一种无RNA纯化步骤TaqMan RT—PCR方法用于快速检测登革病毒 |
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引用本文: | 郑夔,;洪烨,;李小波,;黄吉城,;师永霞,;幸芦琴,;相大鹏,;郭波旋,;胡龙飞. 一种无RNA纯化步骤TaqMan RT—PCR方法用于快速检测登革病毒[J]. 广东卫生防疫, 2008, 0(3): 6-9 |
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作者姓名: | 郑夔, 洪烨, 李小波, 黄吉城, 师永霞, 幸芦琴, 相大鹏, 郭波旋, 胡龙飞 |
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作者单位: | [1]广东检验检疫技术中心卫生检疫实验室,广东广州510700; [2]云浮出入境检验检疫局,广东广州510700; |
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基金项目: | 2005年度国家质检总局科技计划项目(编号2005IK-079) |
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摘 要: | 目的 建立一种简便、快速的登革病毒核酸检测方法。方法 设计合成LNA探针,替代MGB探针,建立一步法TaqManRT—PCR方法,并用已知的登革病毒标准毒株进行方法的优化;用商品化的纯化试剂盒和简单的热释放法获取登革病毒细胞培养液和模拟含登革病毒血清中RNA。以优化的TaqManRT—PCR方法进行检测,比较有或无RNA纯化步骤检测的敏感性和重复性。结果 LNA探针TaqMan RT—PCR有较好的PCR扩增效率(104%)、较广的线性范围(10^0-10^-7样品稀释度)、较高的敏感性(0.003TCID50/反应)和较好的重复性(CV值小于4.53%),新方法检测经简单热释放RNA的细胞培养液和模拟含登革病毒血清中登革病毒也有较高的敏感性(0.03TCID50/反应)和较好的重复性(CV值小于6.36%)。结论 新型LNA探针可替代MGB探针用于TaqMan RT—PCR反应。简便的热释放RNA方法结合高敏感性的TaqMan RT—PCR可满足登革病毒快速检测的需要。
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关 键 词: | 登革病毒 逆转录聚合酶链反应 LNA探针 |
A TaqMan RT - PCR assay without RNA purification for rapid detection of dengue virus |
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Affiliation: | ZHENG Kui, HONG Ye, LI Xiao - bo, et al.( Guangdong Inspection and Quarantine Technology Center Health Quarantine Lab, Guangzhou 510700, China ) |
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Abstract: | Objective To develop a simple and rapid detection method for nucleic acid of dengue virus. Methods A LNA probe was designed and synthesized to replace the MGB probe for the established TaqMan RT- PCR assay, and then optimized with a known dengue virus standard strain. A commercial RNA extraction kit and simple heat -release method were used to obtain dengue virus RNA from cell culture supernate and simulated dengue virus serum, which was detected by the optimized TaqMan RT - PCR assay. The results without RNA purification were compared with methods with RNA purification. Results The LNA probe based TaqMan RT- PCR assay showed good PCR amplification efficiency( 104% ), broad linear range( 10^0 - 10 ^-7), high sensitivity(0.003TCID50/reaction) and repeatability( CV 〈 4.53% ). High sensitivity (0.03TCID50/reaction) and repeatability( CV 〈 6.36% ) were also observed by the new method for detection of the dengue virus in cell culture supernate and simulated serum after heat - releasing RNA. Conclusion The sensitive TaqMan RT - PCR assay, with innovated LNA probe to replace MGB probe, combined with simple heat - release RNA method, satisfied routine dengue virus detection. |
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Keywords: | Dengue virus RT - PCR LNA probe |
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