阅读说明：本技术 一种结核分枝杆菌的快速鉴定与分型方法 (Rapid identification and typing method for mycobacterium tuberculosis ) 是由 徐费凡 秦永伟 于 2019-12-17 设计创作，主要内容包括：本发明公开了一种结核分枝杆菌的快速鉴定与分型方法,包括：S1.标本预处理,利用标本采集装置对标本进行采集,每份标本中加入对硝基苯甲酸,混合充分后静置15-20分钟；S2.涂片；S3.染色；S4.菌体培养,将上述标本分别取出0.5ml接种到固体培养基中,然后器皿口加橡皮塞处于37摄氏度恒温条件下进行培养,每五天观察一次,连续培养八周；S4.菌体在组织磨碎器充分研磨使细胞破裂后,使用BACTEC法,以含14C棕榈酸作碳源底物的7H12培养基；S5.打开橡皮塞通入氧气,继续在37摄氏度条件下培养。该结核分枝杆菌的快速鉴定与分型方法,可以在较短的时间内对结核杆菌进行鉴定和分型,大大降低了鉴定的时间,可以更加快速的出具准确的鉴定报告。(The invention discloses a method for rapidly identifying and typing mycobacterium tuberculosis, which comprises the following steps: s1, pretreating a specimen, namely collecting the specimen by using a specimen collecting device, adding p-nitrobenzoic acid into each specimen, fully mixing, and standing for 15-20 minutes; s2, smearing; s3, dyeing; s4, culturing thalli, namely taking out 0.5ml of the samples respectively, inoculating the samples into a solid culture medium, then culturing the samples under the constant temperature condition of 37 ℃ by adding rubber plugs into the mouth of a vessel, observing the samples once every five days, and continuously culturing the samples for eight weeks; s4, after the thalli are fully ground in a tissue grinder to break cells, a 7H12 culture medium containing 14C palmitic acid as a carbon source substrate is used by a BACTEC method; s5, opening the rubber plug, introducing oxygen, and continuing to culture at 37 ℃. The rapid identification and typing method for the mycobacterium tuberculosis can identify and type the mycobacterium tuberculosis in a short time, greatly reduce the identification time and more rapidly issue an accurate identification report.)

1. A method for rapidly identifying and typing Mycobacterium tuberculosis is characterized by comprising the following steps:

s1, pretreating a specimen, namely collecting the specimen by using a specimen collecting device, adding p-nitrobenzoic acid into each specimen, fully mixing, and standing for 15-20 minutes;

s2, smearing, wherein the smearing comprises a direct smearing and a bacterium collecting smearing;

s3, dyeing, namely, dropwise adding the smear in the S2 to perform primary dyeing, heating for 5-7 minutes, standing and naturally cooling;

s4, culturing thalli, namely taking out 0.5ml of the samples respectively, inoculating the samples into a solid culture medium, then culturing the samples under the constant temperature condition of 37 ℃ by adding rubber plugs into the mouth of a vessel, observing the samples once every five days, and continuously culturing the samples for eight weeks;

s4, after the thalli are fully ground in a tissue grinder to break cells, using a BACTEC method to measure 14C quantity generated in the bacterial metabolism process by using a 7H12 culture medium containing 14C palmitic acid as a carbon source substrate to calculate whether acid-fast bacilli exist in a specimen;

s5, opening the rubber plug, introducing oxygen, and continuing to culture at 37 ℃.

2. The method for rapid identification and typing of Mycobacterium tuberculosis as claimed in claim 1, wherein: the sample collection device comprises a plurality of sample collection bottles, and is used for containing a sputum sample, pleural effusion and urine respectively, the sample collection bottle containing the sputum sample is added with sodium citrate to the concentration of 5%, and the urine in the sample collection bottle containing the urine is 24-hour mixed urine.

3. The method for rapid identification and typing of Mycobacterium tuberculosis as claimed in claim 1, wherein: the step of direct smear in S2 is: 0.01ml of each specimen was smeared with 10mm & times.

4. The method for rapid identification and typing of Mycobacterium tuberculosis as claimed in claim 1, wherein: the step of the S2 bacterial concentration smear is as follows: 2% NaOH solution was added to each specimen, and then the specimens were allowed to stand at 37 ℃ for 30 minutes before smear was performed.

5. The method for rapid identification and typing of Mycobacterium tuberculosis as claimed in claim 1, wherein: in the S4, the fast growing mycobacterium is grown in 7 days, and the slow growing mycobacterium is grown in more than 7 days.

Technical Field

The invention relates to the technical field of mycobacteria, in particular to a method for rapidly identifying and typing mycobacterium tuberculosis.

Background

Mycobacterium tuberculosis (m. tuberculosis), commonly known as mycobacterium tuberculosis, is the causative agent of tuberculosis. It can invade all organs of the body, but pulmonary tuberculosis is the most common. Tuberculosis remains an important infectious disease to date. The medium reports that about 800 new cases occur each year, and at least 300 million people die from the disease. The death rate of 300 people per 10 million before China is built, the death rate of people living in various diseases is the first, the living level of people is improved after China is built, the sanitary state is improved, group prevention and mass control are particularly developed, children are generally inoculated with BCG, and the morbidity and the mortality of tuberculosis are greatly reduced. However, it should be noted that some parts of the world have an increasing incidence due to aids, drug abuse, application of immunosuppressive agents, alcohol abuse, poverty and the like.

