This product includes 350 ul of 10 x Buffer, 350 ul of 10 x MnCl2, 33 ul of 100 x Template, 33 ul of 100 x NTP, 31 ul of 100 x Zika virus RdRp and 330 ul of 10 x fluorescence dye.
The Flaviviridae virus family are positive, single-stranded, enveloped RNA viruses. Zika virus is one of the Flaviviridae family. The RNA-dependent RNA Polymerase (RdRp) is an essential enzyme for the virus and is an attractive drug target. The viral RdRp assay is based on detection of RNA synthesis by the RdRp enzyme. The assay can be performed in a 384-well or 96-well plate format for tests of the enzyme activities of RNA polymerases in the Flaviviridae family and high throughput screening of inhibitors.
The Zika Virus RNA-dependent RNA Polymerase Assay Kit Plus-100 (Catalog No. ZRPA100KE) includes 350 µl of 10 x Buffer, 350 µl of 10 x MnCl2, 33 µl of 100 x Template, 33 µl of 100 x NTP, 31 µl of 100 x Zika virus RdRp and 330 µl of 10 x fluorescence dye. It is for 100 assays of virus RNA polymerase in a 384-well plate format.
Reference
1. Shu Yang et al, Emetine inhibits Zika and Ebola virus infections through two molecular mechanisms: inhibiting viral replication and decreasing viral entry, Cell Discovery, Volume 4, Article number: 31, (05 June 2018).
2. Chen Y. et al, Cinnamic acid inhibits Zika virus by inhibiting RdRp activity. Antiviral Research Volume 192, August 2021, 105117.
Assay Protocol
The following assay protocol is based on the 384-well plate assay format (plate type: Matrix 4318 or alike). The reaction volume is 30 µl and the final assay volume is 60 µl. For 96-well plate assays (plate type: Costar 3915 or alike), the reaction volume is 50 µl and the final assay volume is 100 µl.
1. Reaction: The total volume of each reaction mixture is 30 µl including 23.1 µl of H2O, 3 µl of 10 x Buffer, 3 µl of 10 x MnCl2, 0.3 µl of 100 x template, 0.3 µl of 100 x RNA polymerase and 0.3 µl of 100 x NTPs. Incubate the reaction mixture at 30°C for 90 min.
2. Detection: Dilute the 10x fluorescence dye with water to make the 1 x dye. Mix 30 µl of the 1 x dye with 30 µl of the reaction mixture. Measure the fluorescence intensity at 535 nm using the excitation wavelength at 485 nm in 5 min.
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