Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya.

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Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya Viruses Huixin Chen, Mariya Parimelalagan, Yee Ling Lai, Kim Sung Lee, Evelyn Siew-Chuan Koay, Hapuarachchige C. Hapuarachchi, Lee Ching Ng, Phui San Ho, Justin Jang Hann Chu The Journal of Molecular Diagnostics Volume 17, Issue 6, Pages 722-728 (November 2015) DOI: 10.1016/j.jmoldx.2015.06.008 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Melting curve is used to select the best primer set for detecting and serotyping dengue virus (DENV)–1, -2, -3, and -4. The melting curves of DENV-1 (A), DENV-2 (B), DENV-3 (C), and DENV-4 (D) reveal that the specific melting temperatures (Tms) of the PCR amplicon are 84.86°C (A), 82.61°C (B), 85.60°C (C), and 83.65°C (D), respectively. The Journal of Molecular Diagnostics 2015 17, 722-728DOI: (10.1016/j.jmoldx.2015.06.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 The primer set of the dengue virus (DENV) serotyping primer (DEst1) and the chikungunya virus (CHK1) were tested in combination for simultaneous detection and differentiation of DENV and CHIKV. The melting curve for each targeting virus is well separated, indicating that they not only are able to detect DENV and CHIKV but also have good compatibility in terms of similar annealing temperature and low tendency of forming heterodimers. The results from the combination of DEst1 and CHK1 also reveal good consistency compared with use of DEst1 alone in terms of specific melting temperatures (Tms) of amplicon from DENV-1, -2, -3, and -4. The Journal of Molecular Diagnostics 2015 17, 722-728DOI: (10.1016/j.jmoldx.2015.06.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Temperature range for viral identification. A temperature range for PCR amplicon specific to dengue virus (DENV)–1, -2, -3, and -4 and chikungunya virus (CHIKV) is given. The melting temperatures (Tms) of the amplicons are within the range from 84.3°C to 85.0°C for DENV-1, 81.4°C to 83.0°C for DENV-2, 85.0°C to 86.5°C for DENV-3, 83.0°C to 84.3°C for DENV-4, and 87.0°C to 89.0°C for CHIKV. Samples with a Tm at 81.0°C or below are defined as negative. The Journal of Molecular Diagnostics 2015 17, 722-728DOI: (10.1016/j.jmoldx.2015.06.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Standard curves for dengue virus (DENV)–1, -2, -3, and -4 and chikungunya virus (CHIKV). The standard curves for virus quantitation using SYBR Green I–based quantitative real-time RT-PCR were generated from the amplification of in vitro transcribed RNA-containing primer targeting sites. The cross-point values obtained in the real-time reactions for the known amounts of the RNA template were plotted against the log of the number of genome equivalent copies. The curve is representative of a typical experiment. The equation that describes the graph, data coefficient (R2), and PCR efficiency (E) of the reaction are indicated. Standard curves of DENV-1 (A), DENV-2 (B), DENV-3 (C), DENV-4 (D), and CHIKV (E) are shown. The Journal of Molecular Diagnostics 2015 17, 722-728DOI: (10.1016/j.jmoldx.2015.06.008) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions