CN107937581B - 用于乳酸菌测序的扩增引物对、乳酸菌种属鉴定方法及应用 - Google Patents
用于乳酸菌测序的扩增引物对、乳酸菌种属鉴定方法及应用 Download PDFInfo
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Abstract
本发明公开了一种用于肠道中乳酸菌测序的扩增引物对,所述引物对为Lab‑6,包括核苷酸序列如SEQ ID NO.1所示的Lab‑6F和SEQ ID NO.2所示的Lab‑6R。该扩增引物对可以实现乳酸菌种属鉴定,具有较好的特异性和实用性,能较好反映肠道中乳酸菌的多样性。适用于发酵乳制品、粪便和土壤等复杂样品中乳酸菌的分析。
Description
技术领域
本发明属于基因工程技术领域,特别涉及一种用于肠道中乳酸菌测序的扩增引物对、乳酸菌种属鉴定方法及应用。这种鉴定方法可以对粪便等复杂样品中乳酸菌进行测序。这对引物序列是基于乳酸菌(主要包含乳酸杆菌属、乳球菌属、魏斯氏菌属、肠球菌属、明串球菌属、链球菌属和片球菌属)16S rRNA基因序列设计的,其PCR扩增产物达750bp左右,满足测序要求。
背景技术
乳酸菌(Lactic Acid Bacteria,LAB)是发酵碳水化合物生产乳酸、无芽孢、不运动或很少运动、过氧化氢酶呈阴性的革兰氏阳性菌的总称。乳酸菌分布广泛,在人类胃肠道生态系统中发挥有益作用。乳酸菌可以通过几种抗菌机制防止肠粘膜病原体的粘连和复制,还可以在肠腔释放各种酶,对消化作用产生协同作用和缓解肠道不良反应的症状。食用LAB发酵乳制品还可能会起到抗肿瘤的作用。因此,乳酸菌具有改善肠道环境和维护肠道菌群平衡的作用。
同时,许多研究发现,乳酸菌在粪便中的存在,其含量往往与宿主健康有紧密联系。Schiffrin等人发现,健康志愿者连续食用益生菌发酵制品3周,其外周血细胞的非特异性吞噬活性增强,其粪便中的双歧杆菌属和乳酸杆菌属的数量明显增加,停用益生菌3周后的粪便菌落即刻恢复到最初时刻,吞噬活动的增加开始减少(Schiffrin E J,Brassart D,Servin AL,et al.Immune modulation of blood leukocytes in humans by lacticacid bacteria:criteria for strain selection.[J].American Journal of ClinicalNutrition,1997,66(2):515S.)。Zheng等人以50名患湿疹的婴儿与51名健康婴儿为研究对象,利用高通量测序技术对婴儿粪便中细菌组成进行研究,发现在健康婴儿组有4个属含量丰富,其中包括链球菌属,尤其是已知具有抗炎性的Streptococcus salivarius,在健康的婴儿粪便中含量很高,在患有湿疹的婴儿体内含量很低。而且,乳酸杆菌属的含量在健康婴儿体内高于湿疹婴儿的,但是无法鉴定到具体种(Zheng H,Liang H,Wang Y,etal.Altered Gut Microbiota Composition Associated with Eczema in Infants[J].Plos One,2016,11(11):e0166026.)。
肠道中有数以万计的微生物,采用传统技术可以从粪便中获取部分可培养和相对含量较高的微生物,但很难从中获取曾经存活或是难以分离的部分微生物。随着高通量测序技术的迅速发展,纯培养技术遇到的这些障碍逐渐被克服,使得全面研究肠道微生物多样性成为可能。但是肠道中乳酸菌的相对含量较低,目前用来研究肠道菌群多样性的细菌通用引物无法扩增出肠道中的乳酸菌进行测序,这使肠道中乳酸菌的群落结构及其在人群肠道的分布和多样性研究受到阻碍。深入解析人体肠道中乳酸菌的群落结构及多样性,不仅有助于研究乳酸菌在肠道中的分布及多样性,还有助于研究肠道中乳酸菌与各种疾病的关系,为调节肠道菌群提供科学理论依据。
目前已有的乳酸菌引物多用于乳酸菌的鉴定和定量,扩增片段多集中在200-400bp,而且最多可以扩增出4个乳酸菌属,这不但无法满足高通量测序及鉴定的要求,而且不利于肠道中的乳酸菌群落结构的全面研究。
发明内容
因此,本发明的目的在于提供一种用于肠道中乳酸菌测序的方法及其所用引物序列,弥补了细菌通用引物不能全面扩增肠道中乳酸菌的缺陷,为研究肠道中乳酸菌与人类健康关系奠定了基础。
本发明的第一个目的是提供一种用于肠道中乳酸菌测序的扩增引物对,所述引物对为Lab-6,包括核苷酸序列如SEQ ID NO.1所示的Lab-6F和SEQ IDNO.2所示的Lab-6R:
Lab-6F:5’-GCTCAGGAYGAACGCYGG-3’,其中,Y表示C或T;
Lab-6R:5’-CACCGCTACACATGRADTTC-3’,其中,R表示A或G,D表示A或G或T。
本发明的第二个目的是提供利用上述扩增引物对鉴定乳酸菌种属的方法,包括如下步骤:
S1:提取含有乳酸菌的待测样品的基因组DNA,以获得的基因组DNA为模板,以Lab-6F和Lab-6R为扩增引物进行PCR扩增,得到PCR扩增产物;
S2:序列测定
将PCR扩增产物纯化后,得到大小750bp的扩增片段,对目标扩增片段进行SMRT高通量测序;
S3:使用QIIME(V1.7)平台对扩增序列进行物种注释,得到准确的鉴定结果。
优选地,所述乳酸菌包括乳酸杆菌属、乳球菌属、魏斯氏菌属、肠球菌属、明串球菌属、链球菌属和片球菌属。
优选地,S2中,PCR扩增体系为:
PCR扩增反应程序为:
本发明的第三个目的是提供上述用于肠道中乳酸菌测序的扩增引物对在制备肠道中乳酸菌检测试剂盒中的应用。
本发明的第四个目的是提供上述用于肠道中乳酸菌测序的扩增引物对在发酵乳制品、粪便或土壤样品中乳酸菌种属分析的应用。
本发明提供的用于肠道中乳酸菌测序的扩增引物是基于乳酸菌16SrRNA基因序列设计的,可以扩增出多个乳酸菌属,具有较好的特异性和实用性,能较好反映肠道中乳酸菌的多样性。同时适用于发酵乳制品、粪便和土壤等样品中乳酸菌的分析。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1中14个待测材料的乳酸菌引物Lab-6特异性扩增结果;
图中,从左到右分别依次为Marker、Enterococcus asini(DSM-11492)、Enterococcus faecium(ATCC 19434T)、Enterococcus Italicus(DSM-15952)、Lactobacillus Casei(ATCC-334)、Lactobacillus Salivarius(DSM-20555)、Lactobacillus Helveticus(1.2278),WeissellaBeninensis(DSM-22752)、Weissellacofusa(IMAU10245)、Weissellakandleri(DSM-20593)、PedicoccusAcidilactici(JCM8791)、PedicoccusPentosaceus(DSM-20336)、Leuconostocpsedomesenteroides(DSM-20193)、Streptococcus Thermonphillus(NM-81-2)、Lactococcusplantarum(DSM-20686)、灭菌蒸馏水。
具体实施方式
为了使本领域技术人员更好地理解本发明的技术方案能予以实施,下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。下述实施例中的实验方法,如无特殊说明,均为常规方法,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
本发明提供的用于肠道中乳酸菌测序的扩增引物对,所述引物对的核苷酸序列分别如下:
Lab-6F:5’-GCTCAGGAYGAACGCYGG-3’,其中,Y表示C或T;
Lab-6R:5’-CACCGCTACACATGRADTTC-3’,其中,R表示A或G,D表示A或G或T。
上述扩增引物对的获得方法具体如下:
从NCBI下载乳酸杆菌属、乳球菌属、魏斯氏菌属、肠球菌属、明串球菌属、链球菌属和片球菌属7个属的所有种的16S rRNA基因序列。通过比对寻找7个属共有的16S rRNA基因序列片段,借助引物设计软件Primer 5.0设计PCR扩增产物约700-1000bp的乳酸菌引物,将设计好的乳酸菌引物运用MEGE6.0软件进行搜索和序列对齐,必要时设计简并碱基。将设计好的乳酸菌引物经过琼脂糖凝胶进行特异性评估进行初步筛选,利用初步筛选好的、带有不同Barcode的乳酸菌引物,对含有乳酸菌的不同样品DNA稀释液进行扩增,最后借助Pacific Bioscience公司的SMRT(Single molecule Real-Time)技术对扩增产物进行高通量测序,进一步筛选,得到适用于对肠道中乳酸菌进行测序的上述扩增引物对。
下面提供利用上述用于肠道中乳酸菌测序的扩增引物对,实现乳酸菌种属的精确鉴定的实施例。
实施例1
