A Taq DNA polymerase mutant and its application in the detection of Mycobacterium tuberculosis.

Abstract

The application discloses a Taq DNA polymerase mutant, wherein the amino acid sequence of the Taq DNA polymerase mutant is shown as SEQ ID NO. 1. The application further discloses a polynucleotide, a recombinant expression vector, a genetically engineered bacterium, a kit and a preparation method of the above-mentioned Taq DNA polymerase mutant. The application further discloses application of the above-mentioned Taq DNA polymerase mutant, polynucleotide, recombinant expression vector, genetically engineered bacterium and kit in detection of Mycobacterium tuberculosis. The application further discloses a method for detecting Mycobacterium tuberculosis. The Taq DNA polymerase mutant can resist impurities in a lung lavage fluid and can resist the lung lavage fluid treated by sodium hydroxide digestion, and can directly amplify and detect a target fragment from a sample to be detected to detect Mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a Taq DNA polymerase mutant and its application in the detection of Mycobacterium tuberculosis. Background Technology

[0002] Currently, many kits for detecting Mycobacterium tuberculosis have been developed. However, most existing kits require liquefaction of samples such as sputum and bronchoalveolar lavage fluid before pathogen nucleic acid extraction and purification, which increases the workload of frontline testing personnel.

[0003] Current methods for detecting Mycobacterium tuberculosis mainly include bacteriological, immunological, and molecular biological detection. Bacteriological detection has a long turnaround time, especially for pathogens like Mycobacterium tuberculosis with its long replication cycle; traditional culture-based methods require approximately four weeks, highlighting their low detection efficiency. Immunological detection relies on antigen-antibody assays, requiring sufficient amounts of specific antigens such as MPB64, ESAT-6, and CFP-10. Therefore, in the early stages of tuberculosis infection or at low bacterial loads, antigen detection may fail to detect sufficient antigens, leading to false negatives. Molecular biological detection relies on nucleic acid amplification technology, offering high sensitivity and specificity, significantly improving the accuracy of early diagnosis; however, it demands sophisticated testing environments and skilled personnel. Therefore, there is a need for reliable, inexpensive, and nucleic acid-extraction-free techniques and methods for detecting Mycobacterium tuberculosis.

[0004] qPCR is a relatively mature molecular diagnostic technique. Two primers and a TaqMan probe bind to specific sites, and Taq DNA polymerase uses its polymerase and exonuclease activities to amplify nucleic acids and release fluorescent groups. The qPCR instrument can then display the amplified images in real time. It has the advantages of being rapid and highly specific. However, current qPCR testing methods are complex because clinical samples (such as bronchoalveolar lavage fluid) contain a large number of impurities, proteins, and inhibitors, requiring nucleic acid extraction to remove these interfering substances. Furthermore, strong alkalis (such as sodium hydroxide solution) are often used to digest the samples, but Taq DNA polymerase is intolerant to strong alkalis, leading to inaccurate detection. Summary of the Invention

[0005] Based on the technical problems existing in the background technology, the present invention proposes a Taq DNA polymerase mutant and its application in the detection of Mycobacterium tuberculosis. The Taq DNA polymerase mutant of the present invention can tolerate impurities in bronchoalveolar lavage fluid and bronchoalveolar lavage fluid treated with sodium hydroxide. It can directly amplify the target fragment from the sample to be tested to detect Mycobacterium tuberculosis without the need for nucleic acid purification steps, and has high sensitivity.

[0006] This invention proposes a Taq DNA polymerase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] This invention involves mutating the amino acid sequence of Taq DNA polymerase using P752I, L156I, and L376V to obtain a Taq DNA polymerase mutant that can be used to detect Mycobacterium tuberculosis and is resistant to impurities in bronchoalveolar lavage fluid and to bronchoalveolar lavage fluid treated with sodium hydroxide.

[0008] The present invention also proposes a polynucleotide that encodes the above-mentioned Taq DNA polymerase mutant.

[0009] Preferably, the DNA sequence of the polynucleotide is as shown in SEQ ID NO.2, or a sequence with homology greater than or equal to 98% to the DNA sequence shown in SEQ ID NO.2.

[0010] The present invention also proposes a recombinant expression vector containing the aforementioned polynucleotides.

[0011] The recombinant expression vector described above is obtained by effectively linking the aforementioned polynucleotide molecules to the expression vector. The expression vector is any one of a viral vector, plasmid, bacteriophage, phage particle, sclerosing particle, F sclerosing particle, or phage; more preferably, it is a plasmid, such as the pET-22b plasmid.

