利用重组酶聚合酶扩增技术快速鉴定肉及肉制品中的猪源性成分

Rapid detection of porcine-derived materials in meat and meat products using recombinase polymerase amplification(RPA) technique

根据家猪线粒体细胞色素b的核苷酸序列设计和筛选实时荧光定量重组酶聚合酶扩增(Real time recombinase polymerase amplification,real-time RPA)反应所需的引物和探针,优化反应参数(Mg^(2+)和引物浓度),建立了一套利用real-time RPA技术进行肉及肉制品中猪源性成分的鉴定方法。应用此方法可以检测出肉及肉制品中低至0.2%的猪源性成分,且能够在恒温的条件下(40℃)15min中内完成反应,克服了传统聚合酶链式反应(Polymerase chain reaction,PCR)技术需要精密温度循环控制设备的局限性,是一种新型、简单、高效的检测方法,对实现快捷便携式的猪源性成分鉴定检测具有重要的意义。

In this study, specific primers and probe for real time recombinase polymerase amplification （real-time RPA） were designed and screened, based on the pig mitochondrial cytochrome b nucleotide sequence, then the RPA assay parameters （Mg2＋ and primers concentration） were optimized. As a result, a set of methods for the detection of porcine-derived materials in meat and meat products using recombinase polymerase amplification （RPA） technique were established. The method could detect porcine-derived materials as low as 0.2% in meat and meat products at a constant temperature of 40 ~C within 15 min. It could also work well without precise temperature cycle control equipment, which was necessary for the traditional polymerase chain reaction （PCR） amplification technique. Thus, this method was a novel, simple and efficient detection approach and had a vital significance to realize a fast portable detection of animal-derived materials.