Rapid culture system and culture method of mycoplasma pneumoniae

Abstract

The invention discloses a rapid culture system of mycoplasma pneumoniae. The rapid culture system comprises a growth-promoting additive A, a growth-promoting additive B and a growth-promoting additive C, wherein the growth-promoting additive A is A549 cell lysis buffer; the growth promoting additive B is a mixture of deoxyribonucleoside triphosphate and / or ribonucleoside triphosphate. The rapid culture system can promote rapid growth and reproduction of the mycoplasma pneumoniae, fully meets the nutritional requirements and growth requirements of the mycoplasma pneumoniae, and is of great significance to isolated culture, enrichment culture, epidemic disease prevention and control and molecular diagnosis of the mycoplasma pneumoniae. The invention also provides a culture method of the mycoplasma pneumoniae. The invention also provides application of the rapid culture system of the mycoplasma pneumoniae in in-vitro cell culture of the mycoplasma pneumoniae.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a rapid culture system and method for Mycoplasma pneumoniae. Background Art

[0002] Mycoplasma pneumoniae (MP) is an important pathogen of community-acquired pneumonia (CAP). Epidemiological studies suggest that MP has a high incidence in school-age children (6-12 years old) and may cause refractory or severe mycoplasma pneumoniae pneumonia (MPP), resulting in severe lung damage or even death. Since MP has no cell wall, it is naturally resistant to antibiotics that act on the cell wall. Macrolides are the first choice for the treatment of childhood infections. In recent years, the prevalence of macrolide-resistant MP strains has increased in my country, which has brought difficulties to the clinical treatment of MP infection in children. The diagnosis of MP infection and resistance is crucial for the treatment of MP infection. Although there are methods for detecting MP macrolide resistance mutation sites through molecular biological methods, this method is limited by the content of MP in the test sample.

[0003] Isolation and culture are helpful for the proliferation of MP in the collected samples, and promote the molecular biological detection and drug resistance identification of MP. However, the culture medium composition is complex and the culture time is long during the MP culture process, which makes it difficult to quickly diagnose MP infection. The culture characteristics of MP are related to its genome size. The genome size of MP is about 800Kbp, which is only about 1 / 5 of the genome size of Escherichia coli. This makes the metabolic capacity of MP weak, resulting in an increase in culture time. Mycoplasma contamination in in vitro cell culture experiments is a problem that plagues scientific researchers.

[0004] In view of the above description, a rapid culture method for Mycoplasma pneumoniae is urgently needed. Summary of the invention

[0005] An object of the present invention is to solve at least the above problems and to provide at least the advantages which will be described hereinafter.

[0006] Another object of the present invention is to provide a rapid culture system for Mycoplasma pneumoniae, which can promote the rapid growth and reproduction of Mycoplasma pneumoniae, fully meet the nutritional and growth needs of Mycoplasma pneumoniae, and is of great significance for the isolation and culture, bacterial enrichment and expansion, disease prevention and control, and molecular diagnosis of Mycoplasma pneumoniae.

[0007] Another object of the present invention is to provide a rapid culture method for Mycoplasma pneumoniae.

[0008] Another object of the present invention is to provide a rapid culture system of Mycoplasma pneumoniae for use in in vitro cell culture of Mycoplasma pneumoniae.

[0009] In order to achieve these purposes and other advantages according to the present invention, a rapid culture system for Mycoplasma pneumoniae is provided.

[0011] include:

[0012] Growth-promoting additive A: A549 cell lysate; and

[0013] Growth promoting additive B: deoxyribonucleoside triphosphate and / or ribonucleoside triphosphate.

[0014] Preferably, in the growth-promoting additive B, the ribonucleoside triphosphates contain 16.7 mM adenine triphosphate, 16.7 mM cytidine triphosphate, 16.7 mM guanosine triphosphate and 16.7 mM uridine triphosphate; and / or, the deoxyribonucleoside triphosphates contain 8.3 mM deoxyadenine triphosphate, 8.3 mM deoxycytidine triphosphate, 8.3 mM deoxyguanosine triphosphate and 8.3 mM deoxyuridine triphosphate.

[0015] A method for culturing Mycoplasma pneumoniae comprises the following steps:

[0016] Step 1: A549 cells were cultured in regular culture medium at 3 × 10 5 ~5×10 5 Cells were seeded in cell culture plates and incubated with CO 2 Incubate in the incubator for 12 h to obtain A549 cell culture medium;

[0017] Step 2: Prepare a conventional Mycoplasma pneumoniae test sample containing Mycoplasma pneumoniae into a cell culture medium and obtain Mycoplasma pneumoniae after sterile filtration for later use;

[0018] Step 3: adding the A549 cell culture fluid obtained in step 1 to the Mycoplasma pneumoniae obtained in step 2, and adding the growth-promoting additive A and the growth-promoting additive B as claimed in claim 1, and continuing to incubate at CO 2 The cells were incubated in an incubator for 48 hours, wherein the amount of growth-promoting additive A added was 120-480 μl, and the amount of growth-promoting additive B added was 40-80 μl.