[0012] The present invention also proposes a genetically engineered bacterium containing the above-mentioned recombinant expression vector or having the above-mentioned polynucleotides integrated into the genome of the genetically engineered bacterium.

[0013] The host cells of the above-mentioned genetically engineered bacteria can be Escherichia coli, Agrobacterium spp., Bacillus spp., etc.; preferably Escherichia coli (such as E. coli competent cells E. coli BL21).

[0014] The present invention also proposes a method for preparing the above-mentioned Taq DNA polymerase mutant, comprising the following steps: culturing the above-mentioned genetically engineered bacteria to express the Taq DNA polymerase mutant.

[0015] The preparation method of the above-mentioned Taq DNA polymerase mutant specifically includes the following steps: cloning the DNA sequence shown in SEQ ID NO.2; constructing the DNA sequence into an expression vector to obtain a recombinant expression vector; transforming the recombinant expression vector into a host cell to obtain a genetically engineered bacterium; culturing and inducing the genetically engineered bacterium to express the Taq DNA polymerase mutant; collecting and cleaving the genetically engineered bacterium; heat-treating; centrifuging; collecting the supernatant; and purifying it sequentially using a nickel column and Heparin affinity to obtain the Taq DNA polymerase mutant.

[0016] The specific procedure for inducing the expression of the Taq DNA polymerase mutant through culture is as follows: the genetically engineered bacteria are cultured in LB liquid medium until OD... 600 When the concentration was 0.6-0.8, IPTG (isopropyl-β-D-thiogalactoside) was added to induce expression and obtain Taq DNA polymerase mutant.

[0017] The above-mentioned genetically engineered bacteria can be cultured in LB liquid medium at 37°C and 200 rpm.

[0018] The final concentration of IPTG can be 0.4 mM; it can be induced at 30℃ and 200 rpm for 12 h.

[0019] The present invention also proposes a kit, the raw materials of which include the above-mentioned Taq DNA polymerase mutant.

[0020] Preferably, the raw materials also include: primer IS-F, primer IS-R, probe IS-P, buffer solution, and nuclease-free water; The DNA sequence of primer IS-F is shown in SEQ ID NO.3; The DNA sequence of primer IS-R is shown in SEQ ID NO.4; The DNA sequence of probe IS-P is: 5'-FAM-CCTGGGCAGGGTTCGCCTACGTGG-BHQ-1-3', where FAM represents carboxyfluorescein and BHQ-1 represents a quenching group.

[0021] The primers IS-F and IS-R mentioned above can specifically target IS6110 of the Mycobacterium tuberculosis complex.

[0022] The aforementioned IS6110 is a unique insertion sequence in the genome of the Mycobacterium tuberculosis complex (MTBC). It plays a central role in the molecular diagnosis, strain identification, and epidemiological investigation of tuberculosis and is often regarded as the preferred target for molecular biological detection.

[0023] The probe IS-P is an oligonucleotide sequence containing a fluorescent reporter group and a quencher group, which specifically binds to the target sequence between primers IS-F and IS-R.

[0024] The above buffer contains deoxyribonucleoside triphosphates (dNTPs) and MgCl2; the buffer and nuclease-free water can be purchased commercially.

[0025] The above buffer solution may contain: Tris, KCl, MgCl2, Tween 20, BSA, and dNTPs.

[0026] The present invention also proposes the application of the above-mentioned Taq DNA polymerase mutant, the above-mentioned polynucleotide, the above-mentioned recombinant expression vector, the above-mentioned genetically engineered bacteria, and the above-mentioned kit in the detection of Mycobacterium tuberculosis.

[0027] The samples to be tested for Mycobacterium tuberculosis were bronchoalveolar lavage fluid or bronchoalveolar lavage fluid that had been digested with sodium hydroxide aqueous solution.

[0028] The present invention also proposes a method for detecting Mycobacterium tuberculosis, comprising the following steps: taking the sample to be tested and mixing it with the raw materials of the above-mentioned kit to obtain a reaction system, performing qPCR amplification, collecting fluorescence signals, and determining the detection results based on the fluorescence amplification curve results; The sample to be tested is bronchoalveolar lavage fluid or bronchoalveolar lavage fluid that has been digested with sodium hydroxide aqueous solution; The sodium hydroxide aqueous solution has a mass fraction of 1-5 wt%, more preferably 4 wt%; The volume ratio of sodium hydroxide aqueous solution to bronchoalveolar lavage fluid is 3-5:1; more preferably 4:1.

