CN110530827A - 一种粪菌定量的方法 - Google Patents
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Abstract
本发明涉及一种粪菌定量的方法,包括以下步骤:(1)粪菌液稀释;(2)将步骤(1)所得粪菌稀释液加入染料;(3)使用流式细胞仪测定步骤(2)所得粪菌稀释液,得到粪菌稀释液中细菌浓度。与现有技术相比,本发明粪菌定量方法操作简易快捷,可同时大批量操作几十个样本,且可测定粪菌总数,结果较为准确。
Description
技术领域
本发明属于粪菌移植技术领域,尤其是涉及一种粪菌定量的方法。
背景技术
粪菌移植(FMT),也称为粪菌治疗或肠微生菌移植,是将健康人粪便中的功能菌群,移植到患者胃肠道内,重建具有正常功能的肠道菌群,实现肠道及肠道外疾病的诊疗。目前,粪菌移植已被视为一种特殊的器官移植,用于治疗难辨梭状芽胞杆菌感染、炎症性肠病、肠易激综合征、代谢综合征、神经发育不良与神经退行性疾病、自身免疫性肠病、肠道食物过敏等。现代粪菌移植技术主要是器械介入的方法,比如胃镜、胃管、肠镜.空肠管等,腹泻型IBS.溃疡性结肠炎等治疗需要多次移植,而每次粪便菌群都需要通过导管经过胃、十二指肠,注入十二指肠远端,过程复杂,患者有一定痛苦,应用受到限制。制备的粪菌样本在室温存放时间短,需要立即进行移植,加入无菌甘油后虽然可以置于-80'C冰冻,但是取用不便。
发明内容
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种粪菌定量的方法。
本发明的目的可以通过以下技术方案来实现:
一种粪菌定量的方法,包括以下步骤:
(1)粪菌液稀释;
(2)将步骤(1)所得粪菌稀释液加入染料;
(3)使用流式细胞仪测定步骤(2)所得粪菌稀释液,得到粪菌稀释液中细菌浓度。
优选地,步骤(1)中,粪菌液采用高温灭菌的生理盐水进行稀释。
优选地,步骤(1)中,粪菌液稀释到1000倍。
优选地,步骤(2)中,所述染料选自染料Sybr green,其用于结合于细菌的 dsDNA上激发荧光以供流式细胞仪检测定量。
优选地,步骤(2)中,所述染料稀释到10000倍后与步骤(1)所得粪菌稀释液以1:1000混匀。
优选地,步骤(2)中,所述染料加入步骤(1)所得粪菌稀释液后,37℃孵育 15min。
优选地,步骤(3)中,所述流式细胞仪选择BECKMAN COULTER公司的流式细胞仪。
优选地,步骤(3)中,所述流式细胞仪参数设置为:FSC-100,SSC-100, FITC-100,PE-1,PC5.5-100,PC7-1,APC-30,APC-A750-1。通过设置这些参数,流式细胞仪可以根据细胞的生物物理、生物化学特性分选出粪便菌群并通过收集荧光强度对粪便菌群进行计量。流式细胞仪测定准确度更高的原因主要在于其参数设置所代表的分选功能。
与现有技术相比,本发明的有益效果为:
1、操作简易快捷,可同时大批量操作几十个样本;
2、测定粪菌总数,结果较为准确。
具体实施方式
下面结合具体实施例对本发明进行详细说明。
实施例
一种粪菌定量的方法,包括以下步骤:
1.1.1粪菌液稀释
(1)取10ul粪菌液用990ul高温灭菌的生理盐水稀释;
(2)重复(1)步骤,得1ml稀释了1000倍的粪菌液;
1.1.2粪菌液定量
(1)加染料:往1ml粪菌液中加入1ul稀释过1000倍的Sybr green;
(2)孵育:将粪菌液混匀后37℃孵育15min,避光保存;
(3)参数设置:使用BECKMAN COULTER公司的B49006AC流式细胞仪,将其参数设置为:FSC-100,SSC-100,FITC-100,PE-1,PC5.5-100,PC7-1,APC-30, APC-A750-1。设置每个样本检测5000个细菌,流速为60ul/min;
(4)定量:该样本检测到5000个细菌用时46s,其细菌浓度为5000/(46÷60*60 ÷1000)=1.09*105个/ml。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (8)
1.一种粪菌定量的方法,其特征在于,包括以下步骤:
(1)粪菌液稀释;
(2)将步骤(1)所得粪菌稀释液加入染料;
(3)使用流式细胞仪测定步骤(2)所得粪菌稀释液,得到粪菌稀释液中细菌浓度。
2.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(1)中,粪菌液采用高温灭菌的生理盐水进行稀释。
3.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(1)中,粪菌液稀释到1000倍。
4.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(2)中,所述染料选自染料Sybr green。
5.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(2)中,所述染料稀释到10000倍后与步骤(1)所得粪菌稀释液以1:1000混匀。
6.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(2)中,所述染料加入步骤(1)所得粪菌稀释液后,37℃孵育15min。
7.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(3)中,所述流式细胞仪选择BECKMAN COULTER公司的流式细胞仪。
8.根据权利要求1所述的一种粪菌定量的方法,其特征在于,步骤(3)中,所述流式细胞仪参数设置为:FSC-100,SSC-100,FITC-100,PE-1,PC5.5-100,PC7-1,APC-30,APC-A750-1。
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