基于微滴数字PCR技术的婴幼儿配方乳粉中双歧杆菌定量检测及应用

Establishment and Application of a Quantitative Method for Detecting Bifidobacterium in Infant Formula by Droplet Digital Polymerase Chain Reaction

应用微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)技术,建立婴幼儿配方乳粉中双歧杆菌定量检测方法。以双歧杆菌的单拷贝特异性基因rpsL为目标基因设计引物探针,对ddPCR条件进行优化,考察方法的特异性、灵敏度和重复性,并与平板计数方法进行对照验证。结果表明,建立的方法具有良好的特异性、灵敏性和重复性。细菌纯培养液的检出限为296 CFU/mL,模拟样品检出限为7 300 CFU/g,不与10种近缘乳酸菌发生交叉反应,且重复性较好,采用已建立的ddPCR方法和平板计数方法对市售婴幼儿配方乳粉样品进行检测,2种方法测定值结果偏差小于10%,结果一致性较好。本研究建立的ddPCR方法对婴幼儿配方乳粉中的双歧杆菌定量检测能够更快速、灵敏、准确,具有一定的应用前景。

This study aimed to establish a quantitative method for detecting Bifidobacterium in infant formula by using droplet digital polymerase chain reaction(ddPCR). A pair of specific primers and probes were designed targeting the single copy gene rpsL of Bifidobacterium. The reaction conditions of ddPCR were optimized, and the specificity, sensitivity and repeatability of the developed method were investigated and compared with those of the plate counting method. The limit of detection(LOD) was 296 CFU/mL for pure bacterial culture, and 7 300 CFU/g for a simulated sample, respectively. There was no cross-reaction with 10 related species of Lactobacillus, and this method had good repeatability. When Bifidobacteria in commercial infant formula samples were detected by ddPCR and the plate counting method, the deviation between the results of the two methods was less than 10%, indicating good agreement. The ddPCR method can detect Bifidobacteria in infant formula milk powder more quickly, sensitively and accurately than the plate counting method, and it thus has good application prospects.