Mycobacterium detection method

Abstract

The invention discloses a detection method of mycobacteria. The detection method comprises the following steps: S1, constructing a fusion protein gp23RD-GFP; s2, a target protein is obtained from the fusion protein gp23RD-GFP; s3, mixing the target protein with the mycobacterium culture, and incubating; s4, preparing a microscopic examination sample; s5, carrying out microscopic examination by using a fluorescence microscope; according to the method, a functional truncated body of phage TM4 receptor binding protein gp23 is fused with a green fluorescent protein to construct a fusion protein gp23RD-GFP, the fusion protein gp23RD-GFP is purified to obtain a target protein, the target protein and a mycobacterium culture are mixed and incubated, and finally the incubated mycobacterium culture is subjected to microscopic examination sample preparation. The detection mode is simple and rapid, and support can be provided for the joint development of the subsequent mycobacterium detection technology.

Description

Technical Field

[0001] The invention belongs to the technical field of mycobacterium detection, and particularly relates to a mycobacterium detection method. Background Art

[0002] Traditional methods for detecting mycobacteria include microscopic examination of specimens, bacterial culture, and differentiation and identification through colony morphology and phenotypic characteristics, but these methods are complicated and require a long incubation time. With the development of technology, molecular diagnostic technology has been developed and has become an important method for clinical microbiology laboratories to identify mycobacteria. For example, heat shock protein hsp65 Gene, RNA polymerase ( rpoB ) gene sequencing and whole genome sequencing multiplex qPCR detection, multiplex TaqMan PCR detection kit detection, real-time PCR, 16S rRNA sequencing and other molecular and immunological techniques. In the past decade, many clinical laboratories have introduced MALDI-TOFMS into the mycobacterium detection workflow, identifying it by analyzing the proteome at the species level. Since then, mycobacterium detection technology has been developed at the molecular, protein and immunological levels; to date, the 16S rRNA gene and other conserved genes, such as the heat shock protein gene ( hsp65 ) and RNA polymerase genes ( rpoB ) is considered the gold standard for gene sequencing targets. 16S rRNA is highly conserved in molecular structure, with only a small amount of nucleotide sequence changes at certain positions, and these changes are specific to the genus or species of mycobacteria. At the same time, it contains a lot of information, and the entire nucleic acid sequence of many mycobacterial 16S rRNAs has been determined. The 16S rRNA gene sequence has two hypervariable regions (hypervariable regions A and B). The sequencing of the 16S rRNA gene of mycobacteria shows that most mycobacteria have a characteristic sequence in hypervariable region A. As long as the sequence of the mycobacterium being tested is compared with the characteristic sequence of the suppression, the mycobacteria can be accurately identified.

[0003] Fluorescent trehalose probe for detection of mycobacteria: a trehalose-based fluorescent probe with a bright far-infrared emitting molecular rotor fluorophore that utilizes the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation. Fluorescein, an excellent trehalose analog, can be metabolized to trehalose glycolipids in mycobacteria through the action of substrate-promiscuous mycoacyltransferase, thereby achieving the purpose of detecting mycobacteria.

[0004] Existing mycobacterium detection technologies, such as traditional cell morphology and phenotype analysis or cell culture, all require a long culture time to complete, and the detection cycle is long; and detection technologies that rely on molecular, immune and proteomic information, although they have shortened the detection cycle to a certain extent, have high requirements for the detection platform, have a strong dependence on technology, and have relatively high economic costs. Summary of the invention

[0005] The object of the present invention is to provide a method for detecting mycobacteria, which utilizes the property of receptor binding protein of bacteriophage to specifically bind to host cells and combines the fluorescence property of green fluorescent protein to obtain a target protein from the fusion protein gp23_RD-GFP, and effectively recognizes and binds to the surface of mycobacteria in a short time through the target protein, thereby realizing rapid detection of mycobacteria and reducing the current cost investment in the process of mycobacterium detection.

[0006] The present invention is achieved through the following technical solutions: A method for detecting mycobacteria comprises the following steps: S1: Construction of fusion protein gp23_RD-GFP; S2: Obtain the target protein from the fusion protein gp23_RD-GFP expression strain; S3: mixing the target protein with the mycobacterial culture and incubating; S4: prepare samples for microscopic examination; S5: Microscopic examination using a fluorescence microscope.

