We have developed a simple, user-friendly, and highly sensitive Zika virus (ZIKV) detection method by incorporating optimized reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a lateral flow assay (LFA). The optimized RT-LAMP reaction was carried out using Bst 3.0 polymerase, which has robust and fast isothermal amplification performance even in the presence of high concentrations of inhibitors; this permitted the amplification of ZIKV RNA in pure water and human whole blood. In addition, the strong reverse transcription activity of Bst 3.0 polymerase enabled specific ZIKV RNA amplification without extra addition of reverse transcriptase. The RT-LAMP condition was optimized by adjusting the Mg2+ and dNTP mix concentration to extirpate nontarget amplification, which is caused by nonspecific primer dimers amplification. After 30 min of RT-LAMP reaction, the resultant amplicons were simply and rapidly analyzed by the LFA test in less than 5 min. The optimized RT-LAMP combined with the LFA allowed specific ZIKV RNA detection down to the single copy level within 35 min.
Bibliographical noteFunding Information:
This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (MEST; NRF-2015R1D1A1A01059806 and NRF-2015R1A5A7037674) and by a Grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (Grant No.: HI16C0272). J.H.L. was also supported by a Research Grant from Kwangwoon University in 2016.
© 2016 American Chemical Society.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry