摘要
悬浮芯片与固体芯片、荧光定量PCR并列成为核酸序列鉴定中的重要的分子生物学工具,并在病原菌检测方面显示出不同的应用领域。悬浮芯片能同时检测多种病原菌,具有处理多样本能力、使用灵活、低成本等特点,适合对未知样本检测及环境监控,能够在生物安全、公共卫生、工农业生产中发挥重要作用;而固体芯片能耦联成千上百个探针,但由于在多样本处理、成本方面欠缺,因此适合于对重要的未知病原体的鉴定;荧光定量PCR具较好特异性、灵敏度,以及多样本处理能力,但在高通量方面欠缺,适合有目的地检测已知病原体。目前已建立三种基于悬浮芯片的检测方法:多重PCR扩增、通用引物扩增16S/23SrDNA、直接对实际样本杂交检测。多重PCR具较好特异性,但其多重能力还难以满足悬浮芯片的高通量的需要;通用引物具较好灵敏度及扩增多靶分子能力,但也存在交叉反应等缺陷。同时,采用PCR扩增方法,悬浮芯片检测的是PCR产物,不能客观反应实际样本中存在病原菌数量及是否具生命力。直接杂交环境样本尽管避免了PCR的缺陷,但在灵敏度方面非常欠缺。目前,在环境样本处理上,仍然缺乏有效的、高通量、自动化的方法,不能满足PCR与悬浮芯片多样本检测的需要。
Suspension array, microarray and real-time quantitative PCR have been important tools for genetic sequences characteristic, especially for pathogen detection and identification. Suspension arrays offer the potential for simultaneous detection of many pathogens that are interest in biosecurity, public health, and veterinary diagnostics. It is best suited for detecting the presence or absence of unknown pathogen and environment monitoring. Microarray will be a powerful tool for the rapid screening of thousands of possible pathogens, but drawback of large sample question makes current routine use impossible. Real-time PCR are rapid and the most sensitive methods for determination of whether a given pathogen is present. However, because of limitation of fluorescence, it is unfeasible for large-scale use. Three basic strategies have been described for pathogen detection based on suspension array： multiplex PCR amplification, universal primers amplification and directing hybridization of RNA. Multiplex PCR has the advantage of a high specificity, but associated suspension arrays are limited in scope. Universal primers are capable of high sensitivity and amplification of all bacteria, but has disadvantage of cross-reaction. Moreover, PCR-dependent method is poorly suited for determining pathogen quantity and viability. Direct hybridization of RNA provides the least bias in gene detection, but also the lowest level of assay sensitivity. Ultimately, validation and high-throughput sample preparation will continue to be significant challenges for these detection systems.
出处
《现代生物医学进展》
CAS
2009年第8期1559-1562,共4页
Progress in Modern Biomedicine
基金
国家"863"计划项目(2006AA06Z414)
国家自然科学基金项目(30671824)
