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2009•Journal of Tropical MedicineOpen access

Development of Real- Time Quantitative RT- PCR for the Detection of Dengue Virus

Yulan Zhu, Dianpeng Wang, Zhen ShengXi, Huang ZongYan, Shengya Liu, Gao Zhao-xian

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Abstract

Objective To develop a real-time quantitative RT-PCR method with TaqMan Probe to assess the RNA copy number of Dengue virus. Methods Primers and TaqMan probe targeting the signature and the conserved sequence of four the Dengue virus subtypes were designed. The reaction protocol was optimized. The sensitivity, specificity and reproducibility were then evaluated. Conventional RT-PCR was also used to compare for the sensitivity of this method. Results The sensitivity of this assay was 1×103 copies / mL. The detection method was found to be highly specific and reproducible. The standard deviation from five separate measurements of the same sample was 0.792. Conclusion The developed method can be used to quantitatively measure the copy number of Dengue virus RNA.

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What this paper is about

Objective To develop a real-time quantitative RT-PCR method with TaqMan Probe to assess the RNA copy number of Dengue virus. Methods Primers and TaqMan probe targeting the signature and the conserved sequence of four the Dengue virus subtypes were designed. The reaction protocol was optimized. The sensitivity, specificity and reproducibility were then evaluated. Conventional RT-PCR was also used to compare for the sensitivity of this method. Results The sensitivity of this assay was 1×103 copies / mL. The detection method was found to be highly specific and reproducible. The standard deviation from five separate measurements of the same sample was 0.792. Conclusion The developed method can be used to quantitatively measure the copy number of Dengue virus RNA.

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Available abstract

Objective To develop a real-time quantitative RT-PCR method with TaqMan Probe to assess the RNA copy number of Dengue virus. Methods Primers and TaqMan probe targeting the signature and the conserved sequence of four the Dengue virus subtypes were designed. The reaction protocol was optimized. The sensitivity, specificity and reproducibility were then evaluated. Conventional RT-PCR was also used to compare for the sensitivity of this method. Results The sensitivity of this assay was 1×103 copies / mL. The detection method was found to be highly specific and reproducible. The standard deviation from five separate measurements of the same sample was 0.792. Conclusion The developed method can be used to quantitatively measure the copy number of Dengue virus RNA.

Key concepts: TaqMan, Dengue virus, Dengue fever, Real-time polymerase chain reaction, Virology, Biology, Virus, RNA

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