[0007] Furthermore, in step S1, the specific operation method for constructing the fusion protein gp23_RD-GFP is: fusing the functional truncation of the bacteriophage TM4 receptor binding protein gp23 with the green fluorescent protein.

[0008] Furthermore, in step S2, the specific method of obtaining the target protein from the fusion protein gp23_RD-GFP expression strain is: S21: Induce the expression of target protein through Escherichia coli expression strain BL21 (DE3); S22: collect the bacteria and centrifuge; S23: the supernatant after centrifugation is passed through a nickel column for protein purification; S24: Collect the target protein after removing impurities.

[0009] Furthermore, in step S21, the specific operation method of inducing the expression of the target protein through the Escherichia coli expression strain BL21 (DE3) is as follows: the constructed fusion protein gp23_RD-GFP recombinant plasmid is transferred to the Escherichia coli expression strain BL21 (DE3), and when the strain grows to an OD600 of 0.7-0.9, an inducer is added to induce the expression of the target protein.

[0010] Furthermore, the inducer is isopropyl-β-D-thiogalactoside (IPTG).

[0011] Furthermore, in step S22, the specific operation method of collecting the bacteria for centrifugation is: resuspending the bacteria with a lysis solution, then disrupting them with a cell disruptor, and centrifuging them at 13,000 rpm for 20 minutes.

[0012] Furthermore, in step S24, the specific operation method of collecting the target protein after removing the impurity proteins is: using a lysis solution to wash away the impurity proteins, and then eluting and collecting the target protein with an elution solution.

[0013] Furthermore, in step S3, when the target protein is mixed with the mycobacterium culture for incubation, the incubation temperature is 16° C. and the incubation time is 35 min-45 min.

[0014] Furthermore, in step S4, the specific operation method of preparing the microscopic sample is: centrifuging the incubated mycobacterium culture and removing the supernatant, washing with phosphate buffer, centrifuging again and then repeatedly washing with phosphate buffer to remove excess protein, and finally resuspending with phosphate buffer to prepare the microscopic sample.

[0015] Furthermore, in step S5, when the fluorescence microscope is used for microscopic examination, the microscopic examination sample is examined under an excitation light with a wavelength of 488 nm.

[0016] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1) In the present invention, a functional truncation of bacteriophage TM4 receptor binding protein gp23 is fused with green fluorescent protein to construct a fusion protein gp23_RD-GFP, and then the fusion protein gp23_RD-GFP is purified to obtain the target protein, and the target protein is mixed with mycobacterium culture and incubated, and finally the incubated mycobacterium culture is subjected to microscopic sample preparation, which can be examined by fluorescence microscopy. The detection method is simple and fast, and can provide support for the joint development of subsequent mycobacterium detection technology.

[0017] 2) In the present invention, when performing sample testing, the mycobacterium culture sample only needs to be moderately incubated with the target protein for about 40 minutes, and then washed 2-3 times to prepare the sample for fluorescence microscopy, which shortens the sample testing cycle and is more economical. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 It is a comparison chart of microscopic examination results in the present invention.

[0020] Figure 2 This is a comparison chart of the results of detecting the binding of the target protein to mycobacteria using magnetic beads in the present invention.

[0021] Figure 3 This is a comparison chart of the analysis results of flow cytometry of Mycobacterium smegmatis in the present invention.

[0022] Figure 4 This is a comparison chart of the analysis results of flow cytometry of Mycobacterium Ra in the present invention. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0024] Embodiment 1: A method for detecting mycobacteria comprises the following steps: S1: Construction of fusion protein gp23_RD-GFP; S2: Obtain the target protein from the fusion protein gp23_RD-GFP expression strain; S3: mixing the target protein with the mycobacterial culture and incubating; S4: prepare samples for microscopic examination; S5: Microscopic examination using a fluorescence microscope.

[0025] First, a fusion protein of the functional truncation of the bacteriophage TM4 receptor binding protein gp23 and green fluorescent protein was constructed, namely the fusion protein gp23_RD-GFP. The specific operation was as follows: the coding gene of green fluorescent protein was introduced between the HindIII and NotI restriction sites of the pRSFDuet-1 vector, and the TM4_gp23 (32-123) gene sequence was introduced between the BamHI and HindIII sites of the vector. The 6-histidine tag carried by the N-terminus of the vector was used to finally construct a fusion expression protein of gp23 (32-123) with 6-histidine tags at the N-terminus and green fluorescent protein GFP, namely the fusion protein gp23_RD-GFP.