[0019] Preferably, the preparation method of the growth-promoting additive A comprises the following steps: repeatedly freezing and thawing a monolayer of A549 cells with a growth confluence of 95-100% to obtain a cell lysate, adding an appropriate amount of conventional culture medium to the cell lysate to obtain a resuspended cell lysate; centrifuging the resuspended cell lysate at 3000g for 5 minutes to obtain the supernatant, which is the growth-promoting additive A.

[0020] Preferably, the conventional culture medium is A549 culture medium: RPMI1640+10% FBS+50 μg / ml ampicillin.

[0021] Preferably, in step 1, the inoculation amount of A549 cells is 4×10 5 .

[0022] Preferably, the step 2 further comprises: soaking and shaking the conventional Mycoplasma pneumoniae detection sample in a conventional culture medium to obtain a lavage fluid sample; and concentrating the lavage fluid sample by centrifugation and then diluting it with a conventional culture medium to obtain a cell culture fluid.

[0023] A rapid culture system of Mycoplasma pneumoniae is used in in vitro cell culture of Mycoplasma pneumoniae.

[0024] The present invention has at least the following beneficial effects:

[0025] (1) Compared with the traditional culture method, the MPs in the in vitro cell culture system of the present invention proliferate by infecting cells, which is similar to the in vivo infection environment and helps to improve the efficiency of the initial isolation and culture of MPs.

[0026] (2) The in vitro cell culture system described in the present invention is suitable for the classification culture of samples with low MP content, and a significant increase in MP concentration can be achieved within 48 hours.

[0027] (3) When the MP content in the culture sample is less than 8×10 4 CFU, the MP proliferation rate in the cell culture system is higher than that in the traditional culture method. 4 When CFU is detected, MPs adhering to the cell surface can be directly observed under a microscope.

[0028] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of 48h and 72h culture tests at different MP inoculation concentrations in the cell culture system;

[0030] Figure 2 The results of 48h and 72h culture tests with different MP inoculation concentrations in SP4 medium;

[0031] Figure 3 It is the result of microscopic detection in the cell culture system;

[0032] Figure 4The results of MP culture in the cell culture system under different concentrations of growth-promoting additive A;

[0033] Figure 5 The results of MP culture in the cell culture system at different cell seeding densities;

[0034] Figure 6 The MP culture results in a cell culture system under different concentrations of growth-promoting additive B in one embodiment;

[0035] Figure 7 This is the MP culture result in the cell culture system under different concentrations of the growth-promoting additive B in another embodiment. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0037] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0038] Example 1: Comparison of cell culture and cell-free culture methods

[0039] The culture of Mycoplasma pneumoniae FH strain was used in this experiment after serial dilution and plate counting. A549 cells were used in the cell culture system with 5×10 5 Cells were inoculated into each well of a 6-well cell culture plate, and 120 μl of growth-promoting additive A was added to the culture system 12 h after inoculation. The MP inoculation amount was based on the highest concentration of 2×10 6 CFU gradient 1:5 n Six dilutions (1-6) were used for inoculation, and the same concentration of MP was used to inoculate SP4 medium (tryptone, 10 g; yeast extract, 8.75 g; peptone, 5.3 g; glucose, 5 g; PPLO broth, 3.5 g; 10% yeast extract, 10 ml; 1% phenol red, 1 ml; fetal bovine serum, 170 ml; penicillin, 50 mg; total volume 1 L), and the cultures were placed in CO 2 Cultured in an incubator at 37°C. After 48h and 72h of culture, cells and SP4 cultures were collected for MP counting. MP counting was performed using the plate dilution method. Figure 1 , SP4 medium culture results are shown in Figure 2 The cell culture system was cultured for 48 hours and the results were observed using a microscope. Figure 3 .

[0040] Example 2: Screening of concentration of cell lysate added in cell culture method

[0041] A549 cells were used at 5 × 10 5 Each well of the cell culture plate was inoculated in a 6-well cell culture plate, and growth-promoting additive A was added to the culture system 12 hours after inoculation. Different concentrations of growth-promoting additive A (4-1 / 16) were added to the cultured cells, with the total volume of the culture medium being controlled to be approximately 2.2 ml. 8×10 4 MP of CFU, continued in CO 2 Incubate at 37°C for 48 hours. After the culture is completed, collect the culture for MP counting. Figure 4 .

[0042] Example 3: Screening of cell seeding density in cell culture method

[0043] A549 cells were seeded in 6-well cell culture plates at a seeding density of 5 × 10 5 / well was 1, and cells were inoculated at six concentrations of 50%, 60%, 70%, 80%, 90% and 100%. 120 μl of growth-promoting additive A was added to the culture system 12 hours after cell inoculation. 8×10 4 MP of CFU, continued in CO 2 Incubate at 37°C for 48 hours. After the culture is completed, collect the culture for MP counting. Figure 5 .

