可视化登革热病毒1型～3型RT-LAMP快速分型检测研究

Rapid visual typing detection of Dengue virus type 1-3 by RT-LAMP assays

目的建立快速分型检测登革热病毒(Dengue virus,DEN)1型~3型的RT-LAMP方法。方法设计特异性引物分别建立DEN-1型、DEN-2型和DEN-3型RT-LAMP方法,利用DEN-1型、DEN-2型和DEN-3型RT-LAMP方法分别检测23份DEN(20份DEN-1、2份DEN-2和1份DEN-3)和20份肝炎病毒(10份HBV和10份HCV)阳性血清标本,评估各型DEN RT-LAMP方法的特异性和敏感度;RT-LAMP扩增结果通过肉眼、电泳和酶切来鉴定。结果经过对23份DEN和20份肝炎病毒阳性血清标本的检测,DEN-1型、DEN-2型和DEN-3型RTLAMP方法只针对靶病毒出现阳性扩增结果,结果可以通过肉眼和电泳进行判断,酶切证实了DEN-1型、DEN-2型和DEN-3型RT-LAMP产物的特异性,DEN-1型、DEN-2型和DEN-3型RT-LAMP方法敏感度均为45 pg/反应管。结论建立了可视化的DEN-1型、DEN-2型和DEN-3型RT-LAMP快速分型检测方法。

Objective To develop the reverse transcription loop-mediated isothermal amplification（RT-LAMP） assays for the rapid typing detection of Dengue virus（DEN） type 1-3. Methods Specific oligonucleotide primers were designed for developing the RT-LAMP assay of DEN-1,DEN-2 and DEN-3,respectively,which was adopted in the detection of the positive serum of 23 specimens of DEN（twenty cases of DEN-1,two cases of DEN-2 and one case of DEN-3） and 20 specimens of hepatitis virus positive（ten cases of HBV and ten cases of HCV）,so as to evaluate the specificity and sensitivity of the assay for each DEN type. RT-LAMP amplification results were identified by bare eyes,electrophoretic analysis and restriction digestion. Results According to the above test,RT-LAMP methods were all only positive for the target virus,and the results could be identified by bare eyes and electrophoretic analysis. The specificity of DEN-1,DEN-2 and DEN-3 type RT-LAMP products were also confirmed with enzyme digestion,and the RT-LAMP sensitivity of DEN-1,DEN-2 and DEN-3 were all 45 pg/tube. Conclusion Rapid visual typing methods for DEN-1,DEN-2 and DEN-3 by RT-LAMP were established in this study.