer (as drawn) includes a short sequence complementary to the 8 bp dual-labeled locked nucleic acid probe on its 5' end, followed by the sequence of one of the adaptors ligated to the library molecules on its 3' end. The reverse primer (as drawn) is complementary to the sequence of the other adaptor. As the polymerase encounters the probe during strand extension, its 5' to 3' exonuclease activity cleaves the probe, releasing the fluorophore from its quencher, thus producing fluorescent signal by dequenching. B The assay is performed on a commercial microfluidic digital PCR chip. At the end of the PCR, compartments that contain amplifiable DNA molecules with sequencing adaptors properly appended give positive signal, while compartments that do not remain dark. The count of positive compartments corresponds to the number of productive library molecules in the volume loaded onto the microfluidic chip, thereby allowing measurement of the concentration of amplifiable library molecules.

Article Snippet: PCR reaction mix containing the diluted template was loaded onto Fluidigm's 12.765 Digital Array microfluidic chip.

Techniques: Sequencing, Labeling, Activity Assay, Digital PCR, Concentration Assay

Journal: Journal of Clinical Microbiology

Article Title: Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes

doi: 10.1128/JCM.01684-19

Figure Lengend Snippet: Two-gene association rates of the top seven STEC serogroups and virulence genes tested by Fluidigm dPCR on pure cultures and culture-spiked cattle feces a

Article Snippet: The positive fraction ( F p ) (percent) of a specific gene was calculated as the number of partitions (wells or chambers) with positive hits for the given gene divided by the total number of partitions in a dPCR panel (QuantStudio 3D chip, n = 2,000; Fluidigm 37K chip, n = 770).

Techniques:

Journal: Journal of Clinical Microbiology

Article Title: Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes

doi: 10.1128/JCM.01684-19

Figure Lengend Snippet: Whole-panel heat maps generated with Fluidigm digital PCR 37K IFC analyses of E. coli strains. Each map represents a whole panel containing 770 PCR chambers (11-by-70 grids). The vertical read bars (red, blue, and brown) in each grid indicate positive amplification signals of the target genes. Comparison pairs were (i) rfbEO157 (red) and stx2 (blue) genes carried by a single genome (a1) or by two separate genomes (b1) of pure cultures of E. coli strains; (ii) rfbEO157 and stx2 carried by a single genome (a2) or by two separate genomes (b2) of culture-spiked cattle feces; (iii) rfbEO157, stx2, and eae (brown) carried by a single genome (a3) or by three separate genomes (b3) of culture-spiked cattle feces; and (iv) rfbEO157, stx2, and eae carried by a single genome (a4) or by three separate genomes (b4) of culture-spiked ground beef.

Article Snippet: The positive fraction ( F p ) (percent) of a specific gene was calculated as the number of partitions (wells or chambers) with positive hits for the given gene divided by the total number of partitions in a dPCR panel (QuantStudio 3D chip, n = 2,000; Fluidigm 37K chip, n = 770).

Techniques: Generated, Digital PCR, Amplification, Comparison

Journal: Journal of Clinical Microbiology

Article Title: Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes

doi: 10.1128/JCM.01684-19

Figure Lengend Snippet: Three-gene association rates of the top 7 STEC serogroups and virulence genes tested by Fluidigm dPCR on pure cultures, culture-spiked cattle feces, and culture-spiked ground beef a

Article Snippet: The positive fraction ( F p ) (percent) of a specific gene was calculated as the number of partitions (wells or chambers) with positive hits for the given gene divided by the total number of partitions in a dPCR panel (QuantStudio 3D chip, n = 2,000; Fluidigm 37K chip, n = 770).

Techniques:

Journal: Journal of Clinical Microbiology

Article Title: Single-Cell-Based Digital PCR Detection and Association of Shiga Toxin-Producing Escherichia coli Serogroups and Major Virulence Genes

doi: 10.1128/JCM.01684-19

Figure Lengend Snippet: Partial heat maps from Fluidigm dPCR analysis of E. coli serogroups O103 (a), O45 (b), and O157 (c) and the virulence genes stx1, stx2, and eae from cattle fecal samples. The vertical read bars (red, blue, and brown) in each grid indicate positive amplification signals of the target genes. (a) The eae gene (brown) is associated with the E. coli O103 serogroup (blue) in samples 38, 43, and 44 but separated from stx1-stx2 (red). (b) stx1 (red) is associated with O45 (blue) in samples 45 and 98 but separated in sample 88. (c) stx1-stx2 (red) and eae (brown) are not associated with E. coli rfbEO157 (blue) in samples 30, 32, and 88.

Article Snippet: The positive fraction ( F p ) (percent) of a specific gene was calculated as the number of partitions (wells or chambers) with positive hits for the given gene divided by the total number of partitions in a dPCR panel (QuantStudio 3D chip, n = 2,000; Fluidigm 37K chip, n = 770).

Techniques: Amplification