[0044] A549 cells were used at 5 × 10 5 Each well of the cells was inoculated in a 6-well cell culture plate, and 120 μl of growth-promoting additive A was added to the culture system 12 h after inoculation. At the same time, gradient concentrations of NTP mixtures (containing 25 mM adenine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate) or dNTP mixtures (containing 25 mM deoxyadenine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate and deoxyuridine triphosphate) were added to the culture system. The maximum volume of NTP added was 80 μl (about 340 mg, R1), and the maximum volume of dNTP added was 80 μl (about 320 mg, D1), and then diluted 2 times (R2, D2). The cells continued to be incubated at CO 2 Incubate at 37°C for 48 hours. After the culture is completed, collect the culture for MP counting. The results show that the two highest concentrations of NTP mixture and dNTP mixture have the effect of promoting MP proliferation. Figure 6 .

[0045] Embodiment 4:

[0046] A549 cells were used at 5 × 10 5Each well of the cells was inoculated in a 6-well cell culture plate, and 120 μl of growth-promoting additive A was added to the culture system 12 hours after inoculation. At the same time, a mixture of dNTP (D) and NTP (R) was added to the culture system, wherein the dNTP or NTP was mixed with the other (dNTP or NTP) diluted in turn at the screening concentration of 16.7 mM (1) in Example 3, and the concentrations were 16.7 mM (1), 8.3 mM (2) and 4.2 mM (3). The cells were continued to be incubated with CO 2 The cells were cultured in an incubator at 37°C for 48 hours. After the culture was completed, the cultures were collected for MP counting. The results showed that the MP proliferation rate under the D1R1 and D2R1 culture conditions was higher than that of the other groups and the control group. Figure 7 .

[0047] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A rapid culture system for Mycoplasma pneumoniae, characterized in that: include: Growth-promoting additive A: A549 cell lysate; as well as Growth promoting additive B: deoxyribonucleoside triphosphate and / or ribonucleoside triphosphate.

2. The rapid culture system of Mycoplasma pneumoniae according to claim 1, characterized in that: In the growth-promoting additive B, the ribonucleoside triphosphates contain 16.7 mM adenine triphosphate, 16.7 mM cytidine triphosphate, 16.7 mM guanosine triphosphate and 16.7 mM uridine triphosphate; and / or, the deoxyribonucleoside triphosphates contain 8.3 mM deoxyadenine triphosphate, 8.3 mM deoxycytidine triphosphate, 8.3 mM deoxyguanosine triphosphate and 8.3 mM deoxyuridine triphosphate.

3. A method for culturing Mycoplasma pneumoniae, characterized in that: The following steps are involved: Step 1: A549 cells were cultured in regular culture medium at 3 × 10 5 ~5×10 5 The cells were seeded on a cell culture plate and incubated in a CO2 incubator for 12 h to obtain A549 cell culture fluid; Step 2: Prepare a conventional Mycoplasma pneumoniae test sample containing Mycoplasma pneumoniae into a cell culture medium and obtain Mycoplasma pneumoniae after sterile filtration for later use; Step 3: Add the A549 cell culture fluid obtained in step 1 to the Mycoplasma pneumoniae obtained in step 2, and add the growth-promoting additive A and the growth-promoting additive B as described in claim 1, and continue to incubate in a CO2 incubator for 48 hours, wherein the amount of growth-promoting additive A added is 120-480 μl, and the amount of growth-promoting additive B added is 40-80 μl.

4. The rapid culture system of Mycoplasma pneumoniae according to claim 3, characterized in that: The preparation method of growth-promoting additive A includes the following steps: repeatedly freezing and thawing a monolayer of A549 cells with a growth confluence of 95-100% to obtain a cell lysate, adding an appropriate amount of conventional culture medium to the cell lysate to obtain a resuspended cell lysate; centrifuging the resuspended cell lysate at 3000g for 5 minutes to obtain the supernatant, which is the growth-promoting additive A.

5. The rapid culture system for Mycoplasma pneumoniae according to any one of claims 3 or 4, characterized in that: The conventional culture medium is A549 culture medium: RPMI1640+10% FBS+50 μg / ml ampicillin.

6. The method for culturing Mycoplasma pneumoniae according to claim 3, characterized in that: In step 1, the inoculation amount of A549 cells was 4×10 5 .

7. The method for culturing Mycoplasma pneumoniae according to claim 3, characterized in that: The step 2 also includes: soaking and shaking the conventional Mycoplasma pneumoniae detection sample in a conventional culture medium to obtain an lavage fluid sample; and centrifuging and concentrating the lavage fluid sample and then diluting it with a conventional culture medium to obtain a cell culture fluid.

8. Use of the rapid culture system for Mycoplasma pneumoniae according to claim 1 in in vitro cell culture of Mycoplasma pneumoniae.

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