CN115261271B - 一种肠道菌群的高通量分离培养与筛选方法 - Google Patents
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Abstract
本发明是一种肠道菌群的高通量分离培养与筛选方法,包括以下步骤:步骤1,粪便样筛选与预处理:对获取的粪便样本进行16S扩增子测序,分析粪便样本的菌群丰富度;步骤2,活菌检测:利用活菌检测技术测定步骤1中的粪便样稀释液中活菌总量;步骤3,极限稀释培养:计算稀释倍数,将稀释后的混合细菌培养基转移到96细菌培养板中进行培养;步骤4,转移培养:挑选生长孔数合适的96孔细菌培养板,全部转移至新的96孔中间板;步骤5,菌种鉴定:提取上一骤中的取出的菌液DNA,电泳检测扩增条带并送样测序;检查测序的峰图质量,并进行序列比对;步骤6,菌种保藏。
Description
技术领域
本发明涉及菌群的分离和筛选技术领域,尤其涉及一种肠道菌群的高效分离培养方法和目的细菌的高通量定向筛选方法。
背景技术
国内外许多研究表明肠道菌群对人体健康至关重要,肠道菌群及其代谢产物通过影响肠道通透性、肠黏膜免疫、肠道药物代谢及肠道神经系统活性直接或间接地参与调节胃肠道功能、大脑行为和系统免疫等一系列与人体健康密切相关的过程。
如专利申请202010450572.5公开了一种肠道菌群多基因组学在微生态中药筛选的新方法,涉及药物筛选技术领域。该方法包括:获取粪便样本;对所述粪便样本进行宏代谢组学检测、16S rRNA基因测序、实时荧光定量PCR检测、宏基因组测序,得到检测结果;结合粪便样本所有者的特征、检测结果,基于药理学的清晰信号传导作用机制建立药物有效性筛选模型;利用所述药物有效性筛选模型进行药物筛选。本发明利用肠道菌群检测结果的大数据构建药物筛选模型进行药物筛选,筛选模型可匹配样本所有者的特征,具有针对性,能够更加清晰高效地进行药物筛选。
然而分离筛选肠道菌群的技术手段有限,传统的平板筛选方法成本高,周期长,且由于菌种间竞争抑制或富集过程中生长优势种的存在,导致难以筛选到低丰度的肠道菌种。因此开发一种肠道菌群的高通量分离培养方法是尤为必要的。
发明内容
为解决上述问题,本发明的首要目的是提供一种肠道菌群的高通量分离培养与筛选方法,该方法实现肠道菌群的高效分离和目的细菌的高通量定向筛选,为活菌药物的研发提供丰富的菌种资源。
本发明的另一个目的是提供一种肠道菌群的高通量分离培养与筛选方法,该方法通过临床试验和数据分析发掘与人体疾病相关的关键菌种,开发一种针对目的菌种的高通量定向筛选方法,基于粪便样本活菌计数和粪便样本极限稀释培养的技术基础,可实现肠道菌群的快速分离培养。
本发明的再一个目的是提供一种肠道菌群的高通量分离培养与筛选方法,该方法使细菌分离分散到96孔板中,从而解除不同细菌间的生长竞争或生长抑制,使丰度较低或生长劣势的细菌能够生长,且分离筛选周期短,成本低。
为实现上述目的,本发明采用的技术方案是:
一种肠道菌群的高通量分离培养与筛选方法,包括以下步骤:
步骤1,粪便样筛选与预处理:对获取的粪便样本进行16S扩增子测序,分析粪便样本的菌群丰富度;
具体地说,将含有目的菌种且相对丰度较高的供体粪便样用于分离培养或定向筛选,并按照一定比例往粪便样中加入改良的生理盐水,震荡混匀。
步骤2,活菌检测:利用活菌检测技术测定步骤1中的粪便样稀释液中活菌总量;
步骤3,极限稀释培养:根据活菌总量,计算稀释倍数,并使用改良培养基对步骤1所得的粪便样进行梯度稀释,将稀释后的混合细菌培养基转移到96细菌培养板中,并使用封口膜密封后,放入37℃厌氧培养箱进行培养;
提供极限稀释使细菌分离分散到96孔板中,将细菌进行了分散,给细菌提供了较为宽松的生长环境,从而解除不同细菌间的生长竞争或生长抑制,使丰度较低或生长劣势的细菌能够生长,有利于各种细菌的培育。
步骤4,转移培养:挑选生长孔数不超过40%的96孔细菌培养板,全部转移至新的96孔中间板,再取出部分菌液用于菌液DNA的提取,再向96孔中间板中补充YCFA、BHIS或MRS富营养培养基或定向筛选培养基,继续培养剩余菌液;
转移培养能够实现目的细菌的高通量定向筛选,提供转移培养可以重点培养目的细菌,使其能够快速生长,提高定向筛选的效果,缩短分离筛选周期,提高培养效率。
步骤5,菌种鉴定:提取上一骤中的取出的菌液DNA,PCR扩增16S基因序列,电泳检测扩增条带并送样测序;检查测序的峰图质量,并进行序列比对;
步骤6,菌种保藏:比对所得菌种鉴定结果,筛选需要保藏的菌株编号,按照对应编号保藏步骤4中所述的96孔中间板所培养的菌液。
