CN116837124A - 一种芽孢杆菌属微生物的快速分析方法 - Google Patents
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Abstract
本发明提供了一种芽孢杆菌属微生物的快速分析方法,属于生物技术领域,通过检索500条芽孢杆菌属的16S rRNA基因序列,并进行序列比对,找到分别位于基因序列上游和下游的两段高度保守基因区,以所述基因区为模板设计特异性引物,利用特异性引物扩增样品中的DNA,然后对扩增产物进行高通量测序,能够实现样品中的芽孢杆菌属微生物在种属水平上的快速定量分析,相对于传统的16S rRNA分类测序操作简便快速、灵敏度高、特异性强,也为其它种属的快速分析提供了新思路。
Description
技术领域
本发明涉及生物技术领域,具体是一种芽孢杆菌属微生物的快速分析方法。
背景技术
白酒作为世界三大蒸馏酒之一,在我国已经存在着千年的历史,因其独特的酿造工艺,被誉为中华民族的瑰宝。白酒的酿造离不开微生物的参与,不同的微生物是形成白酒香型的一个重要因素;中国白酒可以分为清香型、浓香型、酱香型、米香型、芝麻香型、凤香型等香型,这些香型的产生都是由于不同微生物的不同代谢产物而产生。
白酒发酵是由多种微生物共同参与一个复杂过程。在发酵过程中,酒曲是酒香的灵魂,白酒首先将原料粉碎,再稍微浸泡一下,最后压制成砖状,在发酵过程中会滋生多种微生物,包括菌株、丝状真菌、细菌和放线菌等多种微生物;白酒发酵过程窖泥微生物也起了决定性作用,微生物以空间位置相对固定的窖泥作为栖息地且经过酿酒环境长时间的驯化,已经形成了一个较为稳定的微生物群落,并形成了一批具有产酒、产香功能的微生物,合理开发利用这些微生物,对白酒品质的提升有重大意义。
芽孢杆菌属微生物包含枯草芽孢杆菌、地衣芽孢杆菌等,芽孢杆菌属在蛋白酶、淀粉酶以及纤维素酶生产方面都具有一定的优势,从固态白酒发酵特点看,这些酶类的生成和富集可以有效降解原料中的蛋白、淀粉以及纤维素类物质,从而可以提高原料利用率,为微生物发酵提供必要的营养元素以及为一些风味物质提供必要的前体。
芽孢杆菌属在培养后均可以直接形成非常复杂的风味,虽然最终风味各有特色,但在一些特征产物诸如3-甲基丁醇、2,3-二丁醇、3-羟基-2-丁酮以及吡嗪类等都有非常相似的代谢产物组分,在芽孢杆菌风味代谢产物机理研究过程中,无论是从酱香型、浓香型还是清香型大曲中所分离的芽孢杆菌,都有一个相同的代谢产物3-羟基-2-丁醇,该产物是芽孢杆菌直接生物代谢所得,是重要的呈香物质;芽孢杆菌除了通过自身发酵所得到的直接风味物质外,还可以产生一些风味合成前体物质,通过一些化学或者生物作用来进一步合成复杂的风味物质。
目前获知样品中的芽孢杆菌属微生物方法主要有两种,一种是利用芽孢杆菌筛选培养基,通过平板涂布的方法进行分析,但该方法耗时长,同时无法对芽孢杆菌进行定量分析;另外,还可以通过高通量测序的方法对样品中所有的微生物群体进行分析,但由于通用引物测序结果不够精确,只能检测到菌属水平,且该方案成本较高,耗时长,也无法对芽孢杆菌进行有效的定量分析。
发明内容
针对现有技术的不足,本发明提供了一种芽孢杆菌属微生物的快速分析方法,通过使用特异性引物扩增样品并进行高通量测序,可以实现样品中芽孢杆菌属微生物在种属水平上的快速定量分析,也为其它种属的快速分析奠定了新思路。
本发明技术方案如下:
一种芽孢杆菌属微生物的高度保守基因区,所述基因区分为上游基因区和下游基因区,所述上游基因区的序列如SEQ ID NO.1所示,所述下游基因区的序列如SEQ ID NO.2所示。
一种用于分析芽孢杆菌属微生物的特异性引物,所述特异性引物的正向序列如SEQ ID NO.3所示,反向序列如SEQ ID NO.4所示。
所述特异性引物在分析芽孢杆菌属微生物中的应用。
所述特异性引物在芽孢杆菌属微生物分析中的应用,应用方法是利用特异性引物扩增样品中的DNA,对扩增产物进行高通量测序,快速分析样品中的芽孢杆菌属微生物。
优选的,所述样品为酿酒过程中的大曲、酒醅或窖泥中的一种。
优选的,应用方法具体包括如下步骤:
(1)提取样品中的微生物菌群总DNA,使用特异性引物对总DNA进行PCR扩增,得PCR扩增产物;
(2)回收步骤(1)中的PCR扩增产物,进行高通量测序。
