CN114686556A - 一种胃内菌群的检测方法 - Google Patents
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Abstract
本发明涉及一种胃内菌群的检测方法，其包括如下步骤：（1）对胃黏膜组织样本进行处理；（2）对处理后的样本中的微生物进行培养；（3）挑取单菌落进行菌株纯化；（4）菌株鉴定。本发明首次提出对胃内多种病原体进行培养鉴定，以达到快速确定胃内菌群构成，动态观察菌群构成变化，并在确定感染病原体型别后进行的药敏实验提供条件。本发明还涉及利用本方法鉴定出的22个菌种的标准菌谱进行分子生物快速检测的方法。
Description
技术领域
本发明属于微生物培养及检测技术领域，具体涉及胃内菌群的检测方法。
背景技术
据第五届口腔微生物规范化研讨会报道，截至目前口腔内微生物已检测到38个菌属的178种菌；人类肠道微生物包含1000多种细菌物种。而上个世纪人们认为胃内的强酸性环境不会有微生物存在。
自1983年Warren和Marshall从胃幽门溃疡患者的胃黏膜活检组织中，发现一种螺旋形细菌后，消化界逐渐对胃内存在幽门螺杆菌和其致病性达成了共识。但目前对胃内细菌的认知、检测和治疗还只局限在幽门螺杆菌，采用C13和C14呼气试验作为幽门螺杆菌感染的诊断方法，利用幽门螺杆菌尿素酶分解尿素产生氨和二氧化碳，同位素标记的二氧化碳可以检测，也是建立在胃内只有幽门螺杆菌这唯一一种含有尿素酶的细菌而构建。近些年国内外学者对胃内幽门螺杆菌之外的细菌和真菌感染也有个例报道，如果临床上只是依据呼气试验阳性就判定为幽门螺杆菌感染，采取现有的一线疗法即四联疗法盲目对受试者进行幽门螺杆菌根除治疗，势必造成抗生素治疗效果不佳和诱导感染菌的耐药菌型。
发明内容
本发明的目的是提供一种可全面、高效的对胃内菌群进行检测的方法。
本发明采用如下技术方案：
一种胃内菌群的检测方法，其包括如下步骤：
（1）采集胃黏膜组织样本；
（2）对胃黏膜组织样本进行处理；
（3）对处理后的样本中的微生物进行培养；
（4）挑取单菌落进行菌株纯化；
（5）菌株鉴定。
步骤（1）中，可选用内镜或磁控胶囊对胃黏膜组织进行样本采集，得到的样本放入盛有BRAIN HEART INFUSION 30%甘油保存液EP管中，2h内进行培养，否则在-20℃至-80℃冷冻保存。
步骤（2）中，使用一次性无菌吸管由保存液EP管中取出胃黏膜组织样本放入灭菌后的研磨器中，加入0.4ml无菌生理盐水进行研磨，得到研磨液。
步骤（3）中，将处理后的胃黏膜组织样本研磨液分别涂布到培养基I~IV的表面，四种培养基均放入三气（10%二氧化碳、5%氧气、85%氮气）培养箱培养48小时。
其中，所述培养基I为含10%无菌脱纤维羊血CAMPYLOBACTER AGAR BASE；培养基II为含10%无菌脱纤维羊血的COLUMBIA BLOOD AGAR BASE；培养基III为含10%无菌脱纤维羊血和2%抗生素混合液的CAMPYLOBACTER AGAR BASE；培养基IV为含10%无菌脱纤维羊血和2%抗生素混合液的COLUMBIA BLOOD AGAR BASE。
其中，所述抗生素混合液由万古霉素（10mg/L） 0.5ml、多粘菌素B（5mg/L）0.25ml、两性霉素B（5mg/L）0.25ml 、TMP（10mg/L）1.5ml和97.5ml无菌纯水制成抗生素混合液。
步骤（4）中，分别从经步骤（3）培养后的培养基I~IV的表面挑取单菌落涂于培养基I或培养基II，三气培养箱48小时培养。
步骤（5）中，由经步骤（4）培养基I或培养基II中得到的纯培养菌种进行菌种鉴定。
一种利用上述检测方法识别的胃内菌群在分子生物快速检测中的应用。
进一步的，将幽门螺杆菌、格雷文氏放线菌、食酸代尔福特菌、光滑念珠菌、热带念珠菌、白色念珠菌、睾丸酮丛毛单胞菌、毗邻颗粒链菌、嗜麦芽窄食单胞菌、食醇鞘氨醇杆菌、少动鞘氨醇单胞菌、类少动鞘氨醇单胞菌、植生拉乌尔菌、土生拉乌尔菌、皮氏罗尔斯顿菌、阿萨希丝孢酵母、摩西假单胞菌、恶臭假单胞菌 、人葡萄球菌、稍变冢村氏菌、schaaliaodontodytica以及mycobacteium abscessus strain构建比对库试剂盒，利用多重核酸探针、免疫发光试剂盒、蛋白芯片或基因芯片等方法进行快速检测。
