Highly sensitive and rapid determination of Coxsackievirus A16 using restriction endonuclease‐mediated reverse transcription multiple cross displacement amplification

Figures

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  Fig. 1. Primers for the VP1 gene used in the CVA16-E-RT-MCDA assay in this study. Position and sequences of the primers used in this research targeting the VP1 gene of CVA16. Right and left arrows indicate the sense and complementary sequences used, respectively

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  Fig. 2. Schematic illustration of CVA16-E-RT-MCDA assay. A real-time fluorescent signal is generated in the CVA16-E-RT-MCDA system by the separation of FAM from BHQ1, which occurs when Nb.BsrDI cleaves its recognition site upon double-strand formation. This system uses a FAM-labeled primer (VP1-D1.#) targeting the VP1 gene, featuring Nb.BsrDI recognition sites and an internal BHQ1 quencher, and amplification is triggered by primer binding under isothermal conditions. A Schematic diagram of MCDA reactions using modified primers. Primer D1 carries a 5’-terminal endonuclease recognition site, a 5’-end FAM fluorophore (F), and an internal BHQ1 quencher (Q). Amplification generates double-stranded products containing the recognition motif. B The procedure of CVA16-E-RT-MCDA assay. Three steps, incorporating rapid viral RNA extraction (15 min), CVA16-E-RT-MCDA reaction (35 min), and results interpretation (2 min), are requested for the CVA16-E-RT-MCDA assay, and the total workflow can be completed within 52 min. The picture was drawn by using Generic BDiagramming Platform (GDP, https://BioGDP. com) [49]

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  Fig. 3. Identification of CVA16-E-RT-MCDA method. A A real-time fluorescence PCR platform was applied to analyze fluctuations in the fluorescent signals of CVA16-E-RT-MCDA amplification products. Curve 1, positive control (PC) of RNA standard template of CVA16; curve 2, negative control (NC) of virus CVA10; curve 3, blank control (BC). B The products of CVA16-E-RT-MCDA were visually observed under blue light. Tube 1, positive control (PC) of RNA standard template of CVA16; tube 2, negative control (NC) of virus CVA10; tube 3, blank control (BC). “ + ”, positive; “-”, negative. C Agarose gel electrophoresis used to detect CVA16-E-RT-MCDA products. Lane M, DL 2,000 bp DNA marker; lane 1, positive control (PC) of RNA standard template of CVA16; lane 2, negative control (NC) of virus CVA10; lane 3, blank control (BC)

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  Fig. 4. Optimal amplification temperature for CVA16-E-RT-MCDA assay. A real-time fluorescent PCR platform was employed to monitor the emission of fluorescent signals during CVA16-E-RT-MCDA amplification reactions. The corresponding curves were shown in the panels. The negative control (NC) was virus CVA10, and the blank control (BC) was RNase-free water. Abscissa represents cycles, ordinate represents fluorescence value. Positive amplification was identified on the basis of an obvious fluorescence measurement. Eight amplification curves (A-H) were revealed from 61 to 68 ℃ (1 ℃ intervals), with 1.4 × 10.6 copies/µL of RNA standard template of CVA16

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  Fig. 5. The sensitivity of CVA16-E-RT-MCDA assay. A series of dilutions (1.4 × 10⁶, 1.4 × 105, 1.4 × 104, 1.4 × 103, 1.4 × 102, 1.4 × 101 and 1.4 × 10.0 copies/µL) of RNA standard template of CVA16 and a blank control (BC) were operated according to standard CVA16-E-RT-MCDA reactions. The fluorescence signals from CVA16-E-RT-MCDA were registered by the real-time fluorescence PCR (A, C) and visually identified under blue light (B). D Ct values were mapped relative to the nucleic acid copy numbers corresponding to positive reaction outcomes. The ordinate implies the number of PCR cycles required to reach the threshold and the abscissa implies the copy number of CVA16 (log10). The error bars symbolize the SDs (n = 3)

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  Fig. 6. Analytic specificity of the CVA16-E-RT-MCDA assay. The specificity was assessed using templates from five CVA16 strains and 19 non-CVA16 pathogens. All products were detected by the real-time fluorescence PCR equipment (A) and observed visually under blue light (B). Curves/tubes 1 − 5, five CVA16 strains; curves/tubes 6 − 24, 19 non-CVA16 pathogens

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  Fig. 7. Comparison of the performance of the qRT-PCR assay and CVA16-E-RT-MCDA assay in detection of 48 clinical anal swab samples and 48 clinical throat swab samples. A1-A48 represented 48 anal swab samples. T1-T48 represented 48 throat swab samples. +, positive; -, negative