Fig 1.
Schematic illustrating the two RT-RPT-VF assays targeting the MERS-CoV UpE and N genes.
(A) The RT-RPA-VF assay was performed at a constant temperature. First, viral RNA is transcribed into cDNA by transcriptase. Second, the recombinase binds to primers/probe to form complexes that scan the template for recombination with cognate sites. Next, the strand-displacing DNA polymerase triggers primer extension events. At the same time, an SSB binds to the non-template strand to prevent the dissociation of the primer. Another enzyme, Nfo, cleaves the THF site when the probe hybridizes to its target sequence, resulting in the loss of the C3-spacer and probe extension. (C) The tube was placed in a closed device of vertical flow visualization strip device to detect the RT-RPA products. The gold-labeled anti-FITC antibody and streptavidin fixed on the test paper will capture the amplicon labeled with FITC and biotin at both ends and accumulate to form a red band.
Table 1.
Respiratory pathogens included in the NATtrolsp RP Multimarker Controls kit.
Fig 2.
Specificity of the primers and probes used in the RT-RPA-VF assays for the MERS-CoV UpE and N genes.
The sequences of genes from MERS-CoV and other CoVs were retrieved from GenBank and aligned using MEGA7 software. Only the target sequences of RT-RPA-VF assays are shown.
Table 2.
Primer and probe sequences for the MERS-CoV RT-qPCR assay.
Table 3.
Sequences of the primers and probes used for the MERS-CoV RT-RPA-VF assay panel.
Table 4.
Optimization of the reaction temperature for RT-RPA-VF assays targeting the MERS-CoV UpE and N genes.
Table 5.
Optimization of the reaction times for RT-RPA-VF assays targeting the MERS-CoV UpE and N genes.
Fig 3.
Quantitative analysis of the MERS-CoV RNA using the absolute RT-qPCR assay targeting UpE gene.
Ten-fold dilutions of synthesized UpE RNA transcripts were used to establish the standard curve, and 100-fold dilutions of total RNA from virus-infected cell suspensions were used as the test template.
Table 6.
Limits of detection of the MERS-CoV RT-RPA-VF assay panel using RNA transcripts.
Fig 4.
Detection of 10-fold serial dilutions of the MERS-CoV RNA using the RT-RPA-VF assays against the UpE (A) and N (B) genes.
Fig 5.
The specificity of the RT-RPA-VF assays against the UpE (A) and N (B) genes. RP1 controls and RP2 controls kits are panels of nucleic acids from multiple respiratory pathogens, including 229E, OC43, NL63, HKU1, influenza A/B, rhinovirus, adenovirus, and parainfluenza.
Table 7.
The sensitivity and specificity of the RT-RPA-VF assay panel were evaluated using spiked extracted RNA specimens.