Establishment and Application of a Reverse Transcription-loop-mediated Isothermal Amplification Method for Detection of Rubella Virus RNA
|Keywords||RT-LAMP Rubella Virus Gene diagnosis|
Objective: To establish a high sensitivity, specificity, simple, rapid and low-cost method to detect rubella virus RNA.Methods: Primers were designed according to the Rubella’s selfish gene E1, and then the system of Reverse Transcription-loop-mediated isothermal amplification (RT-LAMP) to detect Rubella Virus RNA were established and optimized. Meanwhile, the specificity and sensitivity were evaluated. On the basis, a simple method to observe the results with SYBR Green I were set up. 103 peripheral blood serum samples from pregnant women were tested by RT-LAMP system, and the results were compared with RT-PCR and ELISA.Results: According to the optimized results, we found that the most important influencing fact was Mg2+, which was more important than betaine and temperature. The products of RT-LAMP demonstrated the typical ladder patterns only if the sample was rubella virus RNA, but ddH2O, human genome RNA and Hepatitis C virus RNA were failed. Enzyme digestion further proved the specificity of amplification. The detection limit is about 10 copies per reaction. 103 samples of serum from pregnant women were tested for RV by RT-LAMP, RT-PCR and enzyme-linked immunoassay (ELISA). The positive cases was 15 samples by RT-LAMP、14 samples by RT-PCR, and 7 samples by ELISA .Conclusion: The results indicated that RT-LAMP system, which we established to detect Rubella Virus RNA, not only had good specificity, sensitivity, but simple, rapid and low-cost, might play a role in clinical detection.