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微流控芯片恒温扩增技术快速检测多种呼吸道病原体的应用

Combination of micro-fluidics and loop-mediated isothermal amplification method for multiplex-detection of respiratory pathogens

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【作者】 池细俤高世华张忠源叶桂云陈家龙

【Author】 CHI Xi-di;GAO Shi-hua;ZHANG Zhong-yuan;The Affiliated First Hospital of Nanping,Fujian Medical University;

【通讯作者】 高世华;

【机构】 福建医科大学附属南平第一医院检验科

【摘要】 目的 利用微流控芯片恒温扩增技术(LAMP-Disk)建立多种呼吸道病原体快速检测的方法,并对其临床应用的可行性和价值进行评估。方法 设计和筛选针对甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、鼻病毒和肺炎支原体的LAMP引物对,利用盘式微流控芯片建立快速检测方法。以人工构建的参考品确定LAMP-Disk方法的检出限和分析特异性。收集268份呼吸道感染患儿的咽拭子样本,使用荧光定量PCR(qPCR)法与LAMP-Disk进行平行检测,从灵敏度、特异性及总体符合率等方面比较两种方法在临床应用的相关性和差异。结果 LAMP-Disk检测方法对7种呼吸道病原体的检出限为3000~30000 copies/mL。在268份样本中,以qPCR方法为金标准,LAMP-Disk检测甲型流感病毒、乙型流感病毒、呼吸道合胞病毒、腺病毒、副流感病毒、鼻病毒和肺炎支原体的灵敏度分别为90.0%、86.5%、90.6%、64.8%、75.0%、88.2%和91.4%;特异性分别为99.6%、100%、99.2%、99.5%、99.2%、99.6%和100%;总体符合率为98.9%、98.1%、98.1%、92.5%、97.0%、98.9%和98.9%。结论 采用LAMP-Disk方法同时检测多种呼吸道病原体,特异性和灵敏度好,操作简单快速,适合快速检测的应用场景。

【Abstract】 Objective To set-up a loop-mediated isothermal amplification method based on micro-fluidics(LAMP-Disk) for rapid multiplex-detection of respiratory pathogens.Methods Design and selection of primer sets specifically for influenza A virus(Flu A),influenza B virus(Flu B),respiratory syncytial virus(RSV),adenovirus(ADV),Parainfluenza virus(PIV),rhinovirus(HRV),and mycoplasma pneumoniae(MP),respectively.The detection method is a combination of micro-fluidics and LAMP.Artificial references were used for analysis of lower limit of detection(LOD) and specificity.A total of 268 samples from pediatric patients with acute respiratory tract infection were used for comparison the sensitivity, specificity, and consistency between LAMP-Disk and real-time quantitative PCR(qPCR).Results The LODs for the seven pathogens ranged from 3000 to 30000 copies/mL.Among the 268 clinical samples, the sensitivity of LAMP-Disk for FluA,FluB,RSV,ADV,PIV,HRV and MP was 90.0%,86.5%,90.6%,64.8%,75.0%,88.2%,and 91.4%,respectively.The specificity was 99.6%,100%,99.2%,99.5%,99.2%,99.6%,and 100%,respectively.The overall consistency was 8.9%、98.1%、98.1%、92.5%、97.0%、98.9%,and 98.9%,respectively.Conclusion LAMP-Disk is a rapid and simple method for multiplex detection of respiratory pathogens in clinical settings, with satisfactory specificity and sensitivity.

【基金】 福建省自然科学基金资助项目(2019J01609)
  • 【文献出处】 中国实验诊断学 ,Chinese Journal of Laboratory Diagnosis , 编辑部邮箱 ,2023年03期
  • 【分类号】R446.5
  • 【下载频次】81
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