CN110016496A - 利用荧光定量pcr法检测fut2基因类型的方法 - Google Patents
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Abstract
本发明涉及一种利用荧光定量PCR法检测FUT2基因类型的方法，属于分子生物学技术领域。利用荧光定量PCR法检测FUT2基因类型的方法，获取待测样品全血基因组DNA；利用引物及荧光物质进行荧光定量PCR检测：根据PCR检测后熔解曲线的熔解峰位置判断rs1047781位点的A/T突变情况，进而判断FUT2基因的类型为分泌型或非分泌型。本发明所述方法可以特异性的进行中国人群岩藻糖基转移酶基因Fut2单核苷酸位点多态性(SNP)分析，以明确分泌型/非分泌型并预测母乳岩藻糖基化低聚糖(fHMOs)水平及其母乳喂养的新生儿肠道双歧杆菌和乳酸杆菌的丰度。
Description
技术领域
本发明涉及一种利用荧光定量PCR法检测FUT2基因类型的方法，属于分子生物学技术领域。
背景技术
新生儿肠道有益菌群的定植和健康菌群结构的建立显著影响其生长发育[1,2]。研究显示在新生儿肠道中正常菌群的比例分别为：乳酸杆菌1％、双歧杆菌60％—90％、其他细菌10％—40％，其中双歧杆菌(Bifidobacterium spp.)占据绝对优势地位[3]。双歧杆菌是婴儿肠道中早期定植的生理性厌氧菌，是肠道正常菌群中的优势菌，可以帮助维持婴儿肠道稳态，在婴幼儿成长发育、消化吸收、抗感染、免疫调节等诸多方面起到积极的作用[4,5]。因此，双歧杆菌在婴儿肠道的定植和多样性对保证婴幼儿健康有着重要意义。
婴儿肠道菌群的形成主要受分娩方式、喂养方式、抗生素的使用、环境及遗传等因素的影响，具有较大的波动性。其中，喂养方式的不同对婴儿肠道菌群结构的影响巨大[6]。母乳喂养婴儿肠道菌群构成相对简单，形成以双歧杆菌为优势菌的肠道菌群[7]；而人工喂养婴儿则形成更为多样化的肠道菌群，其肠道内双歧杆菌低于母乳喂养婴儿，而拟杆菌、肠杆菌、肠球菌和梭菌等则高于母乳喂养婴儿[8]。近期国际著名期刊《Science》、《Cell》等相继发表了以“母乳—婴儿肠道菌群—婴儿健康”为主线的重要科研成果[9-11]，证明了母乳关键成份对婴儿健康的重要作用和相关机制。研究显示母乳中的低聚糖(HMO)是诱导母乳喂养儿肠道双歧杆菌优势发育的主要因素[12]。母乳喂养的婴儿肠道双歧杆菌可利用HMO成份，特别是岩藻寡糖类(Fucosylated oligosaccharides)作为碳源而生长繁殖[13]。例如，α1,2-fucosylated HMO成份已被证明能促进B.longum subsp.、B.bifidum subsp.和B.breve等双歧杆菌菌株的生长，这些菌株均含有可降解2’-fucosylated HMO的糖基水解酶95(GH95)或岩藻糖苷酶GH29[14-16]。因此，HMO成份的变化可能直接影响子代肠道菌群的发展，作为一类重要的益生元，HMO是“母乳中不该被忽视的关键营养”。
近年来美国学者研究显示，母亲携带岩藻糖基转移酶2(FUT2)基因的类型(分泌型和非分泌型)不同，所分泌母乳中的HMO成份也有所不同，这直接影响了婴儿肠道双歧杆菌的定植时间和发展[17]。母亲乳腺中的FUT2可通过α1,2-linkage将低聚糖底物岩藻糖基化。由于FUT2基因表达于多种ABO血型人群所分泌的体液中(例如眼泪、唾液、乳汁、等等)，因此被称为“分泌基因(secretor gene)”。该基因的突变可导致其编码的FUT2失活，而FUT2基因单核酸位点多态性(SNP)在全球范围内均被发现[18]。在我国母亲人群的研究中显示，rs601338位点的A/G多态性和rs1047781位点的A/T多态性是控制中国母亲母乳FUT2是否处于活化状态的关键，并且可影响母乳中性寡糖的浓度及α1,2岩藻糖寡糖的比例[19,20,21]。FUT2基因处于活化状态的人群归类为分泌型人群，而处于失活状态的人群则为非分泌型，这两类母亲的子代在诸多方面也存在差异，例如，由分泌型母亲母乳喂养的婴儿发生腹泻的几率更小[22]；不同的分泌型母亲其早产儿的生存率也存在差异[23]；特别是，不同分泌型母亲在哺乳期所分泌母乳中乳糖岩藻糖基化的过程会发生变化[24,25]，因此，这些变化可能直接影响到其子代肠道菌群，特别是双歧杆菌的定植和发展，进而影响婴儿健康。
但是，目前关于Fut2基因型的主要方法是利用凝集抑制实验或基因测序，需要采用特定抑制物处理样本并以抗体进行标化[26]，或较长时间的测序及比对过程，费用和时间成本较高；此外也有研究者利用质谱法检测母乳主要的标记性HMO(2′LF等)的水平来区分分泌型与非分泌型[27]，这种方法的费用和时间成本也较高。
【参考文献】
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3.Fukuda S,Toh H,Hase K,Oshima K,Nakanishi Y,Yoshimura K,Tobe T,Clarke JM,Topping DL,Suzuki T,et al:Bifidobacteria can protect fromenteropathogenic infection through production of acetate.Nature 2011,469:543-547.
