SLAMP primer group and kit for detecting stenotrophomonas maltophilia

Abstract

The invention relates to an SLAMP primer group and a kit for detecting stenotrophomonas maltophilia, and relates to the technical field of biological detection. The invention specifically discloses an SLAMP (Single Loop-Mediated Isothermal Amplification) primer group for detecting stenotrophomonas maltophilia, and the SLAMP primer group is a primer group for amplifying a conserved region fragment of the stenotrophomonas maltophilia, and is a primer pair STM-SF / STM-SR; wherein the nucleotide sequence of the STM-SF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the STM-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 stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia. Background Art

[0002] Stenotrophomonas maltophilia belongs to the Xanthomonadaceae family of the order Xanthomonadales. It is widely found in water, soil, and animals. It is a conditional pathogen. With the widespread and high-dose application of clinical antibiotics and immunosuppression, its isolation rate has been on the rise among non-fermenting bacteria. Because the bacteria are resistant to multiple antibiotics, it brings great difficulties to clinical treatment. Stenotrophomonas maltophilia can cause hospital-acquired pneumonia, bloodstream infection, abdominal infection, central nervous system infection, urinary system infection, skin and soft tissue infection, etc.

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

[0004] Among the existing detection methods, PCR, DNA probe technology, etc. Although these technologies can quickly detect the number of Stenotrophomonas maltophilia 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 method for detecting Stenotrophomonas maltophilia. Summary of the invention

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

[0006] Another object of the present application is to provide a kit for detecting Stenotrophomonas maltophilia, 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 Stenotrophomonas maltophilia, wherein the SLAMP primer set is a primer pair STM-SF / STM-SR;

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

[0010] On the other hand, the present application provides a kit for detecting Stenotrophomonas maltophilia, 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 developed SLAMP method (Chinese patent application number: 202411655233.5) 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 and reduce the chance of sample contamination, and is suitable for the rapid diagnosis of Stenotrophomonas maltophilia. 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 Stenotrophomonas maltophilia (GenBank: CP104292.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of 2 primers, including an intra-sequence primer pair (STM-SF / STM-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 Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia. 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 Stenotrophomonas maltophilia, wherein the SLAMP primer set is a primer pair STM-SF / STM-SR;

[0024] The nucleotide sequence of STM-SF is shown in SEQ ID NO.1, and the nucleotide sequence of STM-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]

[0028] A kit for detecting Stenotrophomonas maltophilia, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0029] In some embodiments of the present application, in the above SLAMP primer set, the concentrations of primer STM-SF and primer STM-SR are both 1-2 μM.

[0030] 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.

[0031] In some embodiments of the present application, the color developer is selected from SYBR green I, Eva green, hydroxynaphthol blue, and chrome black T.

[0032] In some embodiments of the present application, the SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH4)2SO4, MgSO4 and TritonX-100.

[0033] 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 STM-SF and 1-2μM primer STM-SR; the reaction solvent is ultrapure water.

[0034] In the present application, the method for detecting Stenotrophomonas maltophilia by the kit comprises the following steps:

[0035] Step 1: Mix the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0036] Step 2: react the amplification reaction solution prepared above at 60-65° C. for 20-80 min, and determine whether the sample contains Stenotrophomonas maltophilia based on the color development result.

[0037] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0038] Example 1

[0039] In this example, Eva Green was used to verify the amplification reaction of the conserved sequence DNA fragment of Stenotrophomonas maltophilia.

[0040] 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.

[0041] 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% Triton X-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), primers: 1600 nM STM-SF (shown in SEQ ID NO.1) and 1600 nM STM-SR (shown in SEQ ID NO.2);

[0042] Target: Extracted genomic DNA from Stenotrophomonas maltophilia (the target fragment sequence is shown in SEQ ID NO.3);

[0043] At the same time, two control groups with and without targets were set up.

[0044] 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 Stenotrophomonas maltophilia, and the application of fluorescence detection therein can achieve the purpose of real-time monitoring, and the result can be judged in advance by observing the real-time amplification curve.

[0045] Example 2

[0046] In this example, Eva Green was used to verify the amplification reaction of Stenotrophomonas maltophilia SLAMP on genomes extracted from various bacteria; the method was the same as that in Example 1.

[0047] 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 STM-SF (shown in SEQ ID NO. 1) and 1600 nM STM-SR (shown in SEQ ID NO. 2);

[0048] Target nucleic acid 1: genomic DNA extracted from Stenotrophomonas maltophilia (the target fragment sequence is shown in SEQ ID NO. 3), set as a positive control;

[0049] Target nucleic acid 2: Genomic DNA extracted from Staphylococcus aureus, set as negative control;

[0050] Target nucleic acid 3: Genomic DNA extracted from Haemophilus influenzae, set as negative control;

[0051] Target nucleic acid 4: Shigella extracted genomic DNA, set as negative control;

[0052] Target nucleic acid 5: genomic DNA extracted from Listeria monocytogenes, set as negative control;

[0053] A non-target control group was also set up.

[0054] 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 showed that the primer set provided by the present invention can effectively distinguish Stenotrophomonas maltophilia from Staphylococcus aureus, Haemophilus influenzae, Shigella, and Listeria monocytogenes, further illustrating the specificity of the SLAMP primer set provided by the present invention.

[0055] Example 3

[0056] In this example, hydroxynaphthol blue (HNB) was used to monitor the endpoint of the SLAMP amplification reaction of Stenotrophomonas maltophilia;

[0057] 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.

[0058] The composition of the reaction solution for SLAMP amplification of Stenotrophomonas maltophilia using hydroxynaphthol blue (HNB) is shown below.

[0059] 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 STM-SF (shown in SEQ ID NO. 1) and 1600 nM STM-SR (shown in SEQ ID NO. 2);

[0060] Target nucleic acid: genomic DNA extracted from Stenotrophomonas maltophilia (the sequence of the target fragment is shown in SEQ ID NO.3).

[0061] 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.

[0062] In summary, the SLAMP primer set and kit for detecting Stenotrophomonas maltophilia in the embodiments of the present application have the following advantages: 1. The present application designs multiple primer sets by targeting the specific conserved region of Stenotrophomonas maltophilia (GenBank: CP104292.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of 2 primers, including an intra-sequence primer pair (STM-SF / STM-SR) which are respectively the upstream and downstream external primers. The primer set provided by the present application has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Stenotrophomonas maltophilia contained in the sample to be tested.

[0063] 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.

[0064] 3. The method or kit provided in this application can complete the rapid and accurate detection of Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia.

[0065] 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 Stenotrophomonas maltophilia, characterized in that: The SLAMP primer set is a primer pair STM-SF / STM-SR; Among them, the nucleotide sequence of STM-SF is shown in SEQ ID NO.1, and the nucleotide sequence of STM-SR is shown in SEQ ID NO.

2.

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

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

4. A kit for detecting Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia 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 Stenotrophomonas maltophilia according to claim 4, characterized in that: The SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH4)2SO4, MgSO4 and Triton X-100.

7. A kit for detecting Stenotrophomonas maltophilia according to claim 4, characterized in that: The reaction system of the kit is composed of: 2-50mM Tris-HCl pH 8.8, 2-20mM KCl, 2-20mM (NH4) 2SO4, 2-20mM MgSO4, 0.1-0.5% TritonX-100, 0.2-1M betaine, 1-1.6mM dNTP, 5-10U Bst DNA polymerase, 100-150μmol / L color developer, 1-2μM primer STM-SF and 1-2μM primer STM-SR; the reaction solvent is ultrapure water.

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