摘要
【目的】开发一种快速高效的结核分枝杆菌(MTB)检测方法,给基层单位、偏远地区的结核病(Tuberculosis)快速筛查提供技术方案,更好地开展结核病的防治工作。【方法】设计RPA引物、crRNA,并引入重力驱动的微流控芯片,建立RPA-CRISPR/Cas12a的片上检测方法,进一步分析该方法的灵敏度与特异性,使用疑似结核病患者的样本进行片上检测和痰培养,比较建立的方法与痰培养法的一致性。【结果】建立的RPA-CRISPR/Cas12a片上检测方法可在30 min内实现MTB检测,LOD为1copies/μL。以痰培养鉴定为参考标准,片上检测法的灵敏度为91.11%,特异度为94.34%,阳性预测值为93.19%,阴性预测值为92.59%,准确性为92.86%,Kappa值为0.856。【结论】建立的RPA-CRISPR/Cas12a的片上检测法的灵敏度、特异性高,并且快速、简单、便捷。
【Objective】To develop a rapid and efficient method for detecting Mycobacterium tuberculosis(MTB),providing a technical solution for rapid screening of tuberculosis(TB)in grassroots units and remote areas,thereby enhancing TB prevention and control efforts.【Method】RPA(recombinase polymerase amplification)primers and crRNA were designed,and a gravity-driven microfluidic chip was introduced to establish an RPA-CRISPR/Cas12a on-chip detection method.The sensitivity and specificity of this method were further analyzed using samples from suspected TB patients for on-chip detection and sputum culture,comparing the established method with the sputum culture method for consistency.【Result】The established RPA-CRISPR/Cas12a on-chip detection method achieves MTB detection within 30 min,with a limit of detection(LOD)of 1 copy/μL.Using sputum culture identification as a reference standard,the on-chip detection method demonstrated a sensitivity of 91.11%,specificity of 94.34%,positive predictive value of 93.19%,negative predictive value of 92.59%,and accuracy of 92.86%,with a Kappa value of 0.856.【Conclusion】The developed RPA-CRISPR/Cas12a on-chip detection method presents high sensitivity and specificity,characterized by rapid,simple,and convenient.
作者
高畅
庄添驰
李宁
刘云
顾鹏飞
赵昕怡
季明辉
GAO Chang;ZHUANG Tian-chi;LI Ning;LIU Yun;GU Peng-fei;ZHAO Xin-yi;JI Ming-hui(Second Clinical Medical College,Nanjing Medical University,Nanjing 211166;School of Nursing,Nanjing Medical University,Nanjing 211166;Jiangning District Center for Disease Control and Prevention,Nanjing 211100;Qinhuai District Center for Disease Control and Prevention,Nanjing 210001;First Clinical Medical College,Nanjing Medical University,Nanjing 211166)
出处
《生物技术通报》
北大核心
2025年第5期62-69,共8页
Biotechnology Bulletin
基金
江苏省卫生健康委员会预防医学科研课题面上项目(Ym2023064)
国家级大学生创新创业训练计划基金项目(202410312005Z)
南京预防医学科研课题(NJYFKT202409)
江苏高校优势学科建设工程四期项目“护理学”(苏教研函[2023]11号)。
作者简介
高畅,女,研究方向:病原体检测,E-mail:1975480691@qq.com;共同第一作者:庄添驰;通信作者:季明辉,男,博士,副教授,研究方向:病原体检测,E-mail:jiminghui@njmu.edu.cn。