4.Lievin V,Peiffer I,Hudault S,Rochat F,Brassart D,Neeser JR,ServinAL:Bifidobacterium strains from resident infant human gastrointestinalmicroflora exert antimicrobial activity.Gut 2000,47:646-652.
5.Huda MN,Lewis Z,Kalanetra KM,Rashid M,Ahmad SM,Raqib R,Qadri F,Underwood MA,Mills DA,Stephensen CB:Stool microbiota and vaccine responses ofinfants.Pediatrics 2014，134:e362-372.
6.Goldsmith F,O'Sullivan A,Smilowitz JT,Freeman SL:Lactation andIntestinal Microbiota:How Early Diet Shapes the Infant Gut.J Mammary GlandBiol Neoplasia 2015,20:149-158.
7.Jimenez E,Delgado S,Maldonado A,Arroyo R,Albujar M,Garcia N,JariodM,Fernandez L,Gomez A,Rodriguez JM:Staphylococcus epidermidis:a differentialtrait of the fecal microbiota of breast-fed infants.BMC Microbiol 2008,8:143.
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发明内容
基于以上技术背景和现状，本发明旨在开发快速、准确反应中国人群Fut2基因关键位点的多态性，并以预测分泌型/非分泌型及母乳岩藻糖基化低聚糖(fHMOs)水平的方法，该方法可在新生儿健康保护、实现个性化营养搭配方面具有深远的社会意义和良好的应用前景。
利用荧光定量PCR法检测FUT2基因类型的方法，所述方法包括下述工艺步骤：
S1，获取待测样品全血基因组DNA；
S2，利用引物及荧光物质进行荧光定量PCR检测，其中，上游引物的序列如SEQ IDNO.1所示；下游引物的序列如SEQ ID NO.2所示：
S3，根据PCR检测后熔解曲线的熔解峰位置判断rs1047781位点的A/T突变情况，进而判断FUT2基因的类型为分泌型或非分泌型。
本发明所述上游引物的序列如SEQ ID NO.1所示；下游引物的序列如SEQ ID NO.2所示，
SEQ ID NO.1：CCTGGCAGAACTACCACCTG。
SEQ ID NO.2：GGCTGCCTCTGGCTTAAAGA。
进一步地，本发明所述引物按如下方式获得：根据从NCBI公开的Fut2基因序列，利用Gene Tools软件自行设计，用于检测rs1047781位点的A/T突变的PCR产物共计570bp。
本发明所述rs1047781位点为A时(未发生A/T突变)，FUT2基因的类型为分泌型；所述rs1047781位点为T时(发生A/T突变)，FUT2基因的类型为非分泌型。
进一步地，所述步骤S3中，判断方法如下：熔解峰位置在89.6℃±0.3，则rs1047781位点未发生A/T突变，FUT2基因的类型为分泌型；熔解峰位置在87℃±0.3，则rs1047781位点发生A/T突变，FUT2基因的类型为非分泌型。
上述方法中，当确定FUT2基因的类型为非分泌型或分泌型后，可进一步确定母乳中岩藻糖基化低聚糖(fHMOs)的含量水平，即当FUT2基因的类型为非分泌型时，岩藻糖基化低聚糖在该哺乳期母乳中的含量较分泌型母乳中岩藻糖基化低聚糖含量低至少50％。