[0026] Then, the constructed fusion protein gp23_RD-GFP recombinant plasmid was transferred to the Escherichia coli expression strain BL21 (DE3), and cultured at 37°C and 160rpm. When the bacteria grew to an absorbance value at 600nm, i.e., OD600 of 0.8, 600μl of 0.5M inducer isopropyl-β-D-thiogalactoside (IPTG) was added to induce the expression of the target protein at 22°C and 160rpm. After induction for 16-20 hours, the bacteria were collected and resuspended with 30 ml of lysis buffer, which was a mixture of 50mM Tris-HCl with a pH of 7.6, 500mM NaCl and 40mM imidazole. The cells were then broken by a cell disruptor with a power of 300W and a breaking time of 25min. After breaking, the cells were centrifuged at 4°C and 13000rpm for 20 min; add the supernatant after centrifugation to the equilibrated nickel column for protein purification, and wash away the impurities with 10-20 column volumes of lysis buffer, then elute and collect the target protein with eluent, which is a mixture of 50 mM Tris-HCl at pH 7.6, 150 mM NaCl and 500 mM imidazole.

[0027] The purified fusion protein gp23_RD-GFP was incubated with the sample solution to be tested, i.e., the mycobacterium culture, at a temperature of 16°C for 40 min. After the incubation was complete, the solution was centrifuged at 9000 rpm for 5 min, the supernatant was removed, and the incubated sample solution was then washed with phosphate buffer, and then centrifuged again at 9000 rpm for 5 min. The above washing and centrifugation steps were repeated to remove excess protein, and finally the solution was resuspended with an appropriate amount of phosphate buffer for preparing microscopic samples.

[0028] Finally, the samples were examined under a microscope. Figure 1 As shown, by using a fluorescence microscope, the sample is examined under a 488nm wavelength excitation light, and mycobacterial cells with green fluorescence attached to the surface can be observed. Figure 1Shown are images of Mycobacterium smegmatis observed under bright field and cells in the same area under 488nm excitation light. A strong green fluorescence signal can be clearly observed on the cell surface, indicating that after the cells in the sample solution are incubated with the target protein, they can be effectively identified and bound, proving that the target protein obtained from the fusion protein gp23_RD-GFP has the ability to detect Mycobacterium smegmatis.

[0029] Embodiment 2: 1. Add an appropriate amount of Ni NTA Magarose Beads to a 2 ml EP tube, remove the storage buffer, and then wash several times with pH 7.6 lysis buffer to balance the magnetic beads; 2. Incubate the purified target protein with Ni NTA Magarose Beads for an appropriate time; then remove excess unbound protein with the help of a magnetic stand and wash twice with lysis buffer; 3. Add an appropriate amount of mycobacterium sample to the EP tube and incubate in a dark environment; 4. After incubation, remove excess bacteria and wash several times with lysis buffer to remove unbound bacteria; 5. Elute the protein with an appropriate amount of PH7.6 elution buffer and transfer to a new EP tube for centrifugation at 9000 rpm for 5 min. Spread the centrifuged bacteria on a 7H10 plate containing ampicillin. Taking advantage of the natural resistance of Mycobacterium smegmatis to ampicillin, the Mycobacterium smegmatis that binds to the target protein on the magnetic beads and is finally enriched.

[0030] from Figure 2 It can be seen that in the control group (i.e., the target protein was not bound to the magnetic beads), the mycobacterium sample was incubated with a single agarose magnetic bead, and then coated on the plate after washing. Since the magnetic beads do not have the ability to bind to mycobacteria, no Mycobacterium smegmatis grew on the plate after culture; on the basis of the control group, the experimental group coupled the target protein with agarose magnetic beads in advance, and incubated and washed under the same conditions. Since the target protein can effectively bind to mycobacteria, the bacteria bound to it are retained during the washing process, and the excess bacteria are removed. Therefore, mycobacteria enriched by the target protein grow on the plate after culture.