本发明的有益效果在于：本发明首次提出对胃内多种病原体进行培养鉴定的方法，以达到可以快速确定胃内菌群构成，动态观察菌群构成变化，并在确定感染微生物型别后进行的药敏实验提供条件。本发明还提出利用22种病原体应用分子生物学技术进行快速检测，使胃内病原体的检测目标由目前的一种幽门螺杆菌拓展到22种菌，通过培养组学、药敏和分子生物学快速鉴定，指导临床精准对受试者进行诊疗，引发医务和科技工作者研发出比呼气试验更能精准检测胃内菌群的诊疗工具，对大众预防疾病发生、增强体质健康有重大意义。
附图说明
图1为可取组织的磁控胶囊结构示意图。
其中，1为磁控胶囊、2为引导软管、3为一次性使用活体取样钳。
图2为样本52在培养基I和培养基II中的培养结果。
图3为样本52在培养基III和培养基IV中的培养结果。
图4为样本94在培养基I和培养基III中的培养结果。
图5为样本54在培养基III和培养基IV中的培养结果。
图6为样本68在培养基I和培养基II中的培养结果。
图7为样本68在培养基III和培养基IV中的培养结果。
图8为胃内菌群的菌属构成比例图。
具体实施方式
下面结合附图及实例进一步详细描述本发明的技术方案，但本发明的保护范围不局限于以下所述。
组织来源
应用普通内镜、无痛内镜及可取组织的磁控胶囊采集的临床受试者的胃黏膜组织，组织采集后放入BRAIN HEART INFUSION （品牌OXOID，货号CM1135）30%甘油保存液EP管中，2h内尽快送实验室进行培养，超过2h建议-20至-80℃冷冻保存。
如图1所示，所述可取组织的磁控胶囊包括磁控胶囊1以及固定设置于磁控胶囊1外壁的引导软管2，所述引导软管可采用可降解医用纤维制成，或者是使用可被胃酸溶解的医用胶固定在磁控胶囊上。受试者吞下磁控胶囊，引导软管随磁控胶囊进入胃内，通过软管使用一次性使用活体取样钳3夹取胃黏膜组织，取出取样钳，得到样本，剪断口腔内软管，磁控胶囊与剩余软管随粪便排出。
2. 培养与鉴定
试验方法
2.1.1组织处理和培养
使用一次性无菌吸管由保存液中取出胃黏膜组织放入灭菌研磨器中，加入0.4毫升无菌生理盐水充分研磨得到组织研磨液，吸取研磨液同时涂布于2.1.2.1和2.1.2.2四种培养皿表面，放入三气培养箱培养48小时。
2.1.2.1和2.1.2.2为四种不同成分的培养基，目的为同时应用不同成分培养基适合多种不同型别的细菌生长，尽可能全面的检出胃内菌群。
培养基制备
2.1.2.1 培养基I：CAMPYLOBACTER AGAR BASE （Karmali），货号CM0935，品牌OXOID + 无菌脱纤维羊血（10%）。培养基II：COLUMBIA BLOOD AGAR BASE，货号CM0331，品牌OXOID + 无菌脱纤维羊血（10%）。
2.1.2.2 培养基III：CAMPYLOBACTER AGAR BASE （Karmali），货号CM0935，品牌OXOID + 无菌脱纤维羊血（10%）+ 抗生素混合液（2%）。培养基IV：COLUMBIA BLOOD AGARBASE，货号CM0331，品牌OXOID + 无菌脱纤维羊血（10%）+ 抗生素混合液（2%）。
2.1.2.3抗生素混合液的制备：由万古霉素（10mg/L） 0.5ml、多粘菌素B（5mg/L）0.25ml、两性霉素B(5mg/L)0.25ml 、TMP（10mg/L）1.5ml和97.5ml无菌纯水制成抗生素混合液。
如图2所示，在相同的培养条件下，相同样本（52#）在培养基I（左皿）和培养基II（右皿）上得到培养结果不同。
图2和图3对比可见，在相同的培养条件下，相同样本（52#）、相同培养基（COLUMBIABLOOD AGAR BASE+ 无菌脱纤维羊血），在不加抗生素混合液培养基II（图2右皿）和加入抗生素混合液培养基IV（图3左皿）得到的培养结果不同。
