鹦鹉热衣原体荧光环介导等温扩增(LAMP)检测方法的建立
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国家重点研发计划专项(2017YFC1200504)


Establishment of a Loop-mediated Isothermal Amplification Method for Chlamydia psittaci
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    摘要:

    鹦鹉热衣原体(Chlamydia psittaci)是一种人兽共患病原体,能引起自然疫源性衣原体病,目前广泛分布于世界各地。本研究应用环介导等温扩增技术(LAMP),针对鹦鹉热衣原体23S RNA基因序列设计引物,并进行筛选以及敏感性和特异性试验,建立了鹦鹉热衣原体恒温荧光扩增方法。该方法在63 ℃恒温条件下1 h内即可显示结果,操作简单、快速,特异性强,灵敏度可达100拷贝/μL,且与流产衣原体、动物布鲁氏菌、牛结核分枝杆菌、沙门氏菌、大肠杆菌、金黄色葡萄球菌无交叉反应;用国产仪器可直接通过检测荧光信号判读结果,既增强了准确性,也避免了开盖污染产生假阳性;应用建立的LAMP方法对实验室保存的临床DNA样品进行检测,发现检测结果与QPCR相同。该方法的建立弥补了传统检测技术的不足,为实现鹦鹉热衣原体的现场快速诊断提供了技术支持。

    Abstract:

    Chlamydia psittaci,a kind of zoonotic pathogen,is widely distributed all over the world,by which,chlamydiosis of natural origin might be caused. In this study,the primers were designed based on 23S RNA gene sequence of Chlamydia psittaci,and then screened. After sensitivity and specificity tests,a thermostatic fluorescence amplification method for Chlamydia psittaci was established according to the loop-mediated isothermal amplification method(LAMP). By the established method,test results could be shown within one hour under the constant temperature of 63 ℃ due to its advantages including simple operation,rapidity,strong specificity,sensitivity of 100 copies/μL and no cross-reaction with Chlamydia abortus,Brucella animalis,Mycobacterium bovis,Salmonella,Escherichia coli and Staphylococcus aureus. The results could be interpreted directly by fluorescence signals with domestic instruments,which not only enhanced the accuracy,but also avoided false positive due to pollution brought by cap opening. The clinical DNA samples stored in laboratories were detected by the LAMP method,it was found that the results were same as that by QPCR. It was concluded that traditional methods were remedied by the established method,which provided technical support for rapid field detection of Chlamydia psittaci.

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孙翔翔,张培培,刘蒙达,樊晓旭,孙世雄,孙明军.鹦鹉热衣原体荧光环介导等温扩增(LAMP)检测方法的建立[J].《中国动物检疫》编辑部,2020,37(9):101-105.

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  • 在线发布日期: 2020-09-02
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