[0031] Embodiment 3: like Figure 3 and Figure 4As shown, the purified target protein and the sample solution to be tested were incubated at 16°C, centrifuged at 9000 rpm for 5 min, and the supernatant was removed. The cells were then washed 2-3 times with phosphate buffer to remove unbound target protein, and the cells were resuspended with an appropriate amount of phosphate buffer for flow cytometer analysis. In the flow cytometer analysis, the 488nm detection channel was used to count the cells with GFP fluorescence signals on the surface of the sample.

[0032] from Figure 3 It can be seen that when the two groups of samples were subjected to flow cytometry analysis (by detecting the GFP fluorescence signal on the cell surface), the untreated single cell (left figure) was used as the control. The value in the upper right corner of the control figure indicates the proportion of cells with detectable GFP fluorescence signals on the surface in the total cells of the sample; the right figure shows the test samples after incubation with the target protein obtained from the fusion protein gp23_RD-GFP. Under the same detection conditions, about 23.6% of the cells in the experimental group could detect GFP fluorescence signals, indicating that the target protein has the ability to detect mycobacteria.

[0033] from Figure 4 It can be seen that through Figure 3 The same detection technology was used to detect the binding ability of the target protein to mycobacterial Ra. Figure 4 (Right picture) shows that GFP fluorescence signals were detected on the surface of about 50% of the cells in the test sample, indicating that the target protein also has the ability to bind to Mycobacterium Ra and can achieve the detection effect.

[0034] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for detecting mycobacteria, characterized in that: The following steps are involved: S1: Construction of fusion protein gp23_RD-GFP; S2: Obtain the target protein from the fusion protein gp23_RD-GFP expression strain; S3: mixing the target protein with the mycobacterial culture and incubating; S4: prepare samples for microscopic examination; S5: Microscopic examination using a fluorescence microscope.

2. The method for detecting mycobacteria according to claim 1, characterized in that: In step S1, the specific operation method for constructing the fusion protein gp23_RD-GFP is: fusing the functional truncated form of the bacteriophage TM4 receptor binding protein gp23 with the green fluorescent protein.

3. The method for detecting mycobacteria according to claim 1, characterized in that: In step S2, the specific method of obtaining the target protein from the fusion protein gp23_RD-GFP expression bacteria is: S21: Induce the expression of target protein through E. coli expression strain BL21; S22: collect bacteria and centrifuge; S23: the supernatant after centrifugation is passed through a nickel column for protein purification; S24: Collect the target protein after removing impurities.

4. The method for detecting mycobacteria according to claim 3, characterized in that: In step S21, the specific operation method of inducing the expression of the target protein by the E. coli expression strain BL21 is: the constructed fusion protein gp23_RD-GFP recombinant plasmid is transferred to the E. coli expression strain BL21, and when the strain grows to an OD600 of 0.7-0.9, an inducer is added to induce the expression of the target protein.

5. The method for detecting mycobacteria according to claim 4, characterized in that: The inducing agent is isopropyl-β-D-thiogalactoside.

6. The method for detecting mycobacteria according to claim 3, characterized in that: In step S22, the specific operation method of collecting the bacterial cells for centrifugation is: resuspending the bacterial cells with a lysis solution, then disrupting them with a cell disruptor, and centrifuging them at 13,000 rpm for 20 minutes.

7. The method for detecting mycobacteria according to claim 3, characterized in that: In step S24, the specific operation method of collecting the target protein after removing the impurity proteins is: using a lysis solution to wash away the impurity proteins, and then eluting and collecting the target protein with an elution solution.

8. The method for detecting mycobacteria according to claim 1, characterized in that: In step S3, when the target protein is mixed with the mycobacterium culture for incubation, the incubation temperature is 16° C. and the incubation time is 35 min-45 min.

9. The method for detecting mycobacteria according to claim 1, characterized in that: In step S4, the specific operation method of preparing the microscopic sample is: centrifuging the incubated mycobacterium culture and removing the supernatant, washing with phosphate buffer, centrifuging again and repeatedly washing with phosphate buffer to remove excess protein, and finally resuspending with phosphate buffer to prepare the microscopic sample.

10. The method for detecting mycobacteria according to claim 1, characterized in that: In step S5, when the fluorescence microscope is used for microscopic examination, the microscopic examination sample is examined under an excitation light with a wavelength of 488 nm.

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