同时为确保菌液无环境污染,使用接种环蘸取部分保藏菌液进行平板划线以验证。
进一步的,步骤1中,所述的粪便样按1g:100-500μl的比例向粪便样中加入改良的生理盐水进行震荡混匀。
进一步的,步骤1中,所述的生理盐水中可添加半胱氨酸盐酸盐、维生素C、维生素E、茶多酚等抗氧化剂中的一种或任意组合,以增加培养环境中的厌氧条件,从而增加厌氧菌存活率。
进一步的,步骤3中,所述的培养基配方应为模拟肠道环境的培养基或定向筛选培养基。优选的,模拟肠道环境培养基包括:YCFA、BHIS、Bryant and Burkey培养基等。定向筛选培养基包括:以黏蛋白为单一碳源的Akkermansia muciniphila(嗜黏蛋白阿克曼菌)筛选培养基、促进Lactobacillus(乳酸杆菌属)生长的M17肉汤或乳酸杆菌选择性肉汤、促进Bifidobacterium(双歧杆菌属)生长的BBL或TPY培养基等。
进一步的,为保证步骤3中梯度稀释的准确性,应以1-10倍的倍数进行梯度稀释至目标浓度,使96孔细菌培养板中仅有10-30%孔数有菌生长。优选的,为保证稀释的准确性,应采用移液枪及量筒等工具进行移液操作,减少取样误差。
由于存在不可培养微生物,实际操作中可培养的细菌种类远小于粪便样中的理论菌量,同时为减少步骤3中稀释和取样操作过程中带来的误差,因此除了将粪便样稀释至目的浓度外,还需稀释至较目的浓度小一倍和较目的浓度倍数大一倍的实验组。
进一步的,步骤3中所述的96孔细菌培养板应采用96深孔板进行,深孔板每孔体积应不小于400μl,每孔的加样体积应不超过80%。
进一步的,为减少步骤3培养过程中培养液的蒸发和96孔板间的污染,步骤3中所述的封口膜应优选为塑封膜、石蜡膜、硅胶膜、铝膜中的一种。
进一步的,为满足后续菌液保藏所需的体积,步骤4中所述的96孔中间板的每孔体积应不小于1.6ml。
进一步的,为满足高效快速进行步骤5中所述菌液DNA的提取,使用96孔磁珠法进行DNA提取,或一步裂解法释放菌液DNA,通过离心收集上清后直接进行16S序列的扩增。
进一步的,步骤5的菌液DNA在进行PCR之前需调整至所需浓度(20-100ng/μl),优选的,使用石英酶标板进行批量检测。
与现有技术相比,本发明的有益效果为:
(1)本方法周期更短,仅需10~12天即可完成一批粪便样本细菌的分离筛选;
(2)本方法成本更低,仅需重新制备培养基,96深孔板和封口膜等材料可经灭菌后反复使用;
(3)本方法效率更高,仅需1人即可完成一套分离培养流程的操作。
(4)本方法通过极限稀释,使细菌分离分散到96孔板中,从而解除不同细菌间的生长竞争或生长抑制,使丰度较低或生长劣势的细菌能够生长。
附图说明
图1为本发明实施例1中涉及的肠道菌群高通量分离培养或定向筛选的流程示意图;
图2为本发明实施例1相关图片。图2中,(1)为使用本专利方法在三个月内筛选得到的肠道菌种的汇总结果,其中包含菌株324株,不同菌种82种,分别为:Bifidobacteriumlongum subsp.suillum、Bacteroides uniformis、Bifidobacterium pseudocatenulatum、Bifidobacterium faecale、Bacteroides fragilis、Bacteroides vulgatus、Parabacteroides distasonis、Phocaeicola dorei、Bifidobacterium longum、Enterococcus faecalis、Enterococcus hirae、Shigella flexneri、Alistipesonderdonkii、Bacteroides cellulosilyticus、Escherichia fergusonii、Klebsiellapneumoniae、Mitsuokella jalaludinii、Paraprevotella clara、Bacteroides faecis、Bifidobacterium bifidum、Citrobacter freundii、Streptococcus salivarius、Bacteroides dorei、Clostridium perfringens、Collinsella aerofaciens、Enterococcus durans、Eubacterium callanderi、Phocaeicola vulgatus、Akkermansiamuciniphila、Bacillus paramycoides、Bacillus subtilis、Bacteroides