TaqMan-MGB探针荧光定量聚合酶链式反应法检测酸奶制品中动物双歧杆菌BB-12

摘 要：【目的】本研究拟建立一种快速、高灵敏性检测和定量酸奶中动物双歧杆菌BB-12的方法。【方法】设计动物双歧杆菌BB-12的16SrRNA序列的MGB探针,利用荧光定量PCR技术,构建目标序列的过表达载体,提取质粒进行标准曲线的制作,最后进行市售酸奶样品检测,定量酸奶中BB-12菌株的数量。【结果】所有载体标准品均产生显著的荧光增幅,Ct值介于16～20之间;而以其他酸奶制品添加菌以及大肠杆菌样品DNA为模板则无荧光扩增信号。在检测灵敏度方面,BB-12载体可被稳定检出的质量浓度低至0.0000584 ng/μL,Ct值平均数为28.73,即检测灵敏度低至为0.05 pg。制作的标准曲线线性良好,扩增效率E为0.39,判定系数R2=0.9999。市售酸奶样品检测特异性好、灵敏度高及定量结果准确。【结论】通过特异性的TaqMan-MGB探针进行荧光定量PCR检测可实现在酸奶中对BB-12菌种的检测及定量。     

荧光定量PCR法计数农家干酪中动物双歧杆菌乳酸亚种

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。     

基于RT-PCR与平板计数法对比分析发酵香肠发酵过程中的乳酸菌数

为快速、准确检测发酵香肠发酵过程中乳酸菌数的动态变化，以自然发酵为对照组，植物乳杆菌F16为发酵剂制作发酵香肠，同时以植物乳杆菌CICC：6238为标准菌株进行标准品的制作，采用实时荧光定量PCR（RT-PCR）技术定量分析发酵香肠发酵过程中的乳酸菌数，并与传统计数方法作对比。结果表明：本方法所建立的标准曲线相关系数R2≧0.99，扩增效率为90%～100%，符合RT-PCR的检测要求；乳酸菌数呈先上升后下降趋势，定量结果与平板计数变化趋势相同，且在各取样点，因存在死亡细胞的DNA，故荧光定量所得结果高于平板计数1lg（CFU/g）左右。利用平板计数和荧光定量数值得出两者间的线性方程y = 0.8116x + 0.4254。任取1批发酵香肠做验证试验，根据线性方程所得活菌数与平板计数值之间的相似度达94%以上，说明可用线性方程预测乳酸菌活菌数。本试验所建立的RT-PCR技术为定量监测发酵香肠发酵过程中乳酸菌的动态变化提供一条快捷途径。     

实时荧光定量PCR检测产琥珀酸丝状杆菌数量方法的研究

为建立产琥珀酸丝状杆菌定性定量研究方法,实现后期该菌种用于纤维素生物可再生能源等方面应用研究。本试验提取15份瘤胃细菌脱氧核糖核酸(Deoxyribonucleic acid,DNA),利用SYBR Premix Ex Taq荧光染料嵌合法和人工合成基因技术,采用实时荧光定量PCR方法检测样品中产琥珀酸丝状杆菌数量;结果:所构建的质粒DNA标准曲线线性关系良好,R2=1,所有样品溶解曲线均为单峰,扩增曲线呈S型,扩增效率为93.9%。表明应用构建实时荧光定量PCR法可对实际样品中产琥珀酸丝状杆菌进行定性定量研究。     

牛奶β-乳球蛋白质实时荧光定量PCR检测方法的建立

建立一种快速、特异、灵敏的Taqman实时荧光定量PCR(real-time PCR)方法,用于牛奶主要过敏原β-乳球蛋白质的检测。根据Gen Bank登录的牛β-乳球蛋白质的DNA序列设计,合成一对特异性引物和探针。将扩增产物连接到p MD19-T载体上,制备质粒标准品并测序鉴定,10倍梯度稀释含有β-乳球蛋白质基因的重组质粒,进行实时荧光定量PCR扩增,绘制标准曲线,检测该方法的特异性、稳定性、灵敏性,同时将建立的方法用于10种市售食品的检测。成功建立了β-乳球蛋白质的实时荧光定量PCR检测方法,标准曲线的Ct值与模板浓度在3.18×103~3.18×107copies范围内线性关系良好,R2值为0.997 8;检测灵敏度高(318 copies/μL);特异性强,对羊奶、豆浆DNA均无扩增反应;稳定性好,组内、组间的变异系数均在5%以内。对10种食品牛奶过敏原的检测结果与标签相符。表明所建立的实时荧光定量PCR方法可应用于食品中牛奶过敏原β-乳球蛋白质的检测并可推广到其它过敏原的检测。     

