A Review on the Advancements in Molecular Point-of-Care Tests
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摘要: 分子即时检测(Point-of-care Test,POCT)是一种快速发展的核酸检验方法,主要基于等温扩增和微流控等技术,具有检验时间短、设备小、操作简单的优点,为“样本进,结果出”的检验需求提供了实现的可能,为新型冠状病毒肺炎等疫情防控和快速筛查提供了有力支撑。分子POCT诊断产品现可用于检测人乳头瘤病毒、人体免疫缺陷病毒、丙型肝炎病毒、淋球菌等病原微生物,具有检测时间短和便携化程度强等特点。但目前该技术还处于产业发展初级阶段,还需要进一步提高分子POCT产品的准确性和一致性,并完善相关质量管理办法和标准,建立标准化验证评价体系。本文综述了基于等温扩增、微流控等原理的分子POCT技术、设备和应用的最新研究进展,并对该技术未来的发展趋势进行了展望。Abstract: The Molecular Point-of-Care Test (POCT) represents a rapidly evolving nucleic acid testing method primarily grounded on isothermal amplification and microfluidic technologies. This technique provides numerous advantages including rapid testing, compact hardware, and simplicity in operation, thereby paving the way for immediate "sample in, result out" diagnostics. It serves as a powerful tool for the containment and management of epidemics such as COVID-19. Currently available molecular POCT diagnostic tools can detect various pathogenic microorganisms like human papillomavirus, human immunodeficiency virus, hepatitis C virus, and Neisseria gonorrhoeae, all while providing fast results and convenient portability. Despite its advantages, this technology remains in the nascent stages of industrial development, necessitating enhancements in the accuracy and consistency of molecular POCT products. Improvement in the relevant quality control measures and standards is required, with the establishment of a standardized validation and assessment system. This paper presents a comprehensive review of the most recent advancements in molecular POCT technologies, devices, and applications, which are underpinned by isothermal amplification and microfluidic principles. It also offers a forecast on the prospective trajectory of this technology.
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表 1 等温扩增微流控芯片性能比较
Table 1. Comparison of performance across different isothermal amplification microfluidic chips
原理 优点 缺点 应用 离心式等温扩
增微流控芯片以离心力为驱动力,通过主轴电机即可驱动多个独立的结构单元,在芯片上对样品分子进行分子诊断[26]。 无需外界驱动泵,
降低芯片复杂程度。难以实现高复杂度、多功能性的反应过程,难以实现上百个样品的高通量或多重检验。 新型冠状肺
炎病毒[27]、人乳头状瘤病毒[11]、大肠杆菌[28]、沙门氏菌[28]等。集成式等温扩
增微流控芯片将样本裂解、核酸提取、核酸扩增及分子检验整合于一体
的微流控芯片[29]。实现“样本入-结果出”的一
体化检验,自动化程度高,降
低环境要求和操作要求。芯片上的试剂存储时间有限,
芯片设计复杂,成本较高,
不利于推广。新型冠状病毒[30]、大肠埃希菌[31]等。 巢式等温扩增
微流控芯片在微流控芯片上集成多步等温扩增反应,第一步为预扩增,为后续反应提供扩增模板[9]。 灵敏度较高、特异性强。 芯片结构设计复杂、
精确度要求高。新型冠状病毒[32]等。 数字液滴等温扩
增微流控芯片样本以液滴形式存在于双夹板结构的芯片之中,通过控制电压来实现对液滴的搬运、分离、混合等操作,在芯片上实现核酸的裂解、提取、扩增、
检验等分析操作。操作简便、检验时间缩短,实现绝对定量检验,灵敏度高。 系统复杂、检验成本高,
技术尚未成熟,稳定型和
重复性有待提高。淋球菌[33]、丙型肝炎病毒[34]、人类免疫缺陷病毒[34]等。 表 2 国内外常见分子POCT检验设备
Table 2. Overview of common molecular POCT diagnostic devices domestically and internationally
仪器名称 公司 原理技术 灵敏度 通量 GeneXpert Dx分子诊断系统 美国Cepheid公司 半巢式qPCR,微流控技术 15.6 CFU/mL(TB) 1,2,4,16,48,80 Cobas Liat医用PCR检验系统 美国Roche公司 qPCR ,气压式微流控技术 10−3~10−1 TCID50/mL 1 BioFire FilmArray 平台 法国梅里埃公司 巢式多重PCR分析技术,
微流控芯片102~103 copies/ml 1 ID NOW快速检验系统 美国雅培公司 切口酶扩增反应技术,
恒温扩增技术102~103 TCID50/mL,
20 000 copies/mL
(2019-nCoV)1 UC0102、UC0104、
UC0108、UC0116 系统中国优思达生物技术公司 交叉引物恒温扩增技术 / 2,4,8,16 EasyNAT平台 中国优思达生物技术公司 交叉引物恒温扩增技术 1 000 copies/mL 2 iPonatic移动分子诊断系统 中国圣湘生物公司 qPCR,一步法核酸免提取技术 200 copies/mL 1 Flash20检验系统 中国卡尤迪生物技术公司 qPCR,微流控技术 400 copies/mL 4 BoxArray 中国万孚倍特生物技术公司 qPCR,微流控技术 / 4 注:CFU/mL表示菌落形成单位;TCID50/mL 表示半数组织感染剂量;“/”表示无相关数据。 -
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