Limit of detection.

<p>Limit of detection of the CCHF RPA assay with a serial (1 in 10) dilution of synthetic RNA template of Europe I strain AY277672. The data is represented graphically as <b>(a)</b> Delta Rn against time (minutes), in the form of a table of time to positive (TTP value) vs target copy number and as TTP vs target copy number, with a regression line indicating data correlation <b>(b)</b>. The data is also shown as a probit analysis performed using the statistical software Matlab for more accurate detection limit approximation <b>(c)</b>, with application of a curve-fitting programme (a shape-preserving piecewise cubic interpolation). Note that the probit analysis uses a lower threshold (Delta Rn 18000) to enable the calculation. The values shown for all LOD data are the mean of 8 independent experiments, each of which was performed with three replicates.</p

Negative viral panel testing.

<p>Table showing the results of a CCHF RPA with a negative viral panel, consisting of a selection of strains from the <i>Alphavirus</i>, <i>Mammarenavirus</i>, <i>Marburgvirus</i>, <i>Flavivirus</i>, <i>Orthohantavirus</i>, <i>Henipavirus</i> and <i>Orthonairovirus</i> genera. Results represent 3 separate experiments, each performed with 3 replicates.</p

A recombinase polymerase amplification assay for rapid detection of Crimean-Congo Haemorrhagic fever Virus infection

<div><p>Background</p><p>Crimean-Congo Haemorrhagic fever Virus (CCHFV) is a rapidly emerging vector-borne pathogen and the cause of a virulent haemorrhagic fever affecting large parts of Europe, Africa, the Middle East and Asia.</p><p>Methodology/principle findings</p><p>An isothermal recombinase polymerase amplification (RPA) assay was successfully developed for molecular detection of CCHFV. The assay showed rapid (under 10 minutes) detection of viral extracts/synthetic virus RNA of all 7 S-segment clades of CCHFV, with high target specificity. The assay was shown to tolerate the presence of inhibitors in crude preparations of mock field samples, indicating that this assay may be suitable for use in the field with minimal sample preparation. The CCHFV RPA was successfully used to screen and detect CCHFV positives from a panel of clinical samples from Tajikistan.</p><p>Conclusions/significance</p><p>The assay is a rapid, isothermal, simple-to-perform molecular diagnostic, which can be performed on a light, portable real-time detection device. It is ideally placed therefore for use as a field-diagnostic or in-low resource laboratories, for monitoring of CCHF outbreaks at the point-of-need, such as in remote rural regions in affected countries.</p></div

Assay design.

<p><b>(a) Primer, probe and synthetic template DNA sequences.</b> Table showing primer and probe sequences and information on the synthetic DNA templates which were used as transcription templates to create the RNA assay controls. Please see supplementary data for the full DNA sequences of the synthetic DNA templates. <b>(b) Alignment of CCHF strains at the assay design region.</b> Alignment of a selection of strains of CCHFV representing all 7 S-segment clades of CCHFV (Africa 1, Africa 2, Africa 3, Asia 1, Asia 2, Europe 1 and Europe 2), with inclusion of the RPA forward primer (CCHF RPA Fw), the reverse primer (CCHF RPA Rev) and the probe (CCHF RPA probe 1).</p

Cross-clade detection of CCHF.

<p>Table showing detection of CCHF viral extracts and synthetic CCHF RNA templates by CCHF RPA assay, in the form of TTP (minutes) and number of replicates detected. The synthetic RNA templates were used at 5X10⁵ copies/reaction and viral extracts were confirmed positive by CCHF RT-PCR. The RT-PCR TTP (minutes) are included in the table. Note that the RPA values shown are the mean of 3 independent experiments, performed with 2–3 replicates.</p

Effect of inhibitors in crude sample preparations.

<p><b>(a) Effect of crude sample dilution on detection in the RPA assay.</b> RPA performed with 5X10⁶ copies/ reaction of synthetic RNA template of Europe I strain AY277672 and a serial (1 in 10) dilution of crude preparations of human male serum, urine and tick pool homogenate. <b>(b) Effect of crude material on assay sensitivity–limit of detection.</b> RPA performed with a serial (1 in 10) dilution of synthetic RNA template of Europe I strain AY277672 and 1 in 10 diluted crude preparations of serum and urine, or 1 in 100 diluted crude preparations of tick pool homogenate. The RPA results are shown as TTP (minutes) and represent the mean of 3–4 independent experiments, each of which was performed with three replicates. RT-PCR data is also included for comparison, with the values shown representing the mean TTP (mins) of 2–3 separate experiments, each of which was performed with 2 replicates.</p

Testing of field samples from Tajikistan CCHF outbreaks between 2013 and 2015.

<p>CCHF RPA testing of a collection of field samples from the 2013–2015 CCHF outbreaks in Tajikistan. Sample types include extracted human sera and extracted tick pool homogenate. The RPA results are shown as TTP (minutes) and number of replicates detected. RT-PCR results are shown as TTP (minutes).</p