阅读说明：本技术 一种粪菌定量的方法 (A kind of method that caprophyl is quantitative ) 是由 严菲月 孟和 郑书钰 徐珂 郑煜明 韩铖潇 秦超 罗凌霄 肖璐 何川 杨凌宇 于 2019-07-16 设计创作，主要内容包括：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。(The present invention relates to a kind of methods that caprophyl is quantitative, comprising the following steps: (1) caprophyl liquid dilutes；(2) dyestuff is added in caprophyl dilution obtained by step (1)；(3) using caprophyl dilution obtained by flow cytometer determination step (2), bacterial concentration in caprophyl dilution is obtained.Compared with prior art, caprophyl quantitative approach easy operation of the present invention high-volume can operate tens samples simultaneously, and can measure caprophyl sum, as a result more accurate.)

1. a kind of method that caprophyl is quantitative, which comprises the following steps:

(1) caprophyl liquid dilutes；

(2) dyestuff is added in caprophyl dilution obtained by step (1)；

(3) using caprophyl dilution obtained by flow cytometer determination step (2), bacterial concentration in caprophyl dilution is obtained.

2. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (1), caprophyl liquid is using high The physiological saline of temperature sterilizing is diluted.

3. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (1), caprophyl liquid is diluted to 1000 times.

4. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (2), the dyestuff is selected from Dyestuff Sybr green.

5. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (2), the dye-dilution It is mixed after to 10000 times with caprophyl dilution obtained by step (1) with 1:1000.

6. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (2), the dyestuff is added After caprophyl dilution obtained by step (1), 37 DEG C of incubation 15min.

7. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (3), the fluidic cell The flow cytometer of instrument selection BECKMAN COULTER company.

8. a kind of quantitative method of caprophyl according to claim 1, which is characterized in that in step (3), the fluidic cell Instrument parameter setting are as follows: FSC-100, SSC-100, FITC-100, PE-1, PC5.5-100, PC7-1, APC-30, APC-A750-1.

Technical field

The invention belongs to caprophyl implantation technique fields, more particularly, to a kind of method that caprophyl is quantitative.

Background technique

Caprophyl transplants (FMT), and the also referred to as micro- raw bacterium transplanting of caprophyl treatment or intestines, is by the function bacterium in healthy human faecal mass Group, is transplanted in patient's gastrointestinal tract, rebuilds the intestinal flora with normal function, realizes the diagnosis and treatment of enteron aisle and parenterally disease. Currently, caprophyl transplanting has been considered as a kind of special organ transplant, for treating clostridium difficile infection, inflammatory bowel Disease, irritable bowel syndrome, metabolic syndrome, neurodevelopment are bad to be eaten with neurodegenerative disease, auto immune enteropathy, enteron aisle Object allergy etc..Modern caprophyl implantation technique is mainly the method, such as gastroscope, stomach tube, colonoscopy jejunum pipe etc. of instrument intervention, abdomen It rushes down the treatments such as type IBS. ulcerative colitis and needs repeatedly transplanting, and faecal microbiota is required through conduit by stomach, ten every time Two duodenum 12 inject distal duodenum, and process is complicated, and patient has certain pain, using being restricted.The caprophyl sample of preparation exists The room temperature resting period is short, needs to be transplanted immediately, although can be placed in -80'C frost after sterile glycerol is added, takes It is inconvenient.

Summary of the invention

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of sides that caprophyl is quantitative Method.

The purpose of the present invention can be achieved through the following technical solutions:

A kind of method that caprophyl is quantitative, comprising the following steps:

(1) caprophyl liquid dilutes；

(2) dyestuff is added in caprophyl dilution obtained by step (1)；

(3) using caprophyl dilution obtained by flow cytometer determination step (2), bacterial concentration in caprophyl dilution is obtained.

Preferably, in step (1), caprophyl liquid is diluted using the physiological saline of high-temperature sterilization.

Preferably, in step (1), caprophyl liquid is diluted to 1000 times.

Preferably, in step (2), the dyestuff is selected from dyestuff Sybr green, is used to be incorporated into the dsDNA of bacterium Upper excitation fluorescence is quantitative for flow cytomery.

Preferably, in step (2), the dye-dilution to after 10000 times with caprophyl dilution obtained by step (1) with 1: 1000 mix.

Preferably, in step (2), after caprophyl dilution obtained by step (1) is added in the dyestuff, 37 DEG C of incubation 15min.

Preferably, in step (3), the flow cytometer of flow cytometer selection BECKMAN COULTER company.

Preferably, in step (3), the flow cytometer parameter setting are as follows: FSC-100, SSC-100, FITC-100, PE-1, PC5.5-100, PC7-1, APC-30, APC-A750-1.By the way that these parameters are arranged, flow cytometer can be according to thin The biophysics of born of the same parents, biochemical characteristic sub-elect faecal microbiota and are measured by collecting fluorescence intensity to faecal microbiota. The higher reason of flow cytometer accuracy of measurement essentially consists in sorting function representated by its parameter setting.

Compared with prior art, the invention has the benefit that

1, easy operation high-volume can operate tens samples simultaneously；

2, measurement caprophyl sum is as a result more accurate.

Specific embodiment

The present invention is described in detail combined with specific embodiments below.