优选的,所述步骤(1)中的PCR扩增程序为:94℃预变性5min;94℃变性1min,55℃退火1min,72℃延伸150s,20个循环;72℃延伸10min。
优选的,所述步骤(2)中PCR扩增产物的回收方法为,首先采用琼脂糖凝胶电泳确认PCR扩增产物的条带大小是否正确,然后将正确的PCR扩增产物利用琼脂糖凝胶DNA提取试剂盒进行回收。
本发明从500条芽孢杆菌属的16S rRNA基因序列中首次筛选出一组在芽孢杆菌属中高度保守的基因区,分别位于芽孢杆菌属16S rRNA基因序列的上游和下游,中间序列为不保守序列;我们在上游基因区和下游基因区各设计一段特异性引物,分别作为正向引物和反向引物,利用所述特异性引物进行样品DNA的PCR扩增,由于样品中庞大的微生物数量,因此在PCR扩增产物中会出现若干种不同的基因序列,其中包括不同种的芽孢杆菌属微生物以及其它微生物种群。
有益效果:
本发明提供了一种芽孢杆菌属微生物的高度保守基因区和特异性引物,并提供了一种芽孢杆菌属微生物的快速分析方法,能够实现样品中的芽孢杆菌属微生物在种属水平上的快速定量分析,且操作简便快速、灵敏度高、特异性强,同时也为其它种属的快速分析鉴定提供了新思路。
附图说明
图1为实施例1中大曲PCR扩增产物的琼脂糖凝胶电泳图;
图2为实施例2中窖泥PCR扩增产物的琼脂糖凝胶电泳图。
具体实施方式
下面结合具体实施例进行说明:
首先,我们通过NCBI GeneBank网站检索了500条芽孢杆菌属的16S rRNA基因,并通过Snapgene软件进行序列比对,找到一组在芽孢杆菌属中高度保守的基因区,其中上游基因区的序列如SEQ ID NO.1所示,下游基因区的序列如SEQ ID NO.2所示;通过Snapgene软件分别在上游基因区和下游基因区设计特异性引物,其中正向引物序列如SEQ ID NO.3所示,反向引物序列如SEQ ID NO.4所示。
以下实施例均采用上述特异性引物进行样品DNA的PCR扩增,使用该特异性引物扩增出的产物大小为626bp。
实施例1:
大曲样品中芽孢杆菌属的快速分析
(1)在酿酒过程中获取大曲样品,使用Sangon Biotech公司Ezup柱式土壤DNA抽提试剂盒对大曲中的总DNA进行提取;
(2)将步骤(1)中提取的总DNA作为模板,使用上述特异性引物进行PCR扩增反应;PCR扩增体系为:模板DNA 2μL,正向引物、反向引物各2μL,灭菌双蒸水19μL,诺唯赞公司Phanta酶25μL;PCR扩增程序为:94℃预变性5min;94℃变性1min,55℃退火1min,72℃延伸150s,20个循环;72℃延伸10min;得PCR扩增产物;
(3)将步骤(2)中的PCR扩增产物进行1%琼脂糖凝胶电泳检测,控制电压为180V,电流为80mA,电泳结束后紫外下拍照并观察是否有条带,结果如图1所示,PCR扩增产物大小为626bp,符合预期条带大小;
(4)利用诺唯赞公司的Gel Extraction Kit试剂盒回收步骤(3)中的PCR扩增产物,将回收后的产物进行高通量测序,检测大曲样品中的各芽孢杆菌属微生物含量;其中,测序工作委托上海生工生物工程公司进行,并由该公司出具测序报告。
测序完成后,检测结果如下表1所示:
表1.利用特异性引物对大曲样品中芽孢杆菌属微生物的检测结果
菌种名称 | 含量(%) |
Bacillus velezensis | 82.07 |
Bacillus paramycoides | 2.89 |
Bacillus licheniformis | 1.6 |
Bacillus sp | 0.25 |
Bacillus coagulans | 0.086 |
Bacillus ginsenggisoli | 0.022 |
由表1可得,通过高通量测序我们发现了在上述大曲样品中芽孢杆菌属种群占测序样本中所有微生物种群的86.918%,具体的,其中样品中芽孢杆菌属微生物包括6种,分别为Bacillus velezensis、Bacillus paramycoides、Bacillus licheniformis、Bacillussp、Bacillus coagulans、Bacillus ginsenggisoli,其中Bacillus velezensis占主导优势;所述6种芽孢杆菌在门水平分类为Firmicutes,在属水平分类均为Bacillus。
对比例1:
