摘要
分别利用荧光定量PCR及平板菌落计数法计数农家干酪中双歧杆菌的菌体数,通过对结果的比较分析,建立一种适用于快速、敏感、特异的检测干酪中双歧杆菌活菌数的方法。利用传统工艺制备双歧杆菌农家干酪,在其贮存期间分别采用荧光定量PCR及平板菌落计数法计数干酪中双歧杆菌的数量。其中,在利用荧光定量PCR法检测时,对影响PCR定量准确的因素进行系统研究,包括设计双歧杆菌引物,并对引物特异性进行评价、考察,从干酪基质中提取DNA的数量和质量,建立标准曲线。引物特异性验证结果表明,引物专一性强。采用试剂盒法从干酪样品中提取DNA的纯度较好,OD_(260)/OD_(280)均在1.75~1.82之间。除贮存第1天外,荧光定量PCR法计数结果比平板计数法高0.39%~2.25%,未见显著差异。荧光定量PCR具有灵敏、特异、简便和快速的特点,可用于干酪中双歧杆菌的定量检测。
To establish a simple, sensitive, accurate and rapid detection method for bifidobacteria in cheese, we compared real-time PCR （qPCR） and plate counts. Bifidobacterium animalis subsp, lactis QYW-BB06 （cfu） enumerated by qPCR were compared to culturable Bifidobacterium animalis subsp, lactis enumerated by plate counts at 1, 5, 10, 15 and 20 days of cheese manufacture. The specificity of each primer set was assessed by qPCR and PCR, the yield and purity of DNA extracted from cheese were evaluated, and the standard curve was established. Target DNA was successfully amplified to show a single peak on the amplieon melting curve, non-target DNA was not amplified. High DNA yield and quality were obtained from cheese sample with mean OD260/OD2s0 ratios ranging from 1.75 to 1.82. Besides of the first day of cheese storage, qPCR counts were higher than plate counts, values ranged from 0.39 to 2.25%. Real-Time PCR assay can be used in detection of bifidobacteria quantitatively in cheese.
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2017年第9期117-123,共7页
Food and Fermentation Industries
基金
黑龙江省教育厅项目(产B族维生素乳酸菌的筛选及生物特性研究2016-KYYWF-0890)
齐齐哈尔市科技局项目(SFGG-201557)
关键词
荧光定量PCR
干酪
双歧杆菌
real-time polymerase chain reaction
cheese
Bifidobacterium