[0029] If the Ct value of the above fluorescence amplification curve is <38 and the amplification curve is "S" shaped, it indicates that the sample to be tested contains Mycobacterium tuberculosis.

[0030] In the above reaction system, the concentrations of primer IS-F, primer IS-R, and probe IS-P can be 0.1-100 μM.

[0031] In the above reaction system, the ratio of the volume of the sample to be tested to the total volume of all raw materials in the kit can be 1-5:23.

[0032] The above reaction system may contain: 2 μL of the sample to be tested, 7.1 μL of nuclease-free water, 0.4 μL of Taq DNA polymerase mutant, 12.5 μL of 2× buffer, 1.25 μL of primer IS-F, 1.25 μL of primer IS-R, and 0.5 μL of probe IS-P.

[0033] The final concentrations of each substance in the buffer solution in the reaction system are 50 mM Tris, 50 mM KCl, 2 mM MgCl2, 0.02% Tween 20, 0.4 mg / mL BSA, 0.2 mM dNTPs, pH=8.5, and the solvent is nuclease-free water.

[0034] The above qPCR amplification program includes: pre-denaturation, 10 cycles of landing amplification, and 40 cycles of master amplification. Pre-denaturation is performed at 94℃ for 600 s. Each cycle of landing amplification is programmed as follows: denaturation at 94℃ for 10 s, gradient annealing starting at 71℃ and decreasing by 1℃ per cycle, holding for 15 s per cycle, and then extension at 72℃ for 15 s. Each cycle of master amplification is programmed as follows: denaturation at 94℃ for 10 s, annealing at 61℃ and acquiring fluorescence signal for 30 s, and extension at 72℃ for 15 s.

[0035] The present invention describes the mutation of the amino acid sequence of Taq DNA polymerase by P752I, L156I, and L376V to obtain a Taq DNA polymerase mutant, which has high sensitivity and can be used to detect Mycobacterium tuberculosis; and can tolerate impurities in bronchoalveolar lavage fluid and can tolerate bronchoalveolar lavage fluid digested with 4 times the volume of 4wt% sodium hydroxide aqueous solution.

[0036] The Taq DNA polymerase mutant described in this invention can directly amplify the target fragment from the sample to be tested (i.e., bronchoalveolar lavage fluid or bronchoalveolar lavage fluid digested with sodium hydroxide aqueous solution) without the need for nucleic acid purification steps, and its detection sensitivity can reach 2 copies / μL.

[0037] The Taq DNA polymerase mutant described in this invention can achieve nucleic acid extraction-free amplification of Mycobacterium tuberculosis complex in bronchoalveolar lavage fluid in clinical practice, reducing detection time and cost. Attached Figure Description

[0038] Figure 1 This is a qPCR curve of Taq DNA polymerase mutant amplifying the target fragment in a test sample containing NaOH. Detailed Implementation

[0039] The technical solution of the present invention will be described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0040] Example 1 A method for preparing a Taq DNA polymerase mutant includes the following steps: The DNA sequence shown in SEQ ID NO.2 was cloned and constructed into the pET-22b plasmid. The recombinant expression vector was obtained by purification using a histidine tag. The recombinant expression vector was transformed into E. coli competent cells E. coli BL21 to obtain genetically engineered bacteria; The genetically engineered bacteria were cultured overnight in 5 mL LB broth at 37°C and 200 rpm, and then transferred to 800 mL LB broth and cultured at 37°C and 200 rpm until OD reached. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.4 mM, induce at 30℃ and 200 rpm for 12 h, then collect the cells, high pressure break them, heat treat at 80℃ for 30 min, and centrifuge to collect the supernatant. The supernatant was loaded into a Ni-IDA affinity chromatography column and washed with 5 column volumes of washing buffer (50 mM Tris-HCl, 100 mM NaCl, pH 8.0); then eluted with an imidazole-containing elution buffer to obtain eluent 1. Eluent 1 was loaded into the heparin chromatography column at a flow rate of 1 mL / min; the column was washed with buffer A (20 mM Tris-HCl, 20 mM NaCl, pH 8.0) at a flow rate of 2 mL / min; then, a NaCl gradient elution was performed with buffer A and buffer B (20 mM Tris-HCl, 1 M NaCl, pH 8.0) at a flow rate of 2 mL / min to obtain eluent 2. Then, elution buffer 2 was dialyzed with dialysis buffer (50 mM Tris-HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM β-ME, pH 8.0); the protein was then concentrated using an ultrafiltration tube to obtain the Taq DNA polymerase mutant, which was stored at -80°C.

