解讀QPCR與PCR的核心差異：從定性到定量

<p>&nbsp;<span style="font-family: Calibri; font-size: 10.5pt;">&nbsp;&nbsp;&nbsp;&nbsp;PCR</span><font face="宋體" style="font-size: 10.5pt;">是</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">1984</font><font face="宋體" style="font-size: 10.5pt;">年開始出現的一種基因檢測技術，由于其操作簡便、靈敏度高，被廣泛應用于基礎研究和臨床檢測，成為分子生物學中必不可少的研究工具。傳統的</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">定量，又稱為終點</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">定量，是通過檢測</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">反應結束后（即平臺期）的擴增產物量來估算初始模板量。通常采用瓊脂糖凝膠電泳分離</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">產物，并使用熒光染料（如溴化乙錠）染色后，通過比較條帶亮度或使用凝膠成像系統進行半定量分析。但在</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">反應中，由于反應體系、反應條件等因素，會影響</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">反應的效率，甚至會出現一些非特異性的擴增產物，因此終點</font><font face="Calibri" style="font-family: Calibri; font-size: 10.5pt;">PCR</font><font face="宋體" style="font-size: 10.5pt;">定量并不準確。</font></p> <p class="MsoNormal"><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;QPCR<font face="宋體">簡介</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋體">實時熒光定量</font>PCR<font face="宋體">（</font><font face="Calibri">Real-timequantitativePCR</font><font face="宋體">，常簡稱為</font><font face="Calibri">qPCR</font><font face="宋體">）是一種常用的定量</font><font face="Calibri">PCR(qPCR)</font><font face="宋體">方法。</font><font face="Calibri">AppliedBiosystems</font><font face="宋體">公司在</font><font face="Calibri">1996</font><font face="宋體">年推出了第一臺商品化的實時熒光定量</font><font face="Calibri">PCR</font><font face="宋體">儀，極大地推動了該技術的普及和應用。相比于傳統的終點</font><font face="Calibri">PCR</font><font face="宋體">，實時熒光定量</font><font face="Calibri">PCR</font><font face="宋體">具有靈敏度高、特異性強、可實現多重反應、自動化程度高、污染風險低、可實時監測結果等特點，在醫學臨床檢測和臨床醫學研究中具有重要意義。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;QPCR<font face="宋體">與</font><font face="Calibri">PCR</font><font face="宋體">區別</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋體">熒光定量</font>PCR<font face="宋體">實時監測與</font><font face="Calibri">DNA</font><font face="宋體">結合的熒光染料激發的熒光；普通</font><font face="Calibri">PCR</font><font face="宋體">通過檢測插入</font><font face="Calibri">DNA</font><font face="宋體">中的染料（通常是紫外線）的量來測量最終</font><font face="Calibri">PCR</font><font face="宋體">產物的量。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 1、<font face="宋體">實時熒光定量</font>PCR<font face="宋體">與傳統</font><font face="Calibri">PCR</font><font face="宋體">反應要求不同</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋體">熒光定量對擴增片段要求較高，一般為</font>100-300bp<font face="宋體">；普通定量可以擴增較長的片段，如果不需要檢測產物分子量，熒光定量可以定量分析產物而不需要電泳，而普通</font><font face="Calibri">PCR</font><font face="宋體">必須進行電泳。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp; &nbsp; 2、<font face="宋體">實時熒光定量</font>PCR<font face="宋體">與傳統</font><font face="Calibri">PCR</font><font face="宋體">反應結果不同</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋體">熒光定量可以做到精準定量，而普通</font>PCR<font face="宋體">做不到。熒光定量可以展示整個擴增過程，擴增效率，溶解溫度，直接得到標準曲線等，這是普通</font><font face="Calibri">PCR</font><font face="宋體">做不到的。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#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:宋體;&#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:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;">&nbsp;&nbsp;&nbsp;&nbsp;<font face="宋體">上述是</font>QPCR<font face="宋體">和</font><font face="Calibri">PCR</font><font face="宋體">區別，</font><font face="Calibri">PCR</font><font face="宋體">技術作為分子生物學基石，為基因研究提供了重要的技術支持。而</font><font face="Calibri">QPCR</font><font face="宋體">在此基礎上發展而來，憑借其定量精準、實時監測等優勢，極大地拓展了</font><font face="Calibri">PCR</font><font face="宋體">技術的應用范圍，尤其在醫學診斷、疾病研究等領域展現出巨大的潛力，未來必將在生命科學領域發揮更重要的作用。</font></span><span style="mso-spacerun:'yes';font-family:Calibri;mso-fareast-font-family:宋體;&#10;mso-bidi-font-family:'Times New Roman';font-size:10.5000pt;mso-font-kerning:1.0000pt;"><o:p></o:p></span></p>