SLAMP primer group and kit for detecting mycobacterium tuberculosis

Abstract

The invention provides an SLAMP primer group and a kit for detecting mycobacterium tuberculosis, and relates to the technical field of biological detection. The invention specifically discloses an SLAMP primer group and a kit for detecting mycobacterium tuberculosis. The SLAMP primer group is a primer group aiming at the amplification of a conserved region fragment of mycobacterium tuberculosis, and is a primer pair MTB-SF / MTB-SR; wherein the nucleotide sequence of the MTB-SF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the MTB-SR is as shown in SEQ ID NO. 2. According to the method or the visual kit provided by the invention, rapid and accurate detection of the mycobacterium tuberculosis can be completed without expensive instruments or complicated operation, so that the method or the visual kit is suitable for on-site rapid detection in a scene with a low professional degree.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a SLAMP primer set and a kit for detecting Mycobacterium tuberculosis. Background Art

[0002] Mycobacterium tuberculosis (MTB) is the pathogen that causes tuberculosis. The bacterium can invade various organs throughout the body, but most commonly causes pulmonary tuberculosis. Tuberculosis is an ancient disease that is widely distributed around the world and is the leading cause of death among bacterial infectious diseases.

[0003] The classic method for detecting Mycobacterium tuberculosis is isolation and culture, which involves inoculating neutralized bacterial collection materials into solid culture medium, adding a rubber stopper to the mouth of the vessel and culturing at 37°C, and observing once a week. However, Mycobacterium tuberculosis grows slowly, and generally takes 2 to 4 weeks to grow into colonies visible to the naked eye. Other relatively fast and accurate methods include RT-PCR-based detection of amplification signals for Mycobacterium tuberculosis DNA, which can detect positive results with only a few bacteria per milliliter, and the results are obtained in 1 to 2 days.

[0004] Among the existing detection methods, PCR, DNA probe technology, etc. Although these technologies can quickly detect the number of Mycobacterium tuberculosis in samples, these methods rely on special and expensive equipment, and their operation process is complicated and time-consuming (including DNA separation, hybridization, colorimetry, and synthesis of probes and primers, etc.). Only technicians who are proficient in their operation skills can complete the test. Therefore, the above methods are not suitable for rapid on-site detection, and it is urgent to establish a rapid, sensitive, and specific Mycobacterium tuberculosis detection method. Summary of the invention

[0005] The purpose of the present application is to provide a SLAMP primer set for detecting Mycobacterium tuberculosis, which has the advantages of high sensitivity and strong specificity.

[0006] Another object of the present application is to provide a kit for detecting Mycobacterium tuberculosis, which has fast detection speed, high accuracy and is easy to use.

[0007] In order to solve the above technical problems, the technical solution adopted in this application is:

[0008] In one aspect, the present application provides a SLAMP primer set for detecting Mycobacterium tuberculosis, wherein the SLAMP primer set is a primer pair MTB-SF / R-SR;

[0009] Among them, the nucleotide sequence of MTB-SF is shown in SEQ ID NO.1, and the nucleotide sequence of MTB-SR is shown in SEQ ID NO.2.

[0010] On the other hand, the present application provides a kit for detecting Mycobacterium tuberculosis, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0011] Short loop mediated isothermal amplification of nucleic acids (SLAMP) is a nucleic acid amplification technology under constant temperature conditions (Chinese patent application number: 202411655233.5) recently improved and developed by the inventor team based on the CDA technology (Chinese patent authorization number: ZL202110473121.8) and the Japanese LAMP nucleic acid amplification method developed in the early stage.

[0012] The SLAMP method (Chinese patent application number: 202411655233.5) developed by the team in the early stage mainly uses two different specific primers to identify specific regions of the target gene and perform amplification reactions under constant temperature conditions. Compared with conventional gene detection methods (such as PCR, etc.), the SLAMP reaction can be completed in a constant temperature water bath, with low instrumentation requirements, much simpler than traditional PCR and culture methods, and can be accurately completed without professionals. The amplification results can be combined with the metal ion indicator hydroxynaphthol blue (HNB), and the color changes can be observed by naked eye to achieve the judgment of the results, making it suitable for primary medical institutions and local inspection and quarantine departments. In addition, SLAMP can also greatly shorten the operation time, reduce the chance of sample contamination, and is suitable for the rapid diagnosis of Mycobacterium tuberculosis. In addition, the key looping primer used in SLAMP is about 30bp, which is shorter than the 40bp looping primer in LAMP. The design of the relevant primers is extremely simple, and only simple rearrangement is required, which will greatly save design time and development costs.

[0013] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0014] 1. The present application designs multiple primer sets for the specific conserved region of Mycobacterium tuberculosis (GenBank: CP136197.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of two primers, including an intrasequence primer pair (MTB-SF / MTB-SR) as upstream and downstream external primers, respectively. The primer set provided by the present application has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Mycobacterium tuberculosis contained in the sample to be tested.

[0015] 2. Since the primer set provided in this application has extremely high specificity, the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation.

