Journal: Science translational medicine

Article Title: An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

doi: 10.1126/scitranslmed.abn4768

Figure Lengend Snippet: (A) Example lateral flow strips for each tested input copy number of gBlock Gene Fragment (gBlock) DNA (HPV16 and 18) and extracted cellular DNA from SiHa (HPV16) and HeLa (HPV18) cells, amplified and detected using commercially available lateral flow strips (Milenia HybriDetect 2). Image contrast for all strips increased +20%. NTC: no-template control. (B). Signal-to-background ratio (SBR) for each condition shown in (A). Mean ± standard deviation, n=3 for each condition. Positivity threshold, indicated by the dashed line, was calculated to be 1.17. The limit of detection was calculated to be 50 copies of gBlock DNA or 500 copies of extracted SiHa cellular DNA for the HPV16 assay and 50 copies of gBlock DNA or extracted DNA from HeLa cells for the HPV18 assay. (C) Gel electrophoresis of the same targets shown in (A) and (B). The “uncut” primer-to-primer product should be 200 base pairs for both assays and is not detected by lateral flow. The “cut” probe-to-primer product is detected by lateral flow and should be 125 base pairs for both assays. The limit of detection on lateral flow is lower for the HPV18 assay in part due favorable formation of the cut product compared to the uncut product. bp: base pairs; L: Low molecular weight ladder. (D) Specificity of the HPV16 and HPV18 assays. Vertical labels refer to HPV type and horizontal labels refer to test (HPV16 or HPV18) or control (C) lines. For each target and off-target HPV type listed next to the lateral flow strips, 106 input copies of gBlock DNA were amplified and detected using commercially available lateral flow strips. Positive signal is only seen at the corresponding test line when the HPV type of the input target corresponded to that targeted in the assay (dashed box), demonstrating high analytical specificity (n=1 for each target).

Article Snippet: Two reference tests were used in this study: GeneXpert HPV and Multiplex High Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping (Atila Biosystems, Mountain View, CA).

Techniques: Amplification, Standard Deviation, Nucleic Acid Electrophoresis, Molecular Weight

Journal: Science translational medicine

Article Title: An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

doi: 10.1126/scitranslmed.abn4768

Figure Lengend Snippet: (A) Starting quantity of HPV16 DNA in SiHa lysate, as determined by qPCR, following sample preparation by either sonication or achromopeptidase (ACP). ACP lyses SiHa cells comparably to probe sonication (p=0.77; no target control (NTC) n=3, remaining conditions n=9 with 3 biological x 3 technical replicates). (B) Images of lateral flow strips containing RPA reactions run with ACP-produced SiHa cell lysate (NTC, +: n=1, SiHa lysate: n=3), and signal-to-background ratios (SBR) of the HPV16 test lines. Signal-to-background ratio (SBR) above the threshold indicates successful amplification of HPV16 DNA from the unpurified lysate. NLC: no lysis control; N.S.: not significant; +: positive control.

Article Snippet: Two reference tests were used in this study: GeneXpert HPV and Multiplex High Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping (Atila Biosystems, Mountain View, CA).

Techniques: Sample Prep, Sonication, Produced, Amplification, Lysis, Positive Control

Journal: Science translational medicine

Article Title: An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

doi: 10.1126/scitranslmed.abn4768

Figure Lengend Snippet: (A) Replicates of three cartridges with increasing copy numbers from 0 (no-target control, NTC) to 10,000 HPV16 and HPV18 copies per reaction using lyophilized amplification reagents. Cartridges were scanned after 15 minutes, and images were cropped around the test windows. (B) Signal-to-background (SBR) analysis of triplicate cartridges in (A). Average ± standard deviation of each test (16, 18) and control (C) line is plotted. Consistent amplification, using lyophilized reagents, and detection are visible at 500–1,000 copies and higher.

Article Snippet: Two reference tests were used in this study: GeneXpert HPV and Multiplex High Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping (Atila Biosystems, Mountain View, CA).

Techniques: Amplification, Standard Deviation

Journal: Science translational medicine

Article Title: An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

doi: 10.1126/scitranslmed.abn4768

Figure Lengend Snippet: (A) Number of samples determined to be positive and negative by the HPV16 assay on the NATflow, stratified by number of starting copies as determined by qPCR. (B) Number of samples determined to be positive and negative by the HPV18 assay on the NATflow, stratified by number of starting copies as determined by qPCR. (C) Log copies of HPV DNA, measured by qPCR, for samples that were true positives (TP) and false negatives (FN) by the NATFlow HPV16 and HPV18 assay. There was a statistically significant difference (*) between mean Ct values for TP and FN samples by the NATflow HPV16 assay (p<0.01); statistical analysis was not conducted between the TP and FN samples by the NATflow HPV18 assay due to small number of positive samples. Arrow indicates increasing amounts of HPV DNA.

Article Snippet: Two reference tests were used in this study: GeneXpert HPV and Multiplex High Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping (Atila Biosystems, Mountain View, CA).

Techniques:

Journal: Science translational medicine

Article Title: An integrated isothermal nucleic acid amplification test to detect HPV16 and HPV18 DNA in resource-limited settings

doi: 10.1126/scitranslmed.abn4768

Figure Lengend Snippet: Cervicovaginal samples collected into PreservCyt buffer (n=55) were tested using NATflow and results compared with GeneXpert HPV test results. Agreement between GeneXpert HPV and the NATflow (A) HPV16 assay and (B) HPV18 assay are stratified by GeneXpert Ct value. (C) GeneXpert Ct values for samples that were true positives (TP) and false negatives (FN) by the NATflow HPV16 and HPV18 assay. There was a statistically significant difference (*) between mean Ct values for TP and FN samples by the NATflow HPV16 assay (p=0.05) but not by the NATflow HPV18 assay (p=0.14). Arrow indicates increasing amounts of HPV DNA.

Article Snippet: Two reference tests were used in this study: GeneXpert HPV and Multiplex High Risk HPV Real Time Fluorescent Detection with HPV16/18 Genotyping (Atila Biosystems, Mountain View, CA).

Techniques: