目的：设计一种筛选高效性多重聚合酶链反应（multiplex polymerase chain reaction，MPCR）公共引物的新方法。方法将3条正向引物和3条反向引物序列分别连接于一段人类β-actin基因序列5'和3'端，连接序列通过pMD19-T载体构建质粒模板，利用此构建的质粒模板可实现9对引物PCR扩增敏感性的筛选，实现了引物的高效筛选；其步骤包括：引物设计、插入序列的选择、酶切转化、质粒提取、敏感性检测。结果通过琼脂糖凝胶电泳分离PCR产物得出实验结果，结果显示9对公共引物的重复敏感性有明显差异，其中2对引物两次敏感性检测均达到10 copies/μL，1对引物两次敏感性均是103 copies/μL。结论该方法适用于从多对引物中排除低效性引物，筛选出敏感性高的引物对。%Objective To design a novel method for screening universal primers efficiently of multiplex polymerase chain reaction (MPCR).MethodsThree forward primers and three reverse primers were connected with the 5' and 3' ends of a sequence of humanβ-actin gene respectively and the sequence was constructed plasmid template by pMD19-T vector. Through the constructed plasmid template, the PCR amplification sensitivity of 9 pairs of primers can be achieved. The method includes primers design, the choice of inserted sequence, restriction enzyme digestion and transformation, plasmid extraction, and sensitivity detection.Results After separating PCR products by agarose gel eletrophoresis, results showed that there was signiifcant discrepancy in the sensitivities among the nine pairs of universal primers, and the sensitivities of two pairs of primers both reached up to 10 copies/μL repeatedly and one other pair was 103 copies/μL.Conclusion This method can be used to screen the primers efifciently and screening out the highly sensitive primers.
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