Detection of measles virus genome by reverse transcription-loop mediated isothermal amplification (RT-LAMP)
Author(s): Li Shuhua, Liu Yan, Cao Guangwen
Pages: 186-189
Year: 2014 Issue: 2
Journal: Chinese Journal of Epidemiology
Keyword: Measles virus; Loop-mediated isothermal amplification; Reverse transcription-polymerase chain reaction;
Abstract: Objective To establish a tool regarding the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the detection of measles virus.Methods Measles virus RNA was extracted by Trizol-LS reagent.The 1 242-1 442 sequence contained 8 primer sites of 6 sets primer.The RT-LAMP gene amplification was detected by a real-time PCR facility with AMV reverse transcriptase at 63 ℃ for 60 min before terminating the amplification at 80 ℃ 2 min.The amplified product was monitored by agarose gel electrophoresis and loop amp fluorescence methods.Sensitivity and specificity of the RT-LAMP assay were subsequently compared with that of conventional RT-PCR.Results The whole procedure of RT-LAMP took about 1 hour.The amplified products appeared to be a ladder-like electrophoresis pattern during the process of agarose gel electrophoresis.The appearance of color change in the reactions with positive controls and positive samples was evident at 20 min after RT-LAMP initiation.The sensitivity of RT-LAMP assay was 100-fold higher than that of the conventional RT-PCR of the real-time RT-PCR.The specificity of MV-specific LAMP assay was conformed by negative amplification of dengue virus and Japanese encephalitis virus.Conclusion RT-LAMP assay appeared rapid,cost-effective,highly sensitive and specific for the detection of genes of imerest and proved to be potentially useful for surveillance on MV,especially in the grass root laboratories or for field studies.
Pages: 186-189
Year: 2014 Issue: 2
Journal: Chinese Journal of Epidemiology
Keyword: Measles virus; Loop-mediated isothermal amplification; Reverse transcription-polymerase chain reaction;
Abstract: Objective To establish a tool regarding the reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for the detection of measles virus.Methods Measles virus RNA was extracted by Trizol-LS reagent.The 1 242-1 442 sequence contained 8 primer sites of 6 sets primer.The RT-LAMP gene amplification was detected by a real-time PCR facility with AMV reverse transcriptase at 63 ℃ for 60 min before terminating the amplification at 80 ℃ 2 min.The amplified product was monitored by agarose gel electrophoresis and loop amp fluorescence methods.Sensitivity and specificity of the RT-LAMP assay were subsequently compared with that of conventional RT-PCR.Results The whole procedure of RT-LAMP took about 1 hour.The amplified products appeared to be a ladder-like electrophoresis pattern during the process of agarose gel electrophoresis.The appearance of color change in the reactions with positive controls and positive samples was evident at 20 min after RT-LAMP initiation.The sensitivity of RT-LAMP assay was 100-fold higher than that of the conventional RT-PCR of the real-time RT-PCR.The specificity of MV-specific LAMP assay was conformed by negative amplification of dengue virus and Japanese encephalitis virus.Conclusion RT-LAMP assay appeared rapid,cost-effective,highly sensitive and specific for the detection of genes of imerest and proved to be potentially useful for surveillance on MV,especially in the grass root laboratories or for field studies.
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