TaqMan-MGB探针实时荧光定量PCR检测克罗诺杆菌MMS基因方法的建立

建立克罗诺杆菌的特异、灵敏的TaqMan-MGB探针实时荧光定量PCR检测方法。方法 根据GenBank公布的克罗诺杆菌MMS基因高保守序列,设计特异引物和TaqMan-MGB探针,建立和优化反应体系,用25种其他常见致病菌评价反应体系的特异性,用克罗诺杆菌MMS基因重组质粒构建实时荧光定量PCR标准曲线,对重组质粒、纯菌和人工模拟污染样本进行灵敏度试验,并与FDA推荐的TaqMan探针实时荧光PCR比较,配对t检验分析两种方法对Ct值和荧光强度的差异。结果 采用TaqMan-MGB探针实时荧光定量PCR检测克罗诺杆菌MMS基因仅需40min,与25种非目标菌无交叉反应,仅对克罗诺杆菌有特异性扩增;所构建方法线性关系良好,相关系数r²=0.999,扩增效率为99.972%,对重组质粒、纯菌、人工模拟污染样品标本的灵敏度分别达10拷贝/反应、3.8和38cfu/ml;与FDA推荐的TaqMan探针实时荧光PCR相比,TaqMan-MGB探针实时荧光定量PCR的Ct值更小、△Rn值更高,灵敏度和分辨率差异均有统计学意义(Ct:t=-14.406,P﹤0.01;△Rn:t=14.230,P﹤0.01)。结论 本研究建立的TaqMan-MGB探针实时荧光定量PCR反应体系能够快速、特异、灵敏地检测克罗诺杆菌MMS基因,可用于婴幼儿奶粉中克罗诺杆菌的快速筛查和鉴定,具有较大的的应用价值和推广价值。     

酸奶中长双歧杆菌PCR计数方法的建立

根据细菌保守序列设计一对通用引物,扩增细菌16S rDNA序列,作为阳性标记;根据长双歧杆菌菌株的16S rRNA和23S rRNA之间的间区基因序列设计定性PCR引物。用BBL琼脂平板对长双歧杆菌混合发酵的酸奶进行培养计数,随机挑选部分长出的菌落,提取DNA做PCR鉴定。将PCR技术与传统平板计数技术结合起来,初步建立酸奶中长双歧杆活菌计数方法。抽提方法操作简便、高效、灵敏,决定着PCR计数方法的准确高效,本研究着重研究了菌体DNA抽提方法的影响因素,以及抽提方法通用性,同时也研究了PCR引物的特异性。将PCR技术与传统平板计数技术结合起来,初步建立酸奶中长双歧杆活菌计数方法。     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应（polymerase chain reaction,PCR）检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25 份标示含双歧杆菌样品进行检测。结果表明：该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到104 CFU/mL。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25 份市售实际样品进行测试,有5 份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

实时荧光PCR法鉴定食品中双歧杆菌

根据双歧杆菌属16S rRNA基因的保守区序设计特异性引物和探针,建立一种鉴定食品中双歧杆菌属的实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法。对方法的特异性、灵敏度、重复性和体系抗干扰能力进行验证,最后采用该方法对市售25份标示含双歧杆菌样品进行检测。结果表明:该检测方法可特异性检测双歧杆菌属细菌,对近缘的乳杆菌属、链球菌属及食品中常见菌群包括大肠杆菌、金黄色葡萄球菌、沙门氏菌等均无扩增。双歧杆菌DNA检测绝对灵敏度可达到2 pg,相对灵敏度可达到10~4 CFU/m L。重复性测试表明相对标准偏差小于1%。同时进行了杂菌干扰检测实验,在培养物水平和纯基因组DNA水平上将青春双歧杆菌ATCC15703与大肠杆菌ATCC25922混合进行检测,检出Ct值较纯菌检测时无显著影响,表明建立的荧光PCR方法抗干扰能力良好。对25份市售实际样品进行测试,有5份标识含有“双歧杆菌”的样品未检测出双歧杆菌成分。本研究所建立的实时荧光PCR法能准确、快速检测食品中双歧杆菌属细菌。     