如图4所示，在相同培养条件下，相同样本（94#）在培养基I（右皿）和培养基III（左皿）上得到的培养结果不同。显示相同培养条件下，同一样本，相同培养基（CAMPYLOBACTERAGAR BASE+ 无菌脱纤维羊血）加入抗生素混合液和不加抗生素混合液生长菌株不同。
如图5所示，在相同培养条件下，相同样本（54#）在培养基III（右皿）和培养基IV（左皿）上得到的培养结果不同。显示，相同培养条件下，同一样本在两种都加入抗生素混合液的不同培养基中生长菌株不同。
上述培养结果表明，同一组织在培养基I、培养基II、培养基III和培养基IV上会得到不同的培养结果，因此，本发明在对胃内菌群进行检测时，同时应用四种培养基分别对同一组织样本进行培养，以达到更接近检出胃内全部菌群的目的。
菌株纯化
由48小时培养后的培养基1~IV四种不同培养皿中挑取单个菌落涂于培养基I或培养基II，三气培养箱48小时培养。
菌株鉴定
（i）全自动细菌鉴定药敏分析系统V2操作规程
i-1）鉴定卡及浓度
GN 革兰阴性杆菌鉴定卡0.5-0.63麦氏浓度。
GP 革兰阳性鉴定卡0.5-0.63 麦氏浓度。
YST 酵母菌鉴定卡1.8-2.2 麦氏浓度。
NH 奈瑟菌、嗜血杆菌和其它苛养菌2.7-3.3 麦氏浓度。
i-2）药敏卡及浓度
AST-GNxx/Nxxx 革兰阴性杆菌药敏卡，3.0ml 盐水+145 ul 0.5-0.63 麦氏单位菌悬液。
AST-GPxx/Pxxx革兰阳性球菌药敏卡，3.0ml盐水+280 ul 0.5-0.63麦氏单位菌悬液。
i-3）菌悬液配制及稀释0.45% NaCl液，pH 4.5-7.2
将2.1.3纯化好的菌株依据革兰染色镜检选定鉴定卡，依据操作指南进行操作，依据鉴定结果选定药敏卡进行药敏实验。
（ii）微生物质谱鉴定系统（IVD MALDI Biotyper）
ii-1）样本制备：取待测样本单菌落或2.1.4纯化好的菌株均匀涂抹在MALDI靶板的样本孔位中，在记录表中记录样本位置和编号。
ii-2）标准品：添加1ul标准品溶液至1个样本孔位中，自然晾干。
ii-3）在干燥后的标准品或样本上加1ul基质溶液（如果样本为难破壁的菌，需先加1ul 70%甲酸，晾干后再加基质溶液），自然晾干后按照IVD MALDI Biotyper 标准操作程序进行菌种鉴定。
鉴定结果
一、利用2.1.4的鉴定方法，对图2~图5所示培养基培养的菌群进行鉴定，结果如表1所示。
表1
由表1可见，在相同的培养条件下，同一样本（52#）在培养基I（图2左皿）和培养基II（图2右皿）生长菌种不同；在相同培养条件下，相同样本（52#）在培养基II（图2右皿）和培养基IV（图3左皿）生长菌种不同；在相同培养条件下，同一样本（94#）在培养基III（图4左皿）和培养基I（图4右皿）生长菌株不同；在相同培养条件下，同一样本（54#）在培养基III（右皿）和培养基IV（左皿）生长菌株不同。
二、不同培养基的培养结果的比较。
在相同的培养条件下，同一样本（68#）在培养基I~II上培养得到的菌种与在培养基I~IV上培养得到的菌种的对比见表2。
表2
由表2可知，同一样本在相同培养条件下，通过四种培养基的共同培养获得了较培养基I~II（均不含抗生素）更多的菌种。
三、胃内菌群的检测结果
发明人从100份胃黏膜组织样本中检出了幽门螺杆菌、23个菌属的84个菌种和其它9种杆菌及schaalia odontodytica、mycobacteium abscessus strain共计96株菌，菌属构成比例如图8所示。
经过对上述96个菌群进行耐酸性（尿素酶实验）、定植能力（鞭毛、粘附力）及致病性的筛选，提出对胃组织样本中检出的22种菌（幽门螺杆菌、格雷文氏放线菌、食酸代尔福特菌、光滑念珠菌、热带念珠菌、白色念珠菌、睾丸酮丛毛单胞菌、毗邻颗粒链菌、嗜麦芽窄食单胞菌、食醇鞘氨醇杆菌、少动鞘氨醇单胞菌、类少动鞘氨醇单胞菌、植生拉乌尔菌、土生拉乌尔菌、皮氏罗尔斯顿菌、阿萨希丝孢酵母、摩西假单胞菌、恶臭假单胞菌 、人葡萄球菌、稍变冢村氏菌、schaalia odontodytica和mycobacteium abscessus strain）进行药敏实验，对比临床现行的只依据呼气实验阳性盲目应用四联疗法根除幽门螺杆菌的临床一线疗法，极大拓展了胃内微生物型别的检测种类和抗生素应用依据，精准应用抗生素对受试者进行感染菌的根除治疗。