caccae、Bacteroides eggerthii、Bifidobacterium longum subsp.suillum、Fusobacteriumvarium、Hungatella effluvii、Prevotella copri DSM、Ruminococcus faecis、Staphylococcus capitis、Stenotrophomonas maltophilia、Alistipes shahii、Bacillusproteolyticus、Bacteroides intestinalis、Bacteroides rodentium、Bacteroidesstercoris、Barnesiella intestinihominis、Butyricimonas faecihominis、Clostridiumhylemonae、Corynebacterium striatum、Cutibacterium acnes、Megasphaera indica、Nosignificant similarity found、Proteus mirabilis、Ruminococcus gnavus、Shigellaboydii、Streptococcus constellatus、Streptococcus pasteurianus、Alistipesindistinctus、Bacteroides massiliensis、Bacteroides thetaiotaomicron、Bifidobacterium stercoris、Blautia producta、Butyricimonas paravirosa、Clostridium sporogenes、Coprobacilluscateniformis、Drancourtella massiliensis、Enterobacter cloacae、Enterococcus faecium、Escherichia marmotae、Faecalicatenacontorta、Flavonifractor plautii、Gemmigerformicilis、Lactonifactorlongoviformis、Lysinibacillus odysseyi、Odoribacter splanchnicus、Paenibacilluslactis、Pantoea agglomerans、Parabacteroides merdae、Shigella sonnei、Staphylococcu saureus、Staphylococcus caprae、Streptococcus rubneri。部分已报道具备有益作用或具有潜在功能的菌种已使用折线特别标注;(2)为部分筛选得到的菌种的平板表型,分别是海氏肠球菌(Enterococcus hirae)、弗氏柠檬酸杆菌(Citrobacterfreundii)、成团泛菌(Pantoea agglomerans)、单形类杆菌(Bacteroides uniformis)、副干酪乳杆菌(Lactobacillus paracasei)和长双岐杆菌乳香型(Bifidobacterium longumsubsp.suillum)。
图3为本发明实施例2中定向筛选嗜黏蛋白阿克曼菌(Akkermansia muciniphila)所用的供体粪便样的菌属相对丰度图,其中Akkermansia菌属占比0.59%,使用折线特别标注。
图4为筛选获得的Akkermansia muciniphila的16S序列的NCBI比对结果,红框内容既为相似性最高的比对结果。
图5为筛选获得的Akkermansia muciniphila 16S rDNA序列的系统进化树。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实例1
一种人体肠道菌群的快速分离培养方法,分离培养流程如图1所示,其步骤如下:
将供体粪便样按1g:100μl的比例加入改良的生理盐水,震荡混匀,并进行梯度稀释。使用移液枪吸取少量粪便样稀释液进行活菌检测,计算并记录粪便原液中的活菌总量。
准备改良的BHIS液体培养基,配方:BHI预制粉末37g,酵母提取物5g,L-半胱氨酸0.5g,刃天青0.1mg,血晶素2.50μg,维生素K1 0.001mg,吐温80 1ml,加水定容至1L,搅拌均匀,使用HCl或NaOH调节pH至6.8,121℃高压灭菌20min,冷却至55℃备用。
