Journal: Science Advances
Article Title: A streamlined CRISPR-based test for tuberculosis detection directly from sputum
doi: 10.1126/sciadv.adx2067
Figure Lengend Snippet: ( A ) Cas13a detecting BCG genomic DNA spiked into sputum:TE matrix and Cas12a detecting internal control. Sp is sputum, and gBCG is genomic BCG. Units are genomes per microliter. Cas13a reporter is FAM, and Cas12a reporter is HEX. ( B ) Comparison of fresh Cas13a (left) or lyophilized Cas13a (right) assays detecting synthetic internal control (IC) and BCG genomic DNA. Synthetic IC and genomic BCG were mixed at a concentration of 10 4 and 10 3 copies per microliter, respectively. ( C ) Lateral flow readout of Cas13a detecting dual targets IS6110 + IS1081 (top) and Cas12a detecting internal control (bottom) in BCG spiked into sputum:TE matrix. Appearance of the top line is a positive readout. Faint lines are interpreted as negative. Disappearance of the bottom line is not necessary. ( D ) Range finding experiment demonstrating the dynamic range of fresh Cas13a dual-detection assay. Cas13a detecting BCG and Cas12a detecting internal control. BCG culture [left; P adj (left to right) is <0.001, <0.001, <0.001, <0.01, <0.05, n.s., n.s., and n.s.] and BCG spiked into sputum:TE [right; P adj (left to right) is <0.0001, <0.0001, <0.0001, <0.05, n.s., n.s., and n.s.]. gBCG is genomic BCG and units are genomes per microliter. All other units are colony-forming units (CFU) per milliliter. ( E ) Testing fresh Cas13a and Cas12a coupled assay against TB-negative clinical sputum samples. Numbers indicate patient number. All samples are n.s. except Sp-17, where P = 0.015. ( F ) LoD for H37Rv. LoD = 69.0 (51.0 to 86.9). ( G ) LoD for BCG. LoD = 80.5 (59.4 to 101.6). In (A), (B), (D), and (E), error bars: SD based on n = 3. (C) A single representative sample. (F) n = 20 and (G) n = 21. In (D) and (E), one-way ANOVA with Dunnett’s test was performed, and P adj values were calculated compared to NTC that contained water. a.u., arbitrary units.
Article Snippet: BCG genomic DNA was ordered from ATCC (catalog no. 35734D-2) and quantified with dPCR.
Techniques: Control, Comparison, Concentration Assay, Detection Assay
ATCC is a verified supplier 
ATCC manufactures this product 
![( A ) Cas13a detecting <t>BCG</t> <t>genomic</t> <t>DNA</t> spiked into sputum:TE matrix and Cas12a detecting internal control. Sp is sputum, and gBCG is genomic BCG. Units are genomes per microliter. Cas13a reporter is FAM, and Cas12a reporter is HEX. ( B ) Comparison of fresh Cas13a (left) or lyophilized Cas13a (right) assays detecting synthetic internal control (IC) and BCG genomic DNA. Synthetic IC and genomic BCG were mixed at a concentration of 10 4 and 10 3 copies per microliter, respectively. ( C ) Lateral flow readout of Cas13a detecting dual targets IS6110 + IS1081 (top) and Cas12a detecting internal control (bottom) in BCG spiked into sputum:TE matrix. Appearance of the top line is a positive readout. Faint lines are interpreted as negative. Disappearance of the bottom line is not necessary. ( D ) Range finding experiment demonstrating the dynamic range of fresh Cas13a dual-detection assay. Cas13a detecting BCG and Cas12a detecting internal control. BCG culture [left; P adj (left to right) is <0.001, <0.001, <0.001, <0.01, <0.05, n.s., n.s., and n.s.] and BCG spiked into sputum:TE [right; P adj (left to right) is <0.0001, <0.0001, <0.0001, <0.05, n.s., n.s., and n.s.]. gBCG is genomic BCG and units are genomes per microliter. All other units are colony-forming units (CFU) per milliliter. ( E ) Testing fresh Cas13a and Cas12a coupled assay against TB-negative clinical sputum samples. Numbers indicate patient number. All samples are n.s. except Sp-17, where P = 0.015. ( F ) LoD for H37Rv. LoD = 69.0 (51.0 to 86.9). ( G ) LoD for BCG. LoD = 80.5 (59.4 to 101.6). In (A), (B), (D), and (E), error bars: SD based on n = 3. (C) A single representative sample. (F) n = 20 and (G) n = 21. In (D) and (E), one-way ANOVA with Dunnett’s test was performed, and P adj values were calculated compared to NTC that contained water. a.u., arbitrary units.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_7463/pmc12327463/pmc12327463__sciadv.adx2067-f3.jpg)
