分子信标-实时 PCR法快速检测双歧杆菌的研究

Establishment of Real-Time PCR and Molecular Beacon Detection Method for Bifidobacteria

摘要:

为建立双歧制品中双歧杆菌快速、敏感、特异的检测方法，根据双歧杆菌16S rRNA/16S rDNA基因设计合成了双歧杆菌属特异性引物和分子信标探针，建立了快速检测双歧杆菌的分子信标-实时PCR检测方法，并对反应条件进行优化。检测方法重复性好，批内和批间变异系数均小于5%；特异性强，扩增曲线呈现明显的S型，无非特异性扩增；灵敏度高，是普通 PCR的100倍，对纯双歧杆菌DNA的检出限为5.7fg/PCR反应体系，纯双歧杆菌菌液的检出限为2×10³CFU/mL；线形范围宽，起始模板数在2×10⁸CFU/mL～2×10⁴CFU/mL之间具有良好的线性关系，相关系数大于97%。该方法具有灵敏、特异、简便和快速的特点，可用于对双歧杆菌原位菌数的定量检测。

Abstract:

To establish a simple，sensitive，accurate and rapid detection method for bifidobacteria. Molecular beacon probe and primers were designed and sythesized according to the conserved gene of Bifidobacterium in GenBank，and then reaction parameters of real-time PCR were optimized. For Real-Time PCR and molecular beacon detection for bifidobacteria, the intra-assay and inter-assay coefficient of variation were both less than 5%, indicating good reproducibility of this method, and no non-specific amplification was observed either. Compared with conventional PCR, this method is more sensitive and faster, and the detection limit was 5.7fg/PCR for bifidobacteria DNA. A linear standard curve was obtained between 2×10⁸CFU/mL and 2×10⁴CFU/mL (R>0.97). The method of Real-time PCR and molecular beacon detection for bifodobacteria has many advantages, such as being sensitive, specific, simple and fast, and this method can be used in situ detection of bifidobacteria quantitatively.