使用上述BHIS液体培养基对粪便样进行精确稀释,并使用排枪吸取1ml菌液移入96深孔板,使96深孔板中每孔生长的菌量不超过1个,且单独每块96孔板生长菌孔的比例不超过20%,96孔板覆膜封口后,放入厌氧培养箱37℃进行分离培养7天。
观察96深孔板中每孔的细菌的生长情况和生长数量,使用移液枪吸取每孔菌液转移至中间培养板,每孔菌液预留600μl用于继续培养(转移后每孔加入1.4ml培养基),剩余400μl菌液转移至DNA批量提取所用的96孔板中,并使用磁珠法DNA提取仪进行DNA批量自动化提取。提取完成后使用排枪吸取2μl DNA滴入石英板中检测DNA浓度,并使用无菌水稀释调整DNA浓度,使其符合后续PCR的使用要求(20-100μl)。
配置PCR反应体系扩增菌种的16S DNA序列,2×Taq Plus Master Mix(诺唯赞,中国)15μl,上游引物27F(5’AGAGTTTGATCCTGGCTCAG 3')0.5μl,下游引物1492R(5’TACGGCTACCTTGTTACGACTT 3')0.5μl,菌种DNA模板2μl,加水补足30μl。放入PCR仪进行反应,95℃预变性3min,28个循环(95℃变性15s,60℃复性15s,72℃延伸30s),72℃彻底延伸5min。
取PCR反应产物2μl进行电泳,检测条带是否符合预期大小。将PCR产物纯化后进行一代测序,将测序结果放入National Center for Biotechnology Information(NCBI)网站的16S ribosomal RNA sequences(Bacteria and Archaea)数据库进行序列比对。将比对所得的新菌种和潜在益生菌进行菌种保藏。
利用本方法在3个月内对13个供体样本进行高通量分离培养,共分离培养得到菌种数量如图2中(1)所示,其中共有菌株324株,不同菌种82种,包含已有相关文献资料报道的具有有益作用的菌种,如:Bifidobacterium longum、Bifidobacteriumpseudocatenulatum、Bifidobacterium bifidum、Akkermansia muciniphila、Alistipesshahii、Parabacteroides distasonis、Bacteroides vulgatus、Odoribactersplanchnicus、Enterococcus hirae、Blautia producta、Faecalicatena contorta和Bacteroides vulgatus等。
对筛选得到的菌种进行划线活化,通过菌落形态再次验证所得菌种的正确性,及是否存在杂菌污染。进行验证的菌种包括:海氏肠球菌(Enterococcus hirae)、弗氏柠檬酸杆菌(Citrobacter freundii)、成团泛菌(Pantoea agglomerans)、单形类杆菌(Bacteroides uniformis)、副干酪乳杆菌(Lactobacillus paracasei)和长双岐杆菌乳香型(Bifidobacterium longum subsp.suillum),其平板表型结果如图2中(2)所示,平板上菌落形态与预期相符,且菌落形态单一,未见杂菌污染。
实例2
一种影响PD-1抗体治疗肿瘤效果的肠道Akkermansia muciniphila菌株的高通量筛选方法,步骤如下:
对供体粪便样进行16S扩增子测序,将含有Akkermansia muciniphila菌种且相对丰度大于0.5%的供体粪便样用于高通量定向筛选。本实施例中供体粪便样的菌属相对丰度如图3所示,供体粪便样中共包含43个不同菌属,其中Akkermansia属相对丰度占比0.591%。
将供体粪便样中出现的Akkermansia muciniphila菌种的16S序列导入KOMODO(Known Media Database)网站(komodo.modelseed.org/),根据其16S rDNA的序列来预测培养该菌的培养基配方。或直接通过BacDive网站(bacdive.dsmz.de/)检索Akkermansiamuciniphila菌种的定向筛选培养基。
根据检索到的配方配置Akkermansia muciniphila菌株的定向筛选液体培养基,配方如下:胰化酪蛋白5g、蛋白胨5g、酵母抽提物10g、牛肉提取物5.0g、葡萄糖5.0g、K2HPO42.0g、Tween 80 1.0ml、盐溶液40.0ml、刃天青1.0mg、维生素K1溶液0.2ml、血红素溶液10.0ml、一水合L-半胱氨酸盐酸0.5g、粘蛋白1.0g、去离子水定容至1L。调整pH至6.8,115℃高压灭菌25min,冷却至55℃备用。