进一步地，所述步骤S2中，PCR反应程序分为4个步骤，具体如下：
①37℃5min，1个循环；
②95℃15min，1个循环；
③95℃15s，55℃20s，72℃20s，40个循环；
④95℃15s，60℃1min，95℃15s，60℃15s，1个循环。
进一步地，所述待测样品为血液、唾液或乳汁；更进一步地，优选为乳汁，如母乳。
进一步地，所述荧光定量PCR反应体系，总体积为25μL，具体如下：2倍Premix Ex TaqTM II PCR缓冲液12.5μL；10mol/L上游引物1μL；10mol/L下游引物1μL；DNA模版2μL(终浓度2μM)；ddH2O 8.5μL。
上述技术方案中，所述Premix Ex TaqTM II PCR缓冲液中含有荧光物质，更进一步地，所述荧光物质为SYBR。
本发明的另一目的是提供利用荧光定量PCR法判断母乳中岩藻糖基化低聚糖(fHMOs)在不同哺乳期的水平的方法。
荧光定量PCR法判断母乳中岩藻糖基化低聚糖(fHMOs)在不同哺乳期的含量的方法，所述方法包括下述工艺步骤：
S1，获取母乳全血基因组DNA；
S2，利用引物及荧光物质进行荧光定量PCR检测，其中，上游引物的序列如SEQ IDNO.1所示；下游引物的序列如SEQ ID NO.2所示：
S3，根据PCR检测后熔解曲线的熔解峰位置判断rs1047781位点的A/T突变情况，进而判断FUT2基因的类型为分泌型或非分泌型；
S4，当FUT2基因的类型为非分泌型时，岩藻糖基化低聚糖在该哺乳期母乳中的含量较FUT2基因的类型为分泌型的母乳中岩藻糖基化低聚糖含量低至少50％。
本发明所述岩藻糖基化低聚糖(fHMOs)，是以α1-2/3糖苷键连接的岩藻糖基化母乳低聚糖，包括2′-岩藻糖基乳糖(2′LF)【2′-fucosyllactose(2′LF)】,3′-唾液酸-3-岩藻 糖基乳糖(3′S-3FL)【3′-sialyl-3-fucosyllactose(3′S-3FL)】,3′-岩藻糖基乳糖(3FL)【3′-fucosyllactose(3FL)】,乳糖二岩藻四糖(LDFT)【lactodifucotetraose(LDFT)】,乳酰-N-岩藻五糖(LNFP)【Lacto-N-fucopentaose(LNFP)】,乳糖-N-新双岩藻基六糖(LNnDFH)【lacto-N-neodifucohexaose(LNnDFH)】,乳新岩藻五糖(LNnFP)【lacto-N-neofucopentaose(LNnFP)】。
本发明所述的岩藻糖基化低聚糖(fHMOs)具有调节肠道菌群、促进特定双歧杆菌菌种增殖的功能、并维持肠道微生态平衡、促进新生儿生长发育。所述的特定双歧杆菌包括青春双歧杆菌、两歧双歧杆菌、短双歧杆菌、婴儿双歧杆菌、婴儿双歧杆菌亚种、假小链双歧杆菌、长双歧杆菌等新生儿肠道常见菌种。
本发明的有益效果为：本发明所述利用荧光定量PCR法检测FUT2基因类型的方法，可以特异性的进行中国人群岩藻糖基转移酶基因Fut2单核苷酸位点多态性(SNP)分析，以明确分泌型/非分泌型并预测母乳岩藻糖基化低聚糖(fHMOs)水平及其母乳喂养的新生儿肠道双歧杆菌和乳酸杆菌的丰度。
附图说明
图1(A)～(C)为荧光定量PCR法检测的熔解曲线示意图；图1(A)为T人群出现87.06℃的熔解峰；(B)为A人群主要出现89.62℃的熔解峰；(C)为阴性对照。
图2不同个体荧光定量PCR法检测的熔解曲线图；(A)为T人群出现87.06℃的熔解峰；(B)为A人群主要出现89.62℃的熔解峰；
图3利用测序法检测的Fut2基因序列；
图4利用测序法检测的不同母亲Fut2基因SNP比较；
图5中国母亲母乳样本的分泌型与非分泌型的分布；
图6哺乳期不同分泌型中国母亲母乳HMO含量比较；(A)6天哺乳期不同分泌型中国母亲母乳HMO含量；(B)42天哺乳期不同分泌型中国母亲母乳HMO含量；