[0041] The amino acid sequence of the above-mentioned Taq DNA polymerase mutant is shown in SEQ ID NO.1, as detailed below: MRGMLPLFEPKGRVLLVDGHHLAYRTFHALKGLTTSRGEPVQAVYGFAKSLLKALKEDGDAVIVVFDAKAPSFRHEAYGGYKAGRAPTPEDFPRQLALIKELVDLLGLARLEVPGYEADDVLASLAKKAEKEGYEVRILTADKDLYQLLSDRIHVIHPEGYLITPAWLWEKYGLRPDQWADYRALTGDESDNLPGVKGIGEKTARKLLEEWGSLEALLKNLDRLKPAIREKILAHMDDLKLSWDLAKVRTDLPLEVDFAKRREPDRERLRAFLERLEFGSLLHEFGLLESPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLVAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMIVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE.

[0042] The DNA sequence of the above Taq DNA polymerase mutant is shown as SEQ ID NO. 2, which is specifically as follows:

[0043] Example 2 A kit comprising the following ingredients: Taq DNA polymerase mutant prepared in Example 1, primers IS-F, IS-R, probe IS-P, buffer, and nuclease-free water; The DNA sequence of primer IS-F is shown in SEQ ID NO.3, specifically: GCAGACCTCACCTATGTGTCG; The DNA sequence of primer IS-R is shown in SEQ ID NO.4, specifically: CAGATGGCTTGCTCGATCG; The DNA sequence of probe IS-P is: 5'-FAM-CCTGGGCAGGGTTCGCCTACGTGG-BHQ-1-3', where FAM represents carboxyfluorescein and BHQ-1 represents a quenching group; The buffer solution contains: Tris, KCl, MgCl2, Tween 20, BSA, and dNTPs.

[0044] The above kit is used to detect Mycobacterium tuberculosis. The specific steps are as follows: Using IS6110 cDNA as a template, 4 volumes of 4wt% NaOH aqueous solution were added to obtain test samples with IS6110 cDNA concentrations of 2×10^8 copies / mL, 2×10^5 copies / mL, 2×10^4 copies / mL and 2×10^3 copies / mL, respectively. Take each sample to be tested, prepare each reaction system according to Table 1, and then perform qPCR amplification. The amplification program is as follows: denaturation at 95℃ for 3 min, followed by 40 cycles: 95℃ for 10 s, 60℃ for 30 s. After the reaction, analyze the amplification curves. The results are as follows: Figure 1 As shown.

[0045] Table 1 25μL reaction system

[0046] The final concentrations of each substance in the buffer solution in the reaction system are 50 mM Tris, 50 mM KCl, 2 mM MgCl2, 0.02% Tween 20, 0.4 mg / mL BSA, 0.2 mM dNTPs, pH=8.5, and the solvent is nuclease-free water.

[0047] Figure 1 This is a qPCR curve of Taq DNA polymerase mutant amplifying the target fragment in a test sample containing NaOH.

[0048] Depend on Figure 1 It can be seen that the Taq DNA polymerase mutant described in this invention can be effectively amplified when the IS6110 cDNA concentration is 2×10^8 copies / mL, 2×10^5 copies / mL, 2×10^4 copies / mL and 2×10^3 copies / mL, with a reaction sensitivity of up to 4 copies / reaction. This indicates that even if the sample contains NaOH, the Taq DNA polymerase mutant has high sensitivity. The Taq DNA polymerase mutant described in this invention is resistant to sodium hydroxide and has good sensitivity to Mycobacterium tuberculosis complex.

[0049] Example 3 According to Table 1, take Taq DNA polymerase mutant, primer IS-F, primer IS-R, probe IS-P, buffer buffer and nuclease-free water, mix them and add them to an 8-tube string and store at -20℃. Take bronchoalveolar lavage fluid samples from tuberculosis patients, add 4 times the volume of 4wt% NaOH aqueous solution for digestion treatment, and obtain the sample to be tested; Add 2 μL of the sample to be tested to a thawed 8-tube and perform qPCR amplification. The qPCR amplification program includes: pre-denaturation, 10 cycles of falling amplification, and 40 cycles of master amplification. Pre-denaturation is performed at 94℃ for 600 s. Each cycle of falling amplification is programmed as follows: denaturation at 94℃ for 10 s, gradient annealing starting at 71℃, decreasing by 1℃ per cycle, holding for 15 s per cycle, and then extension at 72℃ for 15 s. Each cycle of master amplification is programmed as follows: denaturation at 94℃ for 10 s, annealing at 61℃ and acquiring fluorescence signal for 30 s, and extension at 72℃ for 15 s.