不同核酸提取方法用于多重荧光PCR法检测3种混合熟肉的比较分析

目的 探讨不同核酸提取方法以及蒸、煮、烤烹饪方式制作的混合熟肉制品的多重荧光定量PCR检测结果之间的差异。方法 用3种不同的提取方法：抽提法、离心柱法及磁珠法,提取经过蒸、煮、烤烹饪方式制作的牛、鸡、猪、鸭混合样品的DNA,通过对不同方法提取DNA的质量及提取DNA用于多重荧光PCR检测的效果方面进行比较。结果 三种方法提取DNA的浓度及纯度无明显差别,磁珠法提取的混合样品DNA进行多重实时荧光PCR检测的Ct值最小,扩增效果最佳。结论 该研究中磁珠法提取熟肉制品DNA的检测效果更为理想。     

Microbiological evaluation and molecular characterization of bifidobacteria strains in commercial fermented milks

The aim of this study was to evaluate the presence of Bifidobacterium animalis subsp. lactis in commercial dairy products using different molecular techniques. We analyzed the microbiological composition of 13 commercial fermented milks available in the Spanish market. Thirteen strains of genus Bifidobacterium were isolated from these products and were identified by genus-specific PCR, by fluorescence in situ hybridization (FISH), by multiplex PCR and amplified ribosomal DNA restriction analysis (ARDRA). The same sets of strains were typed by randomly amplified polymorphic DNA (RAPD) analysis and by amplified fragment length polymorphism technique (AFLP). All strains were identified as B. animalis subsp. lactis using ARDRA and multiplex PCR techniques. Similarity between strains was evaluated based on RAPD and AFLP profiles. The isolated strains showed similar profiles by using these techniques, revealing the reduced genetic variability existing among commercial strains, and all these profiles were reproducible in repeated analysis. ARDRA and multiplex PCR are techniques that allow differentiation of the bifidobacteria at genus and species level, but do not indicate if they are different strains, for which reason the RAPD technique is very useful. All bifidobacteria isolated from commercial fermented milks in Spain belong to the same species B. animalis subsp. lactis. Our results demonstrate the necessity to control the presence of bifidobacteria in commercial fermented milks, not only at species level but also at strain level. Multiplex PCR and RAPDs are the most suitable, rapid and precise techniques to identify all bifidobacteria contained in fermented milk products at genus-, species-, and strain levels.     

酪蛋白糖巨肽对小鼠肠道双歧杆菌增殖水平的影响

采用实时荧光定量聚合酶链式反应（real-time polymerase chain reaction,real-time PCR）分析不同剂量酪蛋白糖巨肽（casein glycomacropeptide,CGMP）对小鼠肠道双歧杆菌增殖水平的影响,揭示乳源CGMP是否有增殖肠道关键益生菌的功能。选用50 只健康BALB/c小鼠,随机分为对照组,安慰组（灌胃生理盐水0.2 mL）,乳源CGMP低、中、高剂量组（灌胃等体积不同质量浓度的乳源CGMP）,灌胃周期为2 周。于灌胃后第0、3、5、7、9、11、15天及停止灌胃后1 周（第21天）采集的小鼠新鲜粪便,并进行粪便菌体基因组DNA抽提。依据双歧杆菌的16S rRNA基因序列设计5 对种属特异性引物,以两歧双歧杆菌（Bifidobacterium bifidum）CICC6071的基因组DNA作为标准品,进行梯度稀释制作标准曲线;分析样品PCR扩增产物的熔解曲线,评价反应的特异性。结果表明：Real-time PCR可准确定量小鼠肠道内双歧杆菌数量;且灌胃中剂量乳源CGMP可以促进小鼠肠道内双歧杆菌的增殖,在灌胃第11天时小鼠肠道内双歧杆菌数量达到最高值,即乳源CGMP对小鼠肠道双歧杆菌的增殖作用存在剂量选择性。     

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.     

