A SLAMP primer set and kit for detecting Haemophilus influenzae

Abstract

The present application proposes a SLAMP primer set and a kit for detecting Haemophilus influenzae, and relates to the field of biological detection technology. The SLAMP primer set is a primer set for amplifying a conserved region fragment of Haemophilus influenzae, which is a primer pair HI‑SF / HI‑SR; wherein the nucleotide sequence of HI‑SF is shown in SEQ ID NO.1, and the nucleotide sequence of HI