使用本发明上述扩增引物对7个属14个种的乳酸菌进行PCR扩增,具体包括如下步骤:
以常见乳酸菌的7个属共14株细菌的DNA为阳性模板,灭菌过滤超纯水为阴性模板,以Lab-6F和Lab-6R为引物进行PCR扩增,得到PCR扩增产物;
上述引物对的核苷酸序列分别如下:
Lab-6F:5’-GCTCAGGAYGAACGCYGG-3’,其中,Y表示C;
Lab-6R:5’-CACCGCTACACATGRADTTC-3’,其中,R表示A,D表示T。
其中,这14株细菌分别是Enterococcus asini(DSM-11492)、Enterococcusfaecium(ATCC 19434T)、Enterococcus Italicus(DSM-15952)、LactobacillusCasei(ATCC-334)、Lactobacillus Salivarius(DSM-20555)、Lactobacillus Helveticus(1.2278),Weissella Beninensis(DSM-22752)、Weissella cofusa(IMAU10245)、Weissella kandleri(DSM-20593)、Pedicoccus Acidilactici(JCM8791)、PedicoccusPentosaceus(DSM-20336)、Leuconostoc psedomesenteroides(DSM-20193)、Streptococcus Thermonphillus(NM-81-2)、Lactococcus plantarum(DSM-20686);
a.PCR扩增体系为:
b.PCR扩增反应程序为:
将PCR扩增产物于1%琼脂糖凝胶中以5v/cm的电压,电泳20-30min,紫外拍照,检测扩增效果,具体如图1所示。
实施例2
比较本发明乳酸菌扩增引物Lab-6(Lab-6F和Lab-6R)和细菌通用引物27f/1492r对粪便样品中乳酸菌的扩增结果,具体包括如下步骤:
(1):以细菌通用引物(27f/1492r)和S1中提供的扩增引物对(所述引物对的核苷酸序列和实施例1相同)对粪便DNA稀释液进行扩增,PCR扩增体系和PCR扩增反应程序均和实施例1相同,得到PCR扩增产物;
(2):序列测定
PCR扩增产物大小约750bp,将其进行纯化、混样、DNA片段修复、连接接头和文库质控,借助单分子实时合成测序仪(Single molecule Real-Time,SMRT)(PacificBiosciences公司)对扩增片段进行测序;使用太平洋生物技术公司SMRT bell TMtemplate prep kit 1.0试剂盒构建DNA测序文库,具体操作按照试剂盒使用说明书进行。
最后,使用Portal(V2.7)中的RS_ReadsOfinsert.1方案进行测序质量控制;其中,质控参数中的最短插入片段长度和最大插入片段长度,参数设定值为650和850;质控合格的序列进行后续分析;
(3):使用QIIME(V1.7)平台对扩增序列进行物种注释,得到准确的鉴定结果。细菌通用引物(27f/1492r)扩增结果与乳酸菌引物(Lab-6)扩增结果进行比较分析结果见表1和表2。
表1引物27f/1492r和Lab-6在粪便样品中扩增出的乳酸菌属及相对含量
表2引物27f/1492r和Lab-6在粪便样品中扩增出的乳酸菌种及相对含量
在属水平上,通用引物27f/1492r仅扩增出Lactobacillus和Streptococcus共2个属,在粪便中的相对含量分别为2.33%和0.05%。而乳酸菌引物Lab-6除了可以扩增出Lactobacillus和Streptococcus,还可以扩增出Enterococcus和Weissella(表1)。在种水平上,通用引物27f/1492r仅扩增出Lactobacillus rogosae和Streptococcussalivarius,而乳酸菌引物Lab-6可以扩增出7个乳酸菌种,除Lactobacillus rogosae和Streptococcus salivarius外,Lab-6还扩增出Streptococcusinfantis(1.24%)、Streptococcus sanguinis(0.57%)、Streptococcus pleomorphus(0.10%)、Enterococcus avium(0.10%)和Weissella confusa(0.10%)(表2)。
