数字聚合酶链反应在病原微生物检测中的应用进展

The application of digital polymerase chain reaction in pathogen detection

数字聚合酶链反应(PCR)是在实时荧光定量PCR的基础上发展起来的第3代核酸定量分析技术。其不依赖标准曲线、耐受性强、灵敏度高等特性使单分子层面的精准定量得以实现。随着商业平台的开发,数字PCR的可操作性提升,近年来逐渐在临床微生物领域展现出了良好的应用前景,具体包括定量检测结核分枝杆菌及2019新型冠状病毒等病原微生物;直接检测无菌体液(如房水中的人巨细胞病毒);定量检测HIV-DNA以评估疗效及病程发展;检测甲型流感病毒对奥司他韦的耐药性突变;标定实时荧光定量PCR的参考标准品等。该文将对数字PCR的技术原理、技术优势、局限性,以及在临床病原菌检测领域的前沿应用展开论述。

Digital polymerase chain reaction(PCR)is the third generation of nucleic acid quantitative analysis technology based on real-time fluorescent quantitative PCR.It does not depend on the standard curve,has strong tolerance and high sensitivity,which makes the precise quantification of single molecule level possible.With the development of commercial platforms,the operability of digital PCR has been improved.In recent years,it has gradually shown a good application prospect in the field of clinical microbiology,including quantitative detection of Mycobacterium tuberculosis and COVID-19,direct detection of aseptic body fluid,such as human cytomegalovirus in aseptic body fluids,quantitative detection of HIV-DNA to evaluate the efficacy and progression of the disease,detection of resistance mutation of influenza A virus to oseltamivir,and calibration of reference standard for real-time fluorescent quantitative PCR and so on.This article will discuss the technical principle,technical advantages,limitations of digital PCR,as well as the frontier application in the field of clinical pathogen detection.