与实施例1不同的是,本对比例1采用传统的16S rRNA分类测序方法检测大曲样品中的芽孢杆菌属含量,具体操作步骤如下:
将步骤(1)中提取的大曲样品总DNA使用16S rRNA通用引物进行PCR扩增反应,将PCR扩增产物回收后进行高通量测序;所述通用引物序列如下:
338F:5'-ACTCCTACGGGAGGCAGCAG-3';
806R:5'-GGACTACHVGGGTWTCTAAT-3'。
检测结果如下表2所示:
表2.利用通用引物对大曲样品中芽孢杆菌属微生物的检测结果
由表2可得,通过传统的16S rRNA分类测序可以显示更多的门和属类,我们检测到了占测序样本中64.58%的芽孢杆菌属种群;但由于通用引物对具体微生物的检测不够精确,因此无法分析芽孢杆菌属中的具体微生物种属水平。
实施例2:
窖泥样品中芽孢杆菌属的快速分析
在酿酒过程中的窖池内获取窖泥样品,使用Sangon Biotech公司Ezup柱式土壤DNA抽提试剂盒对窖泥样品中的总DNA进行提取;将提取后的总DNA采用特异性引物进行PCR扩增,回收PCR扩增产物进行高通量测序,详细步骤见实施例1。
检测结果如下表3所示:
表3.利用特异性引物对窖泥样品中芽孢杆菌属微生物的检测结果
菌种名称 | 含量(%) |
Bacillus velezensis | 83.09 |
Bacillus paramycoides | 1.54 |
Bacillus licheniformis | 2.18 |
Bacillus coagulans | 0.13 |
Bacillus ginsenggisoli | 0.077 |
Bacillus sp. | 0.042 |
由表3可得,在上述窖泥样品中芽孢杆菌属种群占测序样本中所有微生物种群的87.059%;具体的,其中样品中芽孢杆菌属微生物包括6种,分别为Bacillus velezensis、Bacillus paramycoides、Bacillus licheniformis、Bacillus coagulans、Bacillusginsenggisoli和Bacillus sp.,其中Bacillus velezensis占主导优势;所述6种芽孢杆菌在门水平分类为Firmicutes,在属水平分类均为Bacillus。
对比例2:
与实施例2不同的是,本对比例2采用传统方法检测窖泥样品中的芽孢杆菌属含量,具体操作步骤见对比例1。
通过传统16S rRNA分类测序,我们检测到了占测序样本中67.3%的芽孢杆菌属种群,但无法分析芽孢杆菌属中的具体微生物种属水平。
综上,本发明能够实现样品中的芽孢杆菌属微生物在种属水平上的快速定量分析,且操作简便快速、灵敏度高、特异性强,利用该特异性引物还能够在短时间内低成本扩增出所需要的菌种,同时也为其它种属的快速分析鉴定提供了新思路。
Claims (8)
1.一种芽孢杆菌属微生物的高度保守基因区,其特征在于,所述基因区分为上游基因区和下游基因区,所述上游基因区的序列如SEQ ID NO.1所示,所述下游基因区的序列如SEQ ID NO.2所示。
2.一种用于分析芽孢杆菌属微生物的特异性引物,其特征在于,所述特异性引物的正向序列如SEQ ID NO.3所示,反向序列如SEQ ID NO.4所示。
3.权利要求2所述特异性引物在分析芽孢杆菌属微生物中的应用。
4.如权利要求3所述的应用,其特征在于,应用方法是利用特异性引物扩增样品中的DNA,对扩增产物进行高通量测序,快速分析样品中的芽孢杆菌属微生物。
5.如权利要求4所述的应用,其特征在于,所述样品为酿酒过程中的大曲、酒醅或窖泥中的一种。
6.如权利要求4所述的应用,其特征在于,应用方法具体包括如下步骤:
(1)提取样品中的微生物菌群总DNA,使用特异性引物对总DNA进行PCR扩增,得PCR扩增产物;
(2)回收步骤(1)中的PCR扩增产物,进行高通量测序。
7.如权利要求6所述的应用,其特征在于,所述步骤(1)中的PCR扩增程序为:94℃预变性5min;94℃变性1min,55℃退火1min,72℃延伸150s,20个循环;72℃延伸10min。
8.如权利要求6所述的应用,其特征在于,所述步骤(2)中PCR扩增产物的回收方法为,利用琼脂糖凝胶DNA提取试剂盒进行回收。
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