[0016] 3. The method or kit provided in this application can complete the rapid and accurate detection of Mycobacterium tuberculosis without expensive instruments or complicated operations. Therefore, it is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided in this application will greatly facilitate on-site detection and can achieve rapid and accurate detection of Mycobacterium tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application 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 paying creative work.

[0018] Figure 1 This is a graph showing the change in fluorescence intensity versus reaction time in Example 1 of the present application.

[0019] Figure 2 This is a graph showing the change in fluorescence intensity versus reaction time in Example 2 of the present application.

[0020] Figure 3 This is an endpoint monitoring chart based on color change of the reaction using HNB in ​​Example 3 of the present application. DETAILED DESCRIPTION

[0021] The technical scheme of the present invention is described in detail below in conjunction with the examples. The experimental methods used in the following examples are conventional methods unless otherwise specified, and can be specifically performed with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kit and product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0022] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to specific embodiments.

[0023] A SLAMP primer set for detecting Mycobacterium tuberculosis, wherein the SLAMP primer set is a primer pair MTB-SF / R-SR;

[0024] The nucleotide sequence of MTB-SF is shown in SEQ ID NO.1, and the nucleotide sequence of MTB-SR is shown in SEQ ID NO.2. The specific primer nucleotide sequences are shown in Table 1:

[0025] Table 1 Primer sequences

[0026]

[0027] A kit for detecting Mycobacterium tuberculosis, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0028] In some embodiments of the present application, in the above SLAMP primer set, the concentrations of primer MTB-SF and primer MTB-SR are both 1-2 μM.

[0029] In some embodiments of the present application, the reaction system of the above-mentioned kit further includes Bst polymerase, SLAMP reaction buffer, reaction solvent and color developer.

[0030] In some embodiments of the present application, the color developer is selected from one of SYBR green I, Eva green, hydroxynaphthol blue and chrome black T.

[0031] In some embodiments of the present application, the SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH4)2SO4, MgSO4 and TritonX-100.

[0032] In some embodiments of the present application, the composition of the reaction system of the above-mentioned kit is: 2-50mM Tris-HCl pH 8.8, 2-20mM KCl, 2-20mM (NH4)2SO4, 2-20mM MgSO4, 0.1-0.5% Triton X-100, 0.2-1M betaine, 1-1.6mM dNTP, 5-10U Bst DNA polymerase, 100-150μmol / L color developer, 1-2μM primer MTB-SF and 1-2μM primer MTB-SR; the reaction solvent is ultrapure water.

[0033] In the present application, the method for detecting Mycobacterium tuberculosis using the kit comprises the following steps:

[0034] Step 1: Mix the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0035] Step 2: react the amplification reaction solution prepared above at 60-65° C. for 20-80 min, and determine whether the sample contains Mycobacterium tuberculosis based on the color development result.

[0036] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0037] Example 1

[0038] In this example, Eva Green was used to verify the amplification reaction of the conserved sequence DNA fragment of Mycobacterium tuberculosis.

[0039] Eva Green is similar to SYBR Green I. It is a dye with a green excitation wavelength that binds to all DNA double helix minor groove regions. Its inhibitory effect on nucleic acid amplification reactions such as PCR is much less than that of the latter. In the free state, EvaGreen emits weak fluorescence, but once it binds to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of Eva Green is related to the amount of double-stranded DNA, and the amount of double-stranded DNA in the nucleic acid amplification system can be detected based on the intensity of the fluorescence signal.

[0040] The reaction solution was composed as follows (add ddH2O to make up to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 14 mM MgSO4, 0.1% TritonX-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 1×Eva Green (Biotum), and primers: 1600 nM MTB-SF (shown in SEQ ID NO.1), 1600 nM MTB-SR (shown in SEQ ID NO.2).

[0041] Target: genomic DNA extracted from Mycobacterium tuberculosis (the target fragment sequence is shown in SEQ ID NO.3);

[0042] At the same time, two control groups with and without targets were set up.

[0043] The SLAN 96 real time PCR reaction temperature was set to 63°C and the reaction time was 60 min. The curve of fluorescence intensity versus reaction time is shown in Figure 1 shown. Figure 1 The results show that the primer set provided by the present invention can realize the rapid amplification of the genome extracted from Mycobacterium tuberculosis, and the application of fluorescence detection therein can realize the purpose of real-time monitoring, and the result can be judged in advance by observing the real-time amplification curve.

[0044] Example 2

[0045] In this example, Eva Green was used to verify the amplification reaction of Mycobacterium tuberculosis SLAMP on genomes extracted from various bacteria; the method was the same as that in Example 1.