In the last two decades, due to the prevalence of aids, mycobacterium tuberculosis carriers infected with HIV have 30-50 times higher probability of developing active tuberculosis than those not infected with HIV because the virus destroys the immune function of the body, and the course of tuberculosis develops faster. In addition, tuberculosis is the earliest opportunistic infection in the progression of HIV infection, which burdens HIV-infected or aids patients with disease and makes them more susceptible to death. Approximately 8 million new cases of tuberculosis have occurred worldwide each year since the 21 st century and resulted in approximately 3 million deaths. Chinese dies about 25 million each year from tuberculosis, which is twice as many as the total number of deaths from various infectious diseases. Therefore, tuberculosis becomes a global health problem threatening human health and becomes the leading cause of death for some developing countries and regions, especially people in high-incidence AIDS areas. The existing identification and typing technology for combined bacillus has low efficiency, delays time and has low accuracy, and technical innovation is carried out on the basis of the existing technology aiming at the conditions.

Disclosure of Invention

The invention aims to provide a method for rapidly identifying and typing mycobacterium tuberculosis, which aims to solve the problems of low efficiency, time delay and low accuracy of a combined bacillus identification and typing technology in the background technology.

In order to achieve the purpose, the invention provides the following technical scheme: a method for rapidly identifying and typing Mycobacterium tuberculosis comprises the following steps:

s1, pretreating a specimen, namely collecting the specimen by using a specimen collecting device, adding p-nitrobenzoic acid into each specimen, fully mixing, and standing for 15-20 minutes;

s2, smearing, wherein the smearing comprises a direct smearing and a bacterium collecting smearing;

s3, dyeing, namely, dropwise adding the smear in the S2 to perform primary dyeing, heating for 5-7 minutes, standing and naturally cooling;

s4, culturing thalli, namely taking out 0.5ml of the samples respectively, inoculating the samples into a solid culture medium, then culturing the samples under the constant temperature condition of 37 ℃ by adding rubber plugs into the mouth of a vessel, observing the samples once every five days, and continuously culturing the samples for eight weeks;

s4, after the thalli are fully ground in a tissue grinder to break cells, using a BACTEC method to measure 14C quantity generated in the bacterial metabolism process by using a 7H12 culture medium containing 14C palmitic acid as a carbon source substrate to calculate whether acid-fast bacilli exist in a specimen;

s5, opening the rubber plug, introducing oxygen, and continuing to culture at 37 ℃.

Preferably, the sample collection device includes that the sample collection bottle is a plurality of, is used for holding phlegm sample, pleural effusion and urine respectively, the sample collection bottle that holds phlegm sample adds sodium citrate to concentration and is 5%, the urine in the sample collection bottle that holds urine is 24 hours mixed urine.

Preferably, the direct smearing step in S2 is: 0.01ml of each specimen was smeared with 10mm & times.

Preferably, the step of S2 bacterial colony smear is: 2% NaOH solution was added to each specimen, and then the specimens were allowed to stand at 37 ℃ for 30 minutes before smear was performed.

Preferably, in S4, the fast growing mycobacterium is grown within 7 days, and the slow growing mycobacterium is grown within 7 days.

Compared with the prior art, the invention has the beneficial effects that:

1. the rapid identification and typing method for the mycobacterium tuberculosis can identify and type the mycobacterium tuberculosis in a short time, greatly reduce the identification time and more rapidly issue an accurate identification report;

2. the reagent adopted is cheap, the detection mode is simple and easy to operate, after the thalli are fully ground in a tissue grinder to break the cells, the 14C quantity generated in the bacterial metabolism process is measured by using a 7H12 culture medium containing 14C palmitic acid as a carbon source substrate by using a BACTEC method to calculate whether acid-resistant bacillus exists in the specimen, and the mycobacterium tuberculosis is acid-resistant bacillus;

3. the fast growth mycobacteria grow in 7 days, the slow growth mycobacteria grow in more than 7 days, and the existence of the mycobacterium tuberculosis can be simply judged in 7 days.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a method for rapidly identifying and typing Mycobacterium tuberculosis comprises the following steps:

s1, pretreating a specimen, namely collecting the specimen by using a specimen collecting device, adding p-nitrobenzoic acid into each specimen, fully mixing, and standing for 15-20 minutes;

s2, smearing, wherein the smearing comprises a direct smearing and a bacterium collecting smearing;

s3, dyeing, namely, dropwise adding the smear in the S2 to perform primary dyeing, heating for 5-7 minutes, standing and naturally cooling;

s4, culturing thalli, namely taking out 0.5ml of the samples respectively, inoculating the samples into a solid culture medium, then culturing the samples under the constant temperature condition of 37 ℃ by adding rubber plugs into the mouth of a vessel, observing the samples once every five days, and continuously culturing the samples for eight weeks;

s4, after the thalli are fully ground in a tissue grinder to break cells, using a BACTEC method to measure 14C quantity generated in the bacterial metabolism process by using a 7H12 culture medium containing 14C palmitic acid as a carbon source substrate to calculate whether acid-fast bacilli exist in a specimen;

s5, opening the rubber plug, introducing oxygen, and continuing to culture at 37 ℃.

The sample collection device comprises a plurality of sample collection bottles, and is used for containing a sputum sample, pleural effusion and urine respectively, the sample collection bottle containing the sputum sample is added with sodium citrate to the concentration of 5%, and the urine in the sample collection bottle containing the urine is 24-hour mixed urine.

The direct smear step in S2 is: 0.01ml of each specimen was smeared with 10mm & times.

The step of the bacterium collecting smear in S2 is: 2% NaOH solution was added to each specimen, and then the specimens were allowed to stand at 37 ℃ for 30 minutes before smear was performed.

In S4, the fast growing mycobacteria grow in 7 days, and the slow growing mycobacteria grow in more than 7 days.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.