A Reverse Transcription-Polymerase Spiral Reaction (RT-PSR)-Based Rapid Coxsackievirus A16 Detection Method and Its Application in the Clinical Diagnosis of Hand, Foot, and Mouth Disease

Figures

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  FIGURE 1. Schematic diagram of reverse transcription-polymerase spiral reaction (RT-PSR) amplification. (A) Template RNA; (B) Reverse transcription of template RNA into cDNA; (C) The F segment of Ft specifically recognizes and binds to the Fc segment on the target single-stranded cDNA; (D) Under the action of Bst DNA polymerase and dNTP, the primer extends to the 3’gap to form a double-stranded DNA structure; (E) The Nc segment on the newly formed single-stranded DNA rotate and combine with the N segment to form a U-shaped structure; (F) The Bcr segment of Bt is combined with the B segment of the U-shaped structure and extends around the structure.

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  FIGURE 2. (A) PCR using cDNA as a template. Lane M: DL 1,000 DNA marker. (1) Negative control (NC), diethylpyrocarbonate (DEPC)-treated water; (2) 100 ng/μl cDNA. (B) Polymerase spiral reaction (PSR) fluorescence amplification curve of two sets of primers. (C) Reverse transcription (RT)-PSR fluorescence amplification curve of two sets of primers. (D) One percent agarose gel electrophoresis of RT-PSR amplified products of primer 2. Lane M: DL 1,000 DNA marker. (1) Positive sample; (2) NC, DEPC-treated water. (E) Fluorescence amplification curves of RT-PSR at different temperatures.

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  FIGURE 3. Sensitivity of reverse transcription-polymerase spiral reaction (RT-PSR) for Coxsackievirus A16 (CVA-16) detection. (A) The sensitivity test result of RT-PSR uses hydroxy naphthol blue (HNB) indicator; (1) 2.4 × 108 copies/μL; (2) 2.4 × 107 copies/μL; (3) 2.4 × 106 copies/μL; (4) 2.4 × 105 copies/μL; (5) 2.4 × 104 copies/μL; (6) 2.4 × 103 copies/μL; (7) 2.4 × 102 copies/μL; (8) 2.4 × 101 copies/μL; negative control (NC), diethylpyrocarbonate (DEPC)-treated water. (B) The sensitivity test result of RT-PSR using agarose gel electrophoresis. Lane M: DL 1,000 DNA marker. (1) 2.4 × 108 copies/μL; (2) 2.4 × 107 copies/μL; (3) 2.4 × 106 copies/μL; (4) 2.4 × 105 copies/μL; (5) 2.4 × 104 copies/μL; (6) 2.4 × 103 copies/μL; (7) 2.4 × 102 copies/μL; (8) 2.4 × 101 copies/μL; negative control (NC), diethylpyrocarbonate (DEPC)-treated water.

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  FIGURE 4. Specificity of the reverse transcription-polymerase spiral reaction (RT-PSR) method. (A) The specificity test result of RT-PSR uses hydroxy naphthol blue (HNB) indicator; (1) Coxsackievirus A16 (CVA-16); (2) Norwalk virus genogroup II (NVG II); (3) Rotavirus A (RVA); (4) CVA-6; (5) Enterovirus (EV)-A71; negative control (NC), diethylpyrocarbonate (DEPC)-treated water. (B) The specificity test result of RT-PSR using agarose gel electrophoresis. Lane M: DL 1,000 DNA marker. (1) Coxsackievirus A16 (CVA-16); (2) Norwalk virus genogroup II (NVG II); (3) Rotavirus A (RVA); (4) CVA-6; (5) Enterovirus (EV)-A71; negative control (NC), diethylpyrocarbonate (DEPC)-treated water.