荧光定量PCR技术及其在动物传染病定量检测中的应用

Fluorescent Quantitative PCR and Its Applications in Diagnosing Animal Infectious Diseases

荧光定量PCR是近年发展起来的一 种新的实时定量检测特定核酸技术,它是核 酸探针技术、荧光共振能量传递技术和PCR 技术的有机结合,而荧光探针是荧光定量 PCR的核心。目前报道的荧光探针主要有 TaqMan、Amplisensor、分子信标、Lightcycler 以及在这4种探针基础上发展起来的其他荧 光探针。各种荧光探针在定量PCR中的作 用是识别和报告特定核酸。这些荧光探针与 相应的靶分子杂交时经历一个自发荧光形成 或消失的构象变化,只有与完全互补的靶核 酸杂交时才会出现这种变化,当靶核酸存在 碱基错配或缺失时,荧光探针就不会与之杂 交,也不会出现荧光的变化。在检测时根据 这种荧光变化来确定检测中特定核酸的存在 和量的多少。荧光探针用于定量PCR不但 提高了PCR的检测灵敏度,还能在同一封闭 管中对模板进行准确定量检测,特别适合特 定核酸扩增的实时监测。荧光定量PCR用 于动物传染病的诊断,不但能定量检测病原 体感染的强弱和在机体内的分布,还具有灵 敏、快速、省力的特点。

Fluorescent quantitative PCR is a lately developed new technology that is used to detect specific nucleic acids in real time. It is a ingenious combination of fluorescent probes, fluorescence resonance energy transfer (FRET) technology and PCR. However, fluorescent probes are the most important part of them. At present, there are TaqMan, Amplisensor, Molecular beacon, Lightcycler, and other probes based above four probes. Fluorescent nucleic acid probes recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the targets contain a mismatched nucleotide or a deletion. These probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive , and can be carried out in a sealed tube. When fluorescent quantitative PCR is introduced into diagnosing animal infectious diseases,it should enable infectious strength and distribution of nucleic acids of animal pathogenic microorganism to be traced. It is also sensitive,rapid, and labour-saved.