Innovative Technology

Innovation

Focusing on the research and development and application of isothermal nucleic acid amplification technology

双重荧光RT-RAA检测5种蚊媒病毒方法的建立_周冬根

  • File size: 614.3KB

Abstract: Objective To establish a rapid recombinase-aided amplification (RAA) method for screening five mosquito-borne viruses—Dengue virus (DENV), Japanese encephalitis virus (JEV), Zika virus (ZIKV), Yellow fever virus (YFV), and Chikungunya virus (CHIKV). Methods The conserved genes of the aforementioned five viruses were analyzed as target regions, with the ribonuclease P (RNaseP) gene incorporated as an internal control. Six sets of specific primers and probes were designed, and bioinformatics analysis—including evaluation of primer dimers and mismatches—was used to combine these into three distinct dual-fluorescence RT-RAA detection systems. All systems were then tested under identical reaction conditions. The sensitivity of the three fluorescent RT-RAA detection systems was validated using in vitro-transcribed RNAs of the five viruses, while their specificity was assessed by testing against nucleic acids from Plasmodium parasites, Hantavirus, Seoul virus, and influenza viruses. Additionally, the repeatability of each system was evaluated using medium-concentration (10³ copies/mL) and low-concentration (10² copies/mL) in vitro-transcribed RNAs of the five viruses. Results Bioinformatics analysis successfully grouped the five viruses along with RNaseP into three dual-fluorescence RT-RAA detection systems: DENV-ZIKV, JEV-YFV, and CHIKV-RNaseP. Each system was optimized to perform at 39°C for 15 minutes. Sensitivity assays revealed that all three dual-fluorescence RT-RAA systems could detect as few as 1–10 copies of in vitro-transcribed RNA from the target viruses, with DENV, ZIKV, and YFV achieving detection limits of 1 copy/mL, JEV at 10 copies/mL, and CHIKV at 10² copies/mL. Importantly, none of the three systems exhibited cross-reactivity with other viruses. Furthermore, all three systems demonstrated 100.0% detection rates when analyzing medium-concentration in vitro-transcribed RNAs, while maintaining an impressive 83.3% detection rate even at lower concentrations. Conclusion The developed detection method exhibits excellent sensitivity, specificity, and reproducibility, making it suitable for identifying the five mosquito-borne viruses in both human and mosquito samples.

Previous page

实时荧光逆转录重组酶介导的等温扩增技术检测寨卡病毒方法的建立

塔希纳病毒的重组酶介导扩增快速检测方法-CDC&云南寄生虫防治所

Next page

Previous page

实时荧光逆转录重组酶介导的等温扩增技术检测寨卡病毒方法的建立

Next page

塔希纳病毒的重组酶介导扩增快速检测方法-CDC&云南寄生虫防治所