Fig 1.
Workflow of rapid shrimp HPV test.
Basic workflow of rapid shrimp HPV test including sample processing (A), recombinase polymerase amplification (RPA; B), and lateral flow detection (C) steps. Created with BioRender.com.
Fig 2.
Analytical sensitivity of shrimp HPV RPA-LFD.
Sensitivity testing used capsid protein gene fragment plasmid DNA diluted 10-fold in nuclease-free water for Shrimp HPV RPA-LFD (D). A, Photograph of lateral-flow strips with control bands (all samples) and test bands (positive samples) compared to copy number of serial diluted synthetic template DNA (copies/μL) and water as negative control (NTC). B, Normalised pixel density (normalised black values) from the lateral flow strip displayed in A, which was used to calculate positives (labelled by *) and negatives. C, Positive (Pos.) results compared to number (No.) of samples tested at that dilution. D, Percentage of positive tests performed at that dilution.
Fig 3.
Diagnostic sensitivity and specificity of rapid shrimp HPV test.
Each banana shrimp (F. merguiensis) sample was tested a total of three times in independent test runs showing a representative result. Sample ID: Individual shrimp hepatopancreas with high (H), medium (M), low (L) HPV copy number and uninfected (N) samples; qPCR: concentration (copies/μg) of extracted hepatopancreas sample tested and corresponding test result (+/-); Rapid shrimp HPV test: Image of lateral flow strip result from rapid HPV test (rapid sample processing with TNA-Cifer Reagent followed by shrimp HPV RPA-LFD) [Images of lateral flow strips with two bands (control and test band) indicates the sample is positive for HPV, and single control band indicates a valid reaction with negative sample]; Blank normalised black pixel value from the lateral flow strip with cut-off at 3.96 and corresponding result (+/-); Positive (Pos.) test results compared to total sample number (No.) tested; and Sensitivity and Specificity of the rapid HPV test with 95% confidence interval (CI).
Fig 4.
Detection of HPV-positive sample mixed into HPV-negative wildtype banana shrimp samples with the rapid shrimp HPV test.
Testing mixed HPV-positive banana shrimp sample (Sample ID: H2, Fig 2) into wildtype samples (Sample IDs: N7-9, Fig 2) at a ratio of 1:1 prior to the required dilution step. Each banana shrimp (F. merguiensis) sample was tested a total of three times in independent test runs showing a representative result. A, Photograph of lateral flow strips from rapid shrimp HPV test (rapid sample processing with TNA-Cifer Reagent followed by HPV RPA-LFD) showing control bands (all samples) and test bands (positive samples) compared to qPCR results (copies/μg), positive control (PTC; synthetic template DNA (2.42 × 106 copies/μL)) and water as negative control (NTC). B, Normalised pixel density (normalised black values) from the lateral flow strip displayed in A showing positive samples or positive control labelled by *. C, Positive (Pos.) test results compared to number (No.) of samples tested in individual runs. D, Percentage of positive tests performed for displayed sample.
Fig 5.
Serial dilutions of a HPV-positive banana shrimp sample assessed with the rapid shrimp HPV test.
Testing used 10-fold serial dilutions of a HPV-positive banana shrimp (F. merguiensis) sample (Sample ID: L6, Fig 2). Dilutions and testing were repeated a total of three times in independent experiments showing a representative result. A, Photograph of lateral flow strips with control and test bands compared to qPCR results, water (NTC) or positive control (PTC; 2.42 × 106 copies/μL synthetic template DNA). B, Normalised pixel density (Normalised black values) from the test displayed in A showing positive samples or positive control (both labelled by *) and negative water control. C, Positive (Pos.) test results compared to number (No.) of dilutions tested in individual runs. D, Percentage of positive tests performed for displayed sample.