解读QPCR与PCR的核心差异：从定性到定量

<p>&nbsp;<span style="font-family: Calibri; font-size: 10.5pt;">&nbsp;&nbsp;&nbsp;&nbsp;PCR</span><font face="宋体" style="font-size: 10.5pt;">是</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">1984</font><font face="宋体" style="font-size: 10.5pt;">年开始出现的一种基因检测技术，由于其操作简便、灵敏度高，被广泛应用于基础研究和临床检测，成为分子生物学中必不可少的研究工具。传统的</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">定量，又称为终点</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">定量，是通过检测</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">反应结束后（即平台期）的扩增产物量来估算初始模板量。通常采用琼脂糖凝胶电泳分离</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">产物，并使用荧光染料（如溴化乙锭）染色后，通过比较条带亮度或使用凝胶成像系统进行半定量分析。但在</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">反应中，由于反应体系、反应条件等因素，会影响</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">反应的效率，甚至会出现一些非特异性的扩增产物，因此终点</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋体" style="font-size: 10.5pt;">定量并不准确。</font></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <h3><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;QPCR<font face="宋体">简介</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">实时荧光定量</font>PCR<font face="宋体">（</font><font face="Calibri">Real-timequantitativePCR</font><font face="宋体">，常简称为</font><font face="Calibri">qPCR</font><font face="宋体">）是一种常用的定量</font><font face="Calibri">PCR(qPCR)</font><font face="宋体">方法。</font><font face="Calibri">AppliedBiosystems</font><font face="宋体">公司在</font><font face="Calibri">1996</font><font face="宋体">年推出了第一台商品化的实时荧光定量</font><font face="Calibri">PCR</font><font face="宋体">仪，极大地推动了该技术的普及和应用。相比于传统的终点</font><font face="Calibri">PCR</font><font face="宋体">，实时荧光定量</font><font face="Calibri">PCR</font><font face="宋体">具有灵敏度高、特异性强、可实现多重反应、自动化程度高、污染风险低、可实时监测结果等特点，在医学临床检测和临床医学研究中具有重要意义。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <h3><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;QPCR<font face="宋体">与</font><font face="Calibri">PCR</font><font face="宋体">区别</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></h3> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">荧光定量</font>PCR<font face="宋体">实时监测与</font><font face="Calibri">DNA</font><font face="宋体">结合的荧光染料激发的荧光；普通</font><font face="Calibri">PCR</font><font face="宋体">通过检测插入</font><font face="Calibri">DNA</font><font face="宋体">中的染料（通常是紫外线）的量来测量最终</font><font face="Calibri">PCR</font><font face="宋体">产物的量。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 1、<font face="宋体">实时荧光定量</font>PCR<font face="宋体">与传统</font><font face="Calibri">PCR</font><font face="宋体">反应要求不同</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">荧光定量对扩增片段要求较高，一般为</font>100-300bp<font face="宋体">；普通定量可以扩增较长的片段，如果不需要检测产物分子量，荧光定量可以定量分析产物而不需要电泳，而普通</font><font face="Calibri">PCR</font><font face="宋体">必须进行电泳。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 2、<font face="宋体">实时荧光定量</font>PCR<font face="宋体">与传统</font><font face="Calibri">PCR</font><font face="宋体">反应结果不同</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">荧光定量可以做到精准定量，而普通</font>PCR<font face="宋体">做不到。荧光定量可以展示整个扩增过程，扩增效率，溶解温度，直接得到标准曲线等，这是普通</font><font face="Calibri">PCR</font><font face="宋体">做不到的。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;</span></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋体">上述是</font>QPCR<font face="宋体">和</font><font face="Calibri">PCR</font><font face="宋体">区别，</font><font face="Calibri">PCR</font><font face="宋体">技术作为分子生物学基石，为基因研究提供了重要的技术支持。而</font><font face="Calibri">QPCR</font><font face="宋体">在此基础上发展而来，凭借其定量精准、实时监测等优势，极大地拓展了</font><font face="Calibri">PCR</font><font face="宋体">技术的应用范围，尤其在医学诊断、疾病研究等领域展现出巨大的潜力，未来必将在生命科学领域发挥更重要的作用。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋体;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>