由此可见,乳酸菌引物Lab-6相较通用引物扩增灵敏度更高,可以扩增出大量通用引物无法扩增出的乳酸菌种,特别是一些低丰度的乳酸菌。表明引物Lab-6适用于发酵乳制品、粪便和土壤等复杂样品中乳酸菌的分析。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。以上所述实施例仅是为充分说明本发明而所举的较佳的实施例,其保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换,均在本发明的保护范围之内,本发明的保护范围以权利要求书为准。
序列表
<110> 内蒙古农业大学
<120> 用于乳酸菌测序的扩增引物对、乳酸菌种属鉴定方法及应用
<141> 2017-12-18
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> 人工序列
<400> 1
gctcaggayg aacgcygg 18
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<400> 2
caccgctaca catgradttc 20
Claims (2)
1.一种用于肠道中乳酸菌测序的扩增引物对在制备肠道中乳酸菌检测试剂盒中的应用,其特征在于,所述引物对为Lab-6,包括核苷酸序列如SEQ ID NO.1所示的Lab-6F和SEQID NO.2所示的Lab-6R:
Lab-6F:5’- GCTCAGGAYGAACGCYGG-3’,其中,Y表示C或T;
Lab-6R:5’-CACCGCTACACATGRADTTC-3’,其中,R表示A或G,D表示A或G或T;
所述乳酸菌包括乳酸杆菌属、乳球菌属、魏斯氏菌属、肠球菌属、明串球菌属、链球菌属和片球菌属,具体包括Enterococcus asini、Enterococcus faecium、Enterococcus italicus、Lactobacillus casei、Lactobacillus salivarius、Lactobacillus helveticus,Weissella beninensis、Weissella cofusa、Weissella kandleri、Pedicoccus acidilactici、Pedicoccus pentosaceus、Leuconostoc psedomesenteroides、Streptococcus thermonphillus、Lactococcus plantarum、Lactobacillus rogosae、Streptococcus salivarius、Streptococcus infantis、 Streptococcus sanguinis、Streptococcus pleomorphus和Enterococcus avium。
2.一种用于肠道中乳酸菌测序的扩增引物对在发酵乳制品、粪便或土壤样品中乳酸菌种属分析的应用,其特征在于,所述引物对为Lab-6,包括核苷酸序列如SEQ ID NO.1所示的Lab-6F和SEQ ID NO.2所示的Lab-6R:
Lab-6F:5’- GCTCAGGAYGAACGCYGG-3’,其中,Y表示C或T;
Lab-6R:5’-CACCGCTACACATGRADTTC-3’,其中,R表示A或G,D表示A或G或T;
所述乳酸菌包括乳酸杆菌属、乳球菌属、魏斯氏菌属、肠球菌属、明串球菌属、链球菌属和片球菌属,具体包括Enterococcus asini、Enterococcus faecium、Enterococcus italicus、Lactobacillus casei、Lactobacillus salivarius、Lactobacillus helveticus,Weissella beninensis、Weissella cofusa、Weissella kandleri、Pedicoccus acidilactici、Pedicoccus pentosaceus、Leuconostoc psedomesenteroides、Streptococcus thermonphillus、Lactococcus plantarum、Lactobacillus rogosae、Streptococcus salivarius、Streptococcus infantis、 Streptococcus sanguinis、Streptococcus pleomorphus和Enterococcus avium。
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