实时荧光定量PCR法鉴定发酵乳中嗜热链球菌

该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应(RT-fqPCR)法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,rec A基因具有种间特异性,种间差异率>10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达10³CFU/m L;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。     

Quantitative analysis and molecular identification of bifidobacteria strains in probiotic milk products

The aim of the present study was to evaluate an agar medium for the quantitative analysis of bifidobacteria in probiotic milk products on the market, and to investigate the identities of active strains. For this purpose, three sour milk products and a soft cheese brand were each analysed ten times with Wilkins-Chalgren agar (WCA) supplemented with mupirocin, and with MRS agar supplemented with dicloxacillin at two different concentrations. By statistical analysis of the bacterial counts obtained, WCA was shown to have the best detection rate. All the sour milk products examined conformed to Swiss food legislation and contained more than 10⁶ cfu of bifidobacteria per gram of living bifidobacteria. All the samples of soft cheese analysed revealed counts that were clearly (approximately 2 logs) below this minimum requirement. Isolated bifidobacteria strains were all identical with reference strains from producer companies shown by pulsed-field gel electrophoresis (PFGE) profiling. According to the labels, the three brands of sour milk examined contained different strains of bifidobacteria (Bifidobacterium lactis Bb 12, Bifidobacterium animalis DN 173010 and Bifidobacterium species 420). However, all the strains revealed repeatedly identical PFGE-patterns, thus showing a close relationship. These findings show that a better taxonomical definition of commercially used bifidobacteria strains is needed.     

应用RT-PCR技术定量检测发酵乳中Lactobacillus plantarum活菌数

根据发酵乳中常见乳酸菌种的16S rRNA基因序列设计了L.plantarum的种属特异性引物,应用该引物对植物乳杆菌的模式菌株L.plantarum ATCC14917、L.plantarum非同源性对照菌株及采自西藏地区的自然发酵乳样品进行了种属特异性PCR检测,并以样品RNA转录的cDNA为模板,对检出含有L.plantarum的发酵乳样品进行了Real-Time PCR定量检测。通过Real-Time PCR图谱分析,准确地检测出了该样品中L.plantarum活菌数的含量为6.6lgcfu/mL。结果表明该方法能够简便、快速地检测出发酵乳中是否含有L.plantarum,并能够对其活菌数进行准确地定量,对发酵乳的生产和监管提供了重要的理论和实践依据。     

DNA-based qualitative and quantitative identification of bovine whey powder in goat dairy products

As one of the main ingredients in some milk powders, whey powder is sometimes added to pure goat milk products, which can cause health risks, economic fraud, and unfair competition of food industries. This study is the first to explore qualitative and quantitative methods to identify adulteration of bovine whey powder in goat dairy products based on DNA. We extracted DNA from whey powder using a modified DNA extraction method; this exhibited good quality and integrity, with purity of 1.53 to 1.75 and concentration of 122 to 179 ng/μL. Conventional PCR and real-time PCR were compared for qualitative detection of bovine whey powder; real-time PCR demonstrated sensitivity of 0.01 ng/μL, which was higher than the 0.05 ng/μL detected by the conventional PCR method. Furthermore, real-time PCR was conducted for DNA quantitative detection, with good linearity (R² = 0.9858) obtained for bovine whey powder contents from 0.1% to 30%. Relative error decreased with increase of the mixing proportion of whey powder; the coefficient of variation above 0.1% of the mixing ratio was close to or less than 5%; and the relative standard deviation of repeatability results was less than 5%. Considering the economic costs of testing, conventional PCR could be performed first, and samples with obvious intentional adulteration detected can be further accurately quantified by real-time PCR. Overall, this research provides a realistic and effective method for qualitative and quantitative identification of bovine whey powder in goat dairy products, thus laying a good foundation for verification of goat dairy product label claims and industrial control.     

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.     

乳制品中活双歧杆菌检测试剂盒的研制及应用

在EMAReal-Time PCR检测方法的基础上,组装检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。建立了EMAReal-Time PCR检测乳制品中活双歧杆菌的标准曲线。组装出检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。该试剂盒批内及批间变异系数(CV)均小于5%;对乳制品中常见乳酸菌无交叉反应;最低检测限为2×104CFU/mL。该试剂盒在-20℃下,至少可保存6个月,4℃可保存3个月。分别采用研究所获得的试剂盒和平板菌落计数法检测12份市售酸乳中的活双歧杆菌的数量,2种检测方法计数结果差异不显著(P>0.05)。该方法所建立的标准曲线相关系数大于98%。所研制的试剂盒重复性较好、特异性较强、灵敏度较高,可以准确地定量检测乳制品中的活双歧杆菌。