新冠病毒RT-LAMP可视化检测方法的建立  

Establishment of the RT-LAMP Visual Detection Method for Novel Coronavirus

作　　者：刘树荣 陶飞飞 熊园翔 温林 郭韫丽[1,2] 孔令保[1,2] LIU Shurong;TAO Feifei;XIONG Yuanxiang;WEN Lin;GUO Yunli;KONG Lingbao(School of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China;Nanchang Key Laboratory of Animal Virus and Gene Engineering,Jiangxi Agricultural University,Nanchang 330045,China)

机构地区：[1]江西农业大学生物科学与工程学院,江西南昌330045 [2]江西农业大学南昌市动物病毒与基因工程重点实验室,江西南昌330045

出　　处：《生物灾害科学》2022年第4期415-422,共8页Biological Disaster Science

基　　金：2022年江西省省级大学生创新训练计划项目(S202210410105)。

摘　　要：【目的】为了实现对新冠病毒一步法检测。【方法】采用RT-LAMP技术,利用具有反转录酶活性的Bst3.0 DNA聚合酶,以新冠病毒N蛋白基因为模板设计LAMP引物,成功建立了以新冠N蛋白基因为靶基因的RT-LAMP检测方法。同时对反应条件中的温度、镁离子浓度、NTP浓度、甜菜碱浓度以及酶浓度进行优化。通过加入钙黄绿素和氯化锰显色剂对反应结果进行判定,用ASFV p54质粒和JEV NS1质粒与稀释后模板做对照。【结果】温度为66℃,镁离子浓度为6mmol/L,甜菜碱浓度为1 mol/L,NTP为1.4 mmol/L,酶浓度为160 U/L为最优反应条件。【结论】证明了Bst3.0 DNA聚合酶对新冠N蛋白基因检测是成功的,该方法具有良好的特异性以及灵敏度。[Objective]The experiment was conducted to achieve one-step detection of novel coronavirus.[Method]The RT-LAMP technique was used to design LAMP primers,using the novel coronavirus N protein gene as the template by using Bst3.0 DNA polymerase with reverse transcriptase activity,and the RT-LAMP detection method using the novel coronavirus N protein gene as the target gene was successfully established.At the same time,the temperature,magnesium ion concentration,NTP concentration,betaine concentration and enzyme concentration in the reaction conditions were optimized.The reaction results were evaluated by adding calcein and manganese chloride chromogenic agent,and ASFV p54 and JEV NS1 plasmids were compared with the diluted template.[Results]The optimal reaction conditions were 66℃,6 mmol/L magnesium ion concentration,1 mol/L betaine 1.4 mmol/L NTP and 160 U/L enzyme concentration.[Conclusion]It is proved that Bst3.0 DNA polymerase is successful in the detection of novel coronavirus N protein gene,showing that the method has good specificity and sensitivity.

关 键 词：新型冠状病毒 环介导等温扩增技术 可视化 

分 类 号：R563.1[医药卫生—呼吸系统] R115[医药卫生—内科学]