图7不同分泌型中国母亲母乳喂养的新生儿肠道优势微生物群的差异；(A)不同分泌型妈妈所哺育的新生儿在出生后第六天的肠道菌群科水平差异；(B)不同分泌型妈妈所哺育的新生儿在出生后第六天的肠道菌群属水平差异；(C)不同分泌型妈妈所哺育的新生儿在出生后第六天和第42天的肠道主要的差异菌群；(D)新生儿在出生后第六天和第42天的肠道菌群与母乳HMO成分之间的相关性；
图8标志性岩藻糖基化HMO(fHMO)与新生儿肠道双歧杆菌菌种丰度之间的相关性。
具体实施方式
下述非限制性实施例可以使本领域的普通技术人员更全面地理解本发明，但不以任何方式限制本发明。
下述实施例中所述试验方法，如无特殊说明，均为常规方法；所述试剂和材料，如无特殊说明，均可从商业途径获得。
下述实施例PCR反应所涉及试剂如下：Premix Ex TaqTM II,含荧光物质SYBR。
实施例1
1.中国母亲Fut2基因SNP检测
(1)样本收集
受试母亲：分别于大连市妇幼保健院和绥化市第一医院妇产科选取56名妊娠34周的健康孕妇(编号#1～#56)，填写知情同意书和详细的病史问卷调查表，主要包括年龄、体征、孕产史、当前和以前的健康史、饮食习惯和体力活动水平，以及药物和补充剂摄入史等。记录婴儿的分娩方式(剖腹产或阴道)，婴儿性别，重量，长度，和出生时胎龄，并在跟踪调查中提供有关自己和婴儿的健康调查问卷。受试者须同意接受医生对其在哺乳期的指导和培训，并提供哺乳期的适当样本。
母乳收集：在不同时期给予受试者样品管，记录收集的日期、样本收集前的饮食时间及饮食内容。样品收集时间点分别为产后6、21、60、120、180天。于采集当天上午母乳喂食婴儿2～4小时后，在医生的指导下利用手动吸奶器收集入15毫升的聚丙烯管中。将所采集的母乳反转混合六次随后保立即保存于-20℃冰箱中。样品以干冰运至实验室，并在-80℃储存。
婴儿粪便收集：分别在上述受试母亲产后6、21、60、120、180天收集其婴儿粪便样本。记录婴儿使用抗生素和是否喂食母乳及婴儿配方食物情况。提示家长填写粪便样品瓶的详细标签，记录婴儿摄入的固体，婴儿配方奶粉，药物等。婴儿的粪便样本置于无菌塑料管，立即保存于在20℃冰箱中。样品以干冰运至实验室，并在-80℃储存。
(2)采用本发明所述进荧光定量PCR法行检测Fut2基因SNP
DNA提取：取受检者的母乳2mL，采用离心吸附柱法按照基因组DNA提取试剂盒的操作说明书操作，提取受检者全血基因组DNA，用琼脂糖凝胶(1％)电泳检测。所提取的DNA条带清晰，完整性好，紫外分光光度计检测DNA浓度为50ng/μL。
PCR引物设计：根据从NCBI下载的人Fut2基因序列，利用Gene Tools软件自行设计，产物为共计570bp，可以检测rs1047781位点的突变。所述上游引物的序列如SEQ IDNO.1所示；下游引物的序列如SEQ ID NO.2所示，
SEQ ID NO.1：CCTGGCAGAACTACCACCTG。
SEQ ID NO.2：GGCTGCCTCTGGCTTAAAGA。
PCR反应体系：按照下表进行混匀，然后上机进行检测。
PCR反应条件：温度循环程序分4个步骤：①37℃5min，1个循环；②95℃15min，1个循环；③95℃15s，55℃20s，72℃20s，40个循环；④95℃15s，60℃1min，95℃15s，60℃15s，1个循环，进行熔解曲线分析。
结果体现：PCR反应后出现特异性扩增，各PCR产物的Tm值之间均能很好的进行区分(大于2.5℃)，T人群出现87.06℃的熔解峰(如图2A)；A人群主要出现89.62℃的熔解峰(如图2B)。综合上述检测结果，可以判定在87℃±0.3出现熔解峰，说明有A/T突变，正常人仅在89.6℃±0.3出现熔解峰。
经检测，受试样本共56例中有13例为T型，占23.2％，其余为A型，如图2所示。
(3)采用基因测序法进行基因SNP检测
DNA提取及PCR引物体系与上述荧光定量PCR法一致，所得PCR产物直接进行碱基测序，所得测序结果如图3(编号#1)所示。通过序列分析比较，与荧光定量PCR法检测的结果一致，受试样本共56例中有13例为T型，占23.2％，其余为A型。
