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    朱顶红褪绿环斑病毒RT-LAMP快速检测体系的建立

    Development of an RT-LAMP Assay for Rapid Detection of hippeastrum chlorotic ringspot virus (HCRV)

    • 摘要: 为快速检测朱顶红褪绿环斑病毒(hippeastrum chlorotic ringspot virus, HCRV),根据HCRV的核壳体(N)蛋白基因保守核苷酸序列,设计了5组引物进行筛选,并采用单一变量法对RT-LAMP体系反应温度、时间、甜菜碱浓度、dNTPs浓度、Mg2+浓度、内外引物浓度比等进行逐一优化,建立了HCRV逆转录环介导等温扩增(RT-LAMP)检测体系。结果表明,最佳引物组为P1,最适反应温度为64 ℃,Betaine、dNTPs、Mg2+的最佳终浓度分别为0.8、0.2、6 mmol/L,最佳内外引物浓度比为5∶1,最佳反应时间50 min。检测结果显示优化后的RT-LAMP经SYBR Green I染色可通过肉眼直接判断结果,具有高度特异性,且灵敏度是常规RT-PCR的100倍。本研究为HCRV的检测提供了一种便捷、高效、可靠的方法。

       

      Abstract: In order to quickly detect the hippeastrum chlorotic ringspot virus(HCRV), five sets of primers were designed and screened according to the conserved nucleotide sequence of the nucleocapsid (N) protein gene of HCRV, and the RT-LAMP system reaction temperature, time, betaine concentration, dNTP concentration, Mg2+ concentration, and concentration ratio of internal and external primers were optimized by single variable method. A detection system of HCRV reverse transcription loop mediated isothermal amplification (RT-LAMP) was established. The results showed that the optimal primer group was P1, the optimal reaction temperature was 64 ℃, and the optimal final concentrations of Betaine, dNTPs, and Mg2+ were 0.8, 0.2, 6 mmol/L. The optimal concentration ratio of internal and external primers is 5∶1, and the optimal reaction time is 50 minutes. The test results showed that after staining with SYBR Green I, the optimized RT-LAMP can be directly evaluated by naked eyes, with high specificity and sensitivity, 100 times higher than that of conventional RT-PCR. This study provides a convenient, efficient, and reliable method for the detection of HCRV.

       

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