Journal: medRxiv

Article Title: Rapid and portable reverse-transcription quantitative PCR assays for Bundibugyo ebolavirus detection

doi: 10.64898/2026.08.17.26360605

Figure Lengend Snippet: Standard curves for Bundibugyo ebolavirus (BDBV) and mitochondrial circular DNA (mcirDNA) TaqMan RT-qPCR assays in singleplex and duplex formats. We generated standard curves for A) BDBV and B) the mcirDNA internal control assays using serial dilutions of synthetic RNA or DNA gene fragments. Data points show the mean and standard deviation of triplicate reactions. Solid lines indicate simple linear regressions, and dotted lines indicate the corresponding 95% confidence intervals. E denotes the RT-qPCR amplification efficiency.

Article Snippet: Synthetic double stranded DNA gene fragments (Twist Bioscience, sequences provided in Table A4) targeting specific, unique regions of Z-EBOV (Viral Protein 24), S-EBOV (L segment, RNA-dependent RNA polymerase), BDBV (L segment, RNA-dependent RNA polymerase), and mcirDNA (Cytochrome C Oxidase Subunit III) were utilized as synthetic standards.

Techniques: Quantitative RT-PCR, Generated, Control, Standard Deviation, Amplification

Journal: medRxiv

Article Title: Rapid and portable reverse-transcription quantitative PCR assays for Bundibugyo ebolavirus detection

doi: 10.64898/2026.08.17.26360605

Figure Lengend Snippet: Standard curves for Bundibugyo ebolavirus (BDBV), Zaire ebolavirus (Z-EBOV), Sudan ebolavirus (S-EBOV), and mitochondrial circular DNA (mcirDNA) TaqMan RT-qPCR assays in singleplex and four-target multiplex formats. We generated standard curves for A) BDBV, B) Z-EBOV, C) S-EBOV, and D) the mcirDNA internal control assays using serial dilutions of synthetic RNA or DNA gene fragments. Data points show the mean and standard deviation of triplicate reactions. Solid lines indicate simple linear regressions, and dotted lines indicate the corresponding 95% confidence intervals. E indicates the RT-qPCR amplification efficiency.

Article Snippet: Synthetic double stranded DNA gene fragments (Twist Bioscience, sequences provided in Table A4) targeting specific, unique regions of Z-EBOV (Viral Protein 24), S-EBOV (L segment, RNA-dependent RNA polymerase), BDBV (L segment, RNA-dependent RNA polymerase), and mcirDNA (Cytochrome C Oxidase Subunit III) were utilized as synthetic standards.

Techniques: Quantitative RT-PCR, Multiplex Assay, Generated, Control, Standard Deviation, Amplification

Journal: medRxiv

Article Title: Rapid and portable reverse-transcription quantitative PCR assays for Bundibugyo ebolavirus detection

doi: 10.64898/2026.08.17.26360605

Figure Lengend Snippet: Abridged Limit of detection (LOD) analysis for Bundibugyo ebolavirus (BDBV), Zaire ebolavirus (Z-EBOV), Sudan ebolavirus (S-EBOV), and mitochondrial circular DNA (mcirDNA) across TaqMan singleplex, duplex, four-target multiplex and SYBR Green RT-qPCR assay formats. We determined the 95% limit of detection (LOD 95 ) using synthetic RNA or DNA gene fragments quantified by digital PCR. We defined the LOD 95 as the lowest concentration detected in at least 95% of replicate reactions (n=21). Only results at the concentration meeting the LOD 95 criterion are shown; complete data are in Table A1.

Article Snippet: Synthetic double stranded DNA gene fragments (Twist Bioscience, sequences provided in Table A4) targeting specific, unique regions of Z-EBOV (Viral Protein 24), S-EBOV (L segment, RNA-dependent RNA polymerase), BDBV (L segment, RNA-dependent RNA polymerase), and mcirDNA (Cytochrome C Oxidase Subunit III) were utilized as synthetic standards.

Techniques: Multiplex Assay, SYBR Green Assay, Digital PCR, Concentration Assay

Journal: medRxiv

Article Title: Rapid and portable reverse-transcription quantitative PCR assays for Bundibugyo ebolavirus detection

doi: 10.64898/2026.08.17.26360605

Figure Lengend Snippet: Standard curves for Bundibugyo ebolavirus (BDBV) and mitochondrial circular DNA (mcirDNA) TaqMan RT-qPCR assays in singleplex and duplex formats on the Mic qPCR cycler under rapid-cycling conditions. We generated standard curves for A) BDBV and B) mcirDNA internal control using serial dilutions of synthetic RNA or DNA gene fragments with the modified Mic qPCR protocol. Data points show the mean and standard deviation of triplicate reactions. Solid lines indicate simple linear regressions, and dotted lines indicate the corresponding 95% confidence interval. E denotes the RT-qPCR amplification efficiency.

