Fig 1.
Schematic showing steps involved in Sh-RPA analysis.
Fig 2.
Flow schematic outlining participant recruitment, clinical samples provided by patients, DNA isolation methods used and molecular diagnostic assessment (real-time PCR and Sh-RPA).
DNA isolation and real-time PCR carried out by Sturt et al., [40] is coloured grey. DNA isolation and Sh-RPA carried out here is coloured according to DNA extraction method used: QIAamp Mini Kit: blue, SpeedXtract: orange, Extracta: green. Dashed arrows are used to illustrate that real-time PCR data obtained during [40] was used to determine which VSS and CVL samples would be selected for Sh-RPA analysis. Sh-RPA performance and sample preparation comparisons are detailed in Table 1.
Fig 3.
Example Sh-RPA amplification curves using representative PuCVL samples.
Three strong and early-onset positive Sh-RPA amplification curves with relatively low associated real-time PCR Ct values are shown (samples CVL208, CVL210 and CVL500). One weak but positive Sh-RPA amplification curve with a relatively high associated real-time PCR Ct value is shown (CVL125). One weak but positive Sh-RPA amplification curve with an associated real-time PCR Ct value of >50 (and so deemed negative by real-time PCR [40]) is shown (CVL185). Samples CVL164 and CVL212 were deemed negative by both real-time PCR (Ct >50) and Sh-RPA. Neither sample generated a Sh-RPA amplification curve. All real-time PCR Ct values were generated during [40].
Table 1.
Sh-RPA performance and sample preparation comparisons using DNA isolated from CVL and VSS samples by QIAamp Mini Kit (coloured blue), SpeedXtract (coloured orange) and Extracta (coloured green), extraction methods.