[0050] A total of 31 bronchoalveolar lavage fluid samples were collected from clinical tuberculosis patients. The bacterial load was verified by the gold standard Xpert and classified into four levels: high, medium, low, very low, and trace. The Ct values ​​of the corresponding samples were detected by the kit of this invention, as shown in Table 2.

[0051] Table 2. qPCR detection results of Taq DNA polymerase mutants on bronchoalveolar lavage fluid samples.

[0052] After summarizing the results, the Xpert verification results were compared with the detection results of the kit described in this invention (i.e., the results in Table 2), and the results are shown in Table 3.

[0053] Table 3 Comparison of Xpert validation results with the reagent kit of this invention

[0054] As can be seen from Table 3, the amplification and detection results of the kit containing the Taq DNA polymerase mutant are generally consistent with the Xpert results; and the kit of this invention does not require nucleic acid extraction, making the operation simple.

[0055] In summary, the Taq DNA polymerase mutant described in this invention exhibits high sensitivity to Mycobacterium tuberculosis complex and is resistant to NaOH; the kit described in this invention does not require nucleic acid extraction, is easy to operate, and its overall clinical detection results are consistent with Xpert, and it is compatible with existing PCR instruments, demonstrating great potential for direct sample amplification.

[0056] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A Taq DNA polymerase mutant, characterized in that, The amino acid sequence of the Taq DNA polymerase mutant is shown in SEQ ID NO.

1.

2. A polynucleotide, characterized in that, The polynucleotide encodes the Taq DNA polymerase mutant of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The DNA sequence of the polynucleotide is shown in SEQ ID NO.

2.

4. The polynucleotide according to claim 2, characterized in that, The polynucleotide DNA sequence is a sequence with 98% or greater homology to the DNA sequence shown in SEQ ID NO.

2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide as described in any one of claims 2-4.

6. A genetically engineered bacterium, characterized in that, The genetically engineered bacteria contain the recombinant expression vector of claim 5 or the genome of the genetically engineered bacteria is integrated with the polynucleotide of any one of claims 2-4.

7. A method for preparing the Taq DNA polymerase mutant as described in claim 1, characterized in that, The method includes the following steps: culturing the genetically engineered bacteria of claim 6 to express the Taq DNA polymerase mutant.

8. A reagent kit, characterized in that, Its raw materials include: the Taq DNA polymerase mutant as described in claim 1.

9. The reagent kit according to claim 8, characterized in that, Its raw materials also include: primer IS-F, primer IS-R, probe IS-P, buffer, and nuclease-free water; The DNA sequence of primer IS-F is shown in SEQ ID NO.3; The DNA sequence of primer IS-R is shown in SEQ ID NO.4; The DNA sequence of probe IS-P is: 5'-FAM-CCTGGGCAGGGTTCGCCTACGTGG-BHQ-1-3', where FAM represents carboxyfluorescein and BHQ-1 represents a quenching group.

10. The use of the Taq DNA polymerase mutant of claim 1, the polynucleotide of any one of claims 2-4, the recombinant expression vector of claim 5, and the genetically engineered bacteria of claim 6 in the preparation of a kit for detecting Mycobacterium tuberculosis.

11. A method for detecting Mycobacterium tuberculosis for non-diagnostic purposes, characterized in that, The process includes the following steps: mixing the sample to be tested with the raw materials of the kit described in claim 8 or 9 to obtain a reaction system, performing qPCR amplification, collecting fluorescence signals, and determining the detection results based on the fluorescence amplification curve results.

12. The method for detecting Mycobacterium tuberculosis for non-diagnostic purposes according to claim 11, characterized in that, The sample to be tested was bronchoalveolar lavage fluid.

13. The method for detecting Mycobacterium tuberculosis for non-diagnostic purposes according to claim 11, characterized in that, The sample to be tested was bronchoalveolar lavage fluid that had been digested with sodium hydroxide aqueous solution.

14. The method for detecting Mycobacterium tuberculosis for non-diagnostic purposes according to claim 13, characterized in that, The sodium hydroxide aqueous solution has a mass fraction of 1-5 wt%.

15. The method for detecting Mycobacterium tuberculosis for non-diagnostic purposes according to claim 13, characterized in that, The volume ratio of sodium hydroxide aqueous solution to bronchoalveolar lavage fluid is 3-5:1.

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