快速分子生物学鉴定
对胃黏膜组织进行快速分子生物学检测，可使受试者快速得到检测结果，并根据检测结果动态观察胃内菌群变化，提示早期根除治疗潜在危害的病原微生物。本发明提出了胃内22种菌的快速检测方法。
22种菌包括：幽门螺杆菌、格雷文氏放线菌、食酸代尔福特菌、光滑念珠菌、热带念珠菌、白色念珠菌、睾丸酮丛毛单胞菌、毗邻颗粒链菌、嗜麦芽窄食单胞菌、食醇鞘氨醇杆菌、少动鞘氨醇单胞菌、类少动鞘氨醇单胞菌、植生拉乌尔菌、土生拉乌尔菌、皮氏罗尔斯顿菌、阿萨希丝孢酵母、摩西假单胞菌、恶臭假单胞菌 、人葡萄球菌、稍变冢村氏菌、schaaliaodontodytica和mycobacteium abscessus strain。利用上述22种标准菌组成菌谱，利用多重核酸快速检测、蛋白芯片以及基因芯片探针对胃内组织样品进行检测。
3.1 胃内22种病原体多重核酸快速检测
利用核酸探针以及PCR技术对本发明提出的胃内22种病原体设计快速核酸检测系统。
3.2 胃内22种病原体蛋白芯片
利用蛋白质芯片高通量、特异性强等特点，对本发明提出的胃内22种病原体进行特异性蛋白质芯片快速鉴定。
3.3 胃内22种病原体基因芯片探针
利用基因芯片快速、准确、高效的检测技术，制备本发明提出的22种胃内病原体进行快速鉴定系统。
3.4免疫发光试剂盒
利用化学发光、生物发光、化学发光酶等免疫分析技术，构建本发明提出的22种胃内病原体的发光免疫分析试剂盒。
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。
Claims (8)
1.一种胃内菌群的检测方法，其特征在于，其包括如下步骤：
（1）对胃黏膜组织样本进行处理；
（2）对处理后的样本中的微生物进行培养；
（3）挑取单菌落进行菌株纯化；
（4）菌株鉴定。
2.根据权利要求1所述的检测方法，其特征在于，步骤（1）中，使用一次性无菌吸管由保存液EP管中取出胃黏膜组织样本放入灭菌后的研磨器中，加入0.4ml无菌生理盐水进行研磨，得到研磨液。
3.根据权利要求1所述的检测方法，其特征在于，步骤（2）中，将处理后的胃黏膜组织样本研磨液分别涂布到培养基I~IV的表面，四种培养基均进入三气培养箱培养48小时。
4.根据权利要求3所述的检测方法，其特征在于，所述培养基I为含10%无菌脱纤维羊血的CAMPYLOBACTER AGAR BASE；培养基II为含10%无菌脱纤维羊血的COLUMBIA BLOOD AGARBASE；培养基III为含10%无菌脱纤维羊血和2%抗生素混合液的CAMPYLOBACTER AGAR BASE；培养基IV为含10%无菌脱纤维羊血和2%抗生素混合液的COLUMBIA BLOOD AGAR BASE。
5.根据权利要求4所述的检测方法，其特征在于，所述抗生素混合液由10mg/L万古霉素0.5ml、5mg/L多粘菌素B 0.25ml、5mg/L两性霉素B 0.25ml 、10mg/L TMP 1.5ml和97.5ml无菌纯水混合而成。
6.根据权利要求1所述的检测方法，其特征在于，步骤（3）中，分别从经步骤（2）培养后的培养基I~IV的表面挑取单菌落凃于培养基I或培养基II的表层，三气培养箱48小时培养。
7.一种利用权利要求1~6任一项所述的检测方法识别的胃内22种菌构成的菌群在分子生物快速检测中的应用。
8.根据权利要求7所述的应用，其特征在于，将幽门螺杆菌、格雷文氏放线菌、食酸代尔福特菌、光滑念珠菌、热带念珠菌、白色念珠菌、睾丸酮丛毛单胞菌、毗邻颗粒链菌、嗜麦芽窄食单胞菌、食醇鞘氨醇杆菌、少动鞘氨醇单胞菌、类少动鞘氨醇单胞菌、植生拉乌尔菌、土生拉乌尔菌、皮氏罗尔斯顿菌、阿萨希丝孢酵母、摩西假单胞菌、恶臭假单胞菌 、人葡萄球菌、稍变冢村氏菌、schaalia odontodytica以及mycobacteium abscessus strain构建比对库试剂盒，利用多重核酸探针、免疫发光试剂盒、蛋白芯片或基因芯片进行快速检测。
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