Article Snippet: Synthetic double stranded DNA gene fragments (Twist Bioscience, sequences provided in Table A4) targeting specific, unique regions of Z-EBOV (Viral Protein 24), S-EBOV (L segment, RNA-dependent RNA polymerase), BDBV (L segment, RNA-dependent RNA polymerase), and mcirDNA (Cytochrome C Oxidase Subunit III) were utilized as synthetic standards.

Techniques: Quantitative RT-PCR, Generated, Control, Modification, Standard Deviation, Amplification

Journal: medRxiv

Article Title: Rapid and portable reverse-transcription quantitative PCR assays for Bundibugyo ebolavirus detection

doi: 10.64898/2026.08.17.26360605

Figure Lengend Snippet: Standard curves for Bundibugyo ebolavirus (BDBV) and mitochondrial circular DNA (mcirDNA) TaqMan RT-qPCR assays in singleplex and duplex formats using lyophilized RT-qPCR mastermix on the QuantStudio 6 Flex and Mic qPCR cycler. We generated standard curves for A) BDBV on QuantStudio 6 Flex, B) the mcirDNA internal control on QuantStudio 6 Flex, C) BDBV on Mic qPCR cycler, and D) mcirDNA on the Mic qPCR cycler using serial dilutions of synthetic RNA gene fragments with lyophilized RT-qPCR mastermix. Data points show the mean and standard deviation of duplicate reactions. Solid lines indicate simple linear regressions, and dotted lines indicate the corresponding 95% confidence intervals. E denotes the RT-qPCR standard curve efficiency.

Article Snippet: Synthetic double stranded DNA gene fragments (Twist Bioscience, sequences provided in Table A4) targeting specific, unique regions of Z-EBOV (Viral Protein 24), S-EBOV (L segment, RNA-dependent RNA polymerase), BDBV (L segment, RNA-dependent RNA polymerase), and mcirDNA (Cytochrome C Oxidase Subunit III) were utilized as synthetic standards.

Techniques: Quantitative RT-PCR, Generated, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: EXO1 and CHAF1A are recruited to R-loops and synergistically suppress R-loop accumulation. ( A ) BrdU alkaline comet assay showing that CHAF1A depletion causes increased accumulation of replication-associated single stranded DNA lesions in both EXO1-knockout lines compared to WT HeLa cells. At least 100 nuclei were quantified for each condition. The median values are marked on the graph and listed at the top. Asterisks indicate statistical significance (Mann–Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. ( B, C ) S9.6 PLA experiments showing increased EXO1 ( B ) and CHAF1A ( C ) recruitment to R-loops in HeLa cells. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( D, E ) S9.6-dsDNA PLA experiments showing increased R-loops in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Alkaline Single Cell Gel Electrophoresis, Knock-Out, MANN-WHITNEY, Two Tailed Test, Over Expression

Journal: Nucleic Acids Research

Article Title: Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage

doi: 10.1093/nar/gkag226

Figure Lengend Snippet: Concomitant depletion of EXO1 and CHAF1A causes the accumulation of R-loop-associated DNA damage. ( A ) S9.6-γH2AX PLA experiments showing increased R-loop-associated DNA damage in HeLa cells with concomitant inactivation of EXO1 and CHAF1A. RNAseH1 overexpression suppresses the PLA signal, indicating that it derives from R-loops. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). The siCHAF1A/siControl mean ratios are also presented. ( B–D ) γH2AX immunofluorescence showing that CHAF1A depletion causes increased DNA damage in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( B, D ) and representative micrographs with scale bars representing 10 µm ( C ) are shown. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard errors of the mean, and asterisks indicate statistical significance ( t -test, two-tailed, unpaired). ( E–G ) Neutral comet assays showing that CHAF1A depletion causes increased DSB formation in both EXO1-knockout lines compared to WT HeLa cells, which is suppressed by RNaseH1 overexpression. Quantifications ( E, G ) and representative micrographs with scale bars representing 10 µm ( F ) are shown. At least 100 comets were quantified for each sample. The median values are marked on the graph, and asterisks indicate statistical significance (Mann–Whitney, two-tailed).

Article Snippet: Antibodies used were: S9.6 (Kerafast ENH001), double-stranded DNA (dsDNA; Novus NBP3-07302), γH2AX (Abcam ab2893), EXO1 (Novus NBP2-16391), and CHAF1A (Cell Signaling Technology 5480s).

Techniques: Over Expression, Two Tailed Test, Immunofluorescence, Knock-Out, MANN-WHITNEY