2.哺乳期不同分泌型中国母亲母乳HMO含量比较
对不同分泌型母亲母乳HMO进行了质谱法检测，发现在哺乳初期(6天)两种分泌型妈妈母乳中主要差异的HMO为H2F1,H3N1F2(如图6A)；在哺乳中期(42天)两种分泌型妈妈母乳中主要差异的HMO为H2F1,H2F2,H3N1F2,H4N2,H4N2A1(如图6B)。
3.不同分泌型中国母亲母乳喂养的新生儿肠道优势微生物群的差异
进一步提取婴儿粪便DNA，对不同分泌性妈妈所母乳哺育的新生儿肠道菌群进行了16S rDNA高通量测序分析，发现双歧杆菌在分泌型妈妈所哺育的新生儿肠道中占有绝对优势(如图7ABC)，而母乳HMO，特别是岩藻糖基化HMO与分泌型妈妈哺育的新生儿肠道菌群之间存在强正相关性(图7D)。
4.标志性岩藻糖基化HMO(fHMO)与新生儿肠道双歧杆菌菌种丰度之间的相关性
进一步以Spearman法分析了母乳HMO与新生儿肠道微生物之间的相关性，发现H2F1,H2F2等岩藻糖基化HMO与新生儿肠道假小链双歧杆菌、加氏乳杆菌、动物双歧杆菌等微生物的相对丰度之间具有显著相关性。
Claims (7)
1.利用荧光定量PCR法检测FUT2基因类型的方法，其特征在于：所述方法包括下述工艺步骤：
S1，获取待测样品全血基因组DNA；
S2，利用引物及荧光物质进行荧光定量PCR检测，其中，上游引物的序列如SEQ ID NO.1所示；下游引物的序列如SEQ ID NO.2所示：
S3，根据PCR检测后熔解曲线的熔解峰位置判断rs1047781位点的A/T突变情况，进而判断FUT2基因的类型为分泌型或非分泌型。
2.根据权利要求1所述的方法，其特征在于：所述步骤S3中，判断方法如下：熔解峰位置在89.6℃±0.3，则rs1047781位点未发生A/T突变，FUT2基因的类型为分泌型；熔解峰位置在87℃±0.3，则rs1047781位点发生A/T突变，FUT2基因的类型为非分泌型。
3.据权利要求1所述的方法，其特征在于：所述步骤S2中，PCR反应程序分为4个步骤，具体如下：
①37℃5min，1个循环；
②95℃15min，1个循环；
③95℃15s，55℃20s，72℃20s，40个循环；
④95℃15s，60℃1min，95℃15s，60℃15s，1个循环。
4.据权利要求1所述的方法，其特征在于：所述待测样品为血液、唾液或乳汁。
5.据权利要求1所述的方法，其特征在于：所述荧光定量PCR反应体系，具体如下：2倍Premix Ex TaqTMII PCR缓冲液12.5μL；10mol/L上游引物1μL；10mol/L下游引物1μL；终浓度2μM DNA模版2μL；ddH2O 8.5μL，总体积25μL。
6.荧光定量PCR法判断母乳中岩藻糖基化低聚糖含量的方法，所述方法包括下述工艺步骤：
S1，获取母乳全血基因组DNA；
S2，利用引物及荧光物质进行荧光定量PCR检测，其中，上游引物的序列如SEQ ID NO.1所示；下游引物的序列如SEQ ID NO.2所示：
S3，根据PCR检测后熔解曲线的熔解峰位置判断rs1047781位点的A/T突变情况，进而判断FUT2基因的类型为分泌型或非分泌型；
S4，当FUT2基因的类型为非分泌型时，岩藻糖基化低聚糖在该哺乳期母乳中的含量较FUT2基因的类型为分泌型的母乳中岩藻糖基化低聚糖含量低至少50％。
7.根据权利要求6所述的方法，其特征在于：所述岩藻糖基化低聚糖是以α1-2/3糖苷键连接的岩藻糖基化母乳低聚糖，包括2′-岩藻糖基乳糖(2′LF),3′-唾液酸-3-岩藻糖基乳糖(3′S-3FL),3′-岩藻糖基乳糖(3FL),乳糖二岩藻四糖(LDFT),乳酰-N-岩藻五糖(LNFP),乳糖-N-新双岩藻基六糖(LNnDFH),乳新岩藻五糖(LNnFP)。
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