其中盐溶液成分如下:CaCl2·2H2O 0.25g、MgSO4·7H2O 0.50g、K2HPO4 1.0g、KH2PO4 1.0g、NaHCO3 10.0g、NaCl 2.0g、去离子水定容至1L。0.22μm滤膜过滤除菌,4℃低温冷藏。
血红素溶液成分如下:血红素50mg、1M NaOH 1ml、去离子水定容至100ml。0.22μm滤膜过滤除菌,4℃低温冷藏。
维生素K1溶液:维生素K1 0.1ml、95%乙醇20.0ml。0.22μm滤膜过滤除菌,4℃低温冷藏。
如实施1所示,进行供体粪便样的预处理,并通过活菌检测技术测定并计算粪便样中的活菌总量。使用定向筛选培养基对粪便样精确稀释至目的浓度,使每个96深孔板生长菌量不超过15%。
将稀释后的菌液转移至96深孔板中,覆盖不透气膜封口密闭,放入厌氧培养箱,37℃培养3-7天。观察96孔板中菌液的浑浊情况,挑选出现菌液生长的菌孔,使用磁珠法批量提取仪进行细菌基因组DNA的提取,PCR扩增16S基因序列并测序。
经鉴定发现一株Akkermansia muciniphila,命名为Akkermansia muciniphilaTG2022,结果如图4所示,该菌株与Akkermansia muciniphila ATCC BAA-835Muc的序列相似度最高,为99.5%,与Akkermansia glycaniphila Pyt的序列相似度为93.49%,与其余非Akkermansia属的菌种序列相似度低于87%。其16S基因的系统发育树如图5所示。
以上结果表明,本发明可利用定向筛选培养基进行特定菌种的高效定向筛选。
以上仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种肠道菌群的高通量分离培养与筛选方法,其特征在于该方法包括以下步骤:
步骤1,粪便样筛选与预处理:对获取的粪便样本进行16S扩增子测序,分析粪便样本的菌群丰富度;
将供体粪便样按1 g:100 μl的比例加入改良的生理盐水,震荡混匀,并进行梯度稀释;所述改良的生理盐水为生理盐水中添加半胱氨酸盐酸盐、维生素C、维生素E、茶多酚中的一种或任意组合;
步骤2,活菌检测:利用活菌检测技术测定步骤1中的粪便样稀释液中活菌总量;吸取粪便样稀释液进行活菌检测,计算并记录粪便原液中的活菌总量;
步骤3,极限稀释培养:根据活菌总量,计算稀释倍数,并使用改良培养基对步骤1所得的粪便样进行梯度稀释,将稀释后的混合细菌培养基转移到96孔细菌培养板中,并使用封口膜密封后,放入37℃厌氧培养箱进行培养;
所述的改良培养基配方为模拟肠道环境的培养基或定向筛选培养基,模拟肠道环境培养基为改良的BHIS液体培养基;定向筛选培养基包括:以黏蛋白为单一碳源的嗜黏蛋白阿克曼菌筛选培养基、促进乳杆菌生长的M17肉汤或乳酸杆菌选择性肉汤、促进双歧杆菌生长的BBL或TPY培养基;
所述的改良的BHIS液体培养基,配方:BHI预制粉末37 g,酵母提取物5 g,L-半胱氨酸0.5 g,刃天青0.1 mg,血晶素2.50 μg,维生素K1 0.001 mg,吐温80 1ml,加水定容至1 L,搅拌均匀,使用HCl或NaOH调节pH至6.8,121℃高压灭菌20 min,冷却至55℃备用;
步骤4,转移培养:挑选生长孔数不超过40%的96孔细菌培养板,全部转移至新的96孔中间板,再取出部分菌液用于菌液DNA的提取,再向96孔中间板中补充YCFA、BHIS或MRS富营养培养基或定向筛选培养基,继续培养剩余菌液;
步骤5,菌种鉴定:提取上一步骤中的取出的菌液DNA,PCR扩增16S基因序列,电泳检测扩增条带并送样测序;检查测序的峰图质量,并进行序列比对;
步骤6,菌种保藏:比对所得菌种鉴定结果,筛选需要保藏的菌株编号,按照对应编号保藏步骤4中所述的96孔中间板所培养的菌液。
2.根据权利要求1所述的肠道菌群的高通量分离培养与筛选方法,其特征在于步骤3中所述的封口膜为塑封膜、石蜡膜、硅胶膜、铝膜中的任意一种。
3.根据权利要求1所述的肠道菌群的高通量分离培养与筛选方法,其特征在于步骤4中所述的96孔中间板的每孔体积应不小于1.6 ml。
4.根据权利要求1所述的肠道菌群的高通量分离培养与筛选方法,其特征在于步骤5中,所述菌液DNA的提取,使用96孔磁珠法进行DNA提取,或一步裂解法释放菌液DNA,通过离心收集上清后直接进行16S 序列的扩增。