[0046] The reaction solution was composed as follows (add ddH2O to make up to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 14 mM MgSO4, 0.1% TritonX-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), and primers: 1600 nM MTB-SF (shown in SEQ ID NO. 1) and 1600 nM MTB-SR (shown in SEQ ID NO. 2);

[0047] Target nucleic acid 1: genomic DNA extracted from Mycobacterium tuberculosis (the target fragment sequence is shown in SEQ ID NO. 3), set as a positive control;

[0048] Target nucleic acid 2: Genomic DNA extracted from Haemophilus influenzae, set as negative control;

[0049] Target nucleic acid 3: genomic DNA extracted from Listeria monocytogenes, set as negative control;

[0050] Target nucleic acid 4: Salmonella extracted genomic DNA, set as negative control;

[0051] Target nucleic acid 5: Genomic DNA extracted from Staphylococcus aureus, set as negative control;

[0052] A non-target control group was also set up.

[0053] The SLAN 96 real time PCR reaction temperature was set to 61°C and the reaction time was 60 min. The curve of fluorescence intensity versus reaction time is shown in Figure 2 shown. Figure 2 The results show that the primer set provided by the present invention can effectively distinguish Mycobacterium tuberculosis from Haemophilus influenzae, Listeria monocytogenes, Salmonella, and Staphylococcus aureus, further illustrating the specificity of the SLAMP primer set provided by the present invention.

[0054] Example 3

[0055] In this example, hydroxynaphthol blue (HNB) was used to monitor the endpoint of the SLAMP amplification reaction of Mycobacterium tuberculosis.

[0056] Hydroxynaphthol blue (HNB) is a metal ion indicator that displays different indicator colors to judge the results according to the changes in the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate in the reaction.

[0057] The composition of the reaction solution for SLAMP amplification of Mycobacterium tuberculosis using hydroxynaphthol blue (HNB) is shown below.

[0058] The reaction solution was composed as follows, and the rest was added with ddH2O to 25 μL: 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 14 mM MgSO4, 0.1% TritonX-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 120 μM HNB, and primers: 1600 nM MTB-SF (shown in SEQ ID NO. 1) and 1600 nM MTB-SR (shown in SEQ ID NO. 2);

[0059] Target nucleic acid: genomic DNA extracted from Mycobacterium tuberculosis (the sequence of the target fragment is shown in SEQ ID NO.3).

[0060] The amplification reaction was set up with 8 positive controls and 8 negative controls. The water bath reaction temperature was set to 61°C and the reaction time was 60 min. Figure 3 As shown, violet indicates negative, and sky blue indicates positive. The experimental results show that the application of HNB can realize the judgment of the reaction result by observing the color change with naked eyes.

[0061] In summary, the SLAMP primer set and kit for detecting Mycobacterium tuberculosis in the embodiment of the present application have the following advantages: 1. The present application designs multiple primer sets by the specific conserved region of Mycobacterium tuberculosis (GenBank: CP136197.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of 2 primers, including an intra-sequence primer pair (MTB-SF / MTB-SR) as upstream and downstream external primers, respectively. The primer set provided by the present application has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Mycobacterium tuberculosis contained in the sample to be tested.

[0062] 2. Since the primer set provided in this application has extremely high specificity, the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation.

[0063] 3. The method or kit provided in this application can complete the rapid and accurate detection of Mycobacterium tuberculosis without expensive instruments or complicated operations. Therefore, it is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided in this application will greatly facilitate on-site detection and can achieve rapid and accurate detection of Mycobacterium tuberculosis.

[0064] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present application.

Claims

1. A SLAMP primer set for detecting Mycobacterium tuberculosis, characterized in that: The SLAMP primer set is a primer pair MTB-SF / R-SR; Among them, the nucleotide sequence of MTB-SF is shown in SEQ ID NO.1, and the nucleotide sequence of MTB-SR is shown in SEQ ID NO.

2.

2. A kit for detecting Mycobacterium tuberculosis, characterized in that: The reaction system of the kit comprises the SLAMP primer set according to claim 1.

3. A kit for detecting Mycobacterium tuberculosis according to claim 2, characterized in that: In the SLAMP primer set, the concentrations of primer MTB-SF and primer MTB-SR are both 1 to 2 μM.

4. A kit for detecting Mycobacterium tuberculosis according to claim 2, characterized in that: The reaction system of the kit also includes Bst polymerase, SLAMP reaction buffer, reaction solvent and color developer.

5. A kit for detecting Mycobacterium tuberculosis according to claim 4, characterized in that: The color developer is selected from one of SYBR green I, Eva green, hydroxynaphthol blue and chrome black T.

6. A kit for detecting Mycobacterium tuberculosis according to claim 4, characterized in that: The SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH4)2SO4, MgSO4 and TritonX-100.

7. A kit for detecting Mycobacterium tuberculosis according to claim 2, characterized in that: The reaction system of the kit comprises: 2-50 mM Tris-HCl pH 8.8, 2-20 mM KCl, 2-20 mM (NH4)2SO4, 2-20 mM MgSO4, 0.1-0.5% Triton X-100, 0.2-1 M betaine, 1-1.6 mM dNTP, 5-10 U Bst DNA polymerase, 100-150 μmol / L color developer, 1-2 μM primer MTB-SF and 1-2 μM primer MTB-SR; and the reaction solvent is ultrapure water.

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