5.根据权利要求4所述的肠道菌群的高通量分离培养与筛选方法,其特征在于步骤5中,步骤5的菌液DNA在进行PCR之前需调整至20-100 ng/μl,同时,使用石英酶标板进行批量检测。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105385682A (zh) * | 2015-12-29 | 2016-03-09 | 杭州谷坤生物技术有限公司 | 快速提取人粪便细菌dna的简易方法 |
CN105886382A (zh) * | 2016-04-27 | 2016-08-24 | 烟台民望机电技术有限公司 | 一种肠道内容物菌群提取装置 |
CN107058564A (zh) * | 2017-05-18 | 2017-08-18 | 济南市儿童医院(山东大学齐鲁儿童医院) | 儿童自闭症肠道菌群多样性的分析方法及特异性引物对 |
CN110093276A (zh) * | 2019-06-03 | 2019-08-06 | 新乡医学院 | 一种定向选择性分离肠道细菌的方法 |
CN110097928A (zh) * | 2019-04-17 | 2019-08-06 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种基于肠道菌群预测组织微量元素含量的预测方法和预测模型 |
CN112666287A (zh) * | 2020-12-22 | 2021-04-16 | 上海鹿明生物科技有限公司 | 基于gcmsms高通量分析动物肠道菌群代谢物的方法及应用 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2915884B1 (en) * | 2014-03-03 | 2017-12-06 | Fundacio Institut d'Investigació Biomèdica de Girona Dr. Josep Trueta | Method for diagnosing colorectal cancer from a human feces sample by quantitive PCR |
US10982290B2 (en) * | 2014-07-25 | 2021-04-20 | Global Life Sciences Solutions Operations UK Ltd | Screening and monitoring the progression of type 2 diabetes by the molecular identification of human gut flora using FTA as a faecal collection device |
-
2022
- 2022-08-01 CN CN202210917227.7A patent/CN115261271B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105385682A (zh) * | 2015-12-29 | 2016-03-09 | 杭州谷坤生物技术有限公司 | 快速提取人粪便细菌dna的简易方法 |
CN105886382A (zh) * | 2016-04-27 | 2016-08-24 | 烟台民望机电技术有限公司 | 一种肠道内容物菌群提取装置 |
CN107058564A (zh) * | 2017-05-18 | 2017-08-18 | 济南市儿童医院(山东大学齐鲁儿童医院) | 儿童自闭症肠道菌群多样性的分析方法及特异性引物对 |
CN110097928A (zh) * | 2019-04-17 | 2019-08-06 | 广东省微生物研究所(广东省微生物分析检测中心) | 一种基于肠道菌群预测组织微量元素含量的预测方法和预测模型 |
CN110093276A (zh) * | 2019-06-03 | 2019-08-06 | 新乡医学院 | 一种定向选择性分离肠道细菌的方法 |
CN112666287A (zh) * | 2020-12-22 | 2021-04-16 | 上海鹿明生物科技有限公司 | 基于gcmsms高通量分析动物肠道菌群代谢物的方法及应用 |
Non-Patent Citations (2)
Title |
---|
人肠道微生物中抗菌活性菌株的筛选及其代谢产物研究;李德利;丁鹏敏;刘双月;杨秀伟;王如峰;;中国现代中药;22(01);34-42 * |
精神分裂症患者肠道菌群多态性与代谢产物的关系;余卓恒等;《国际检验医学杂志》;第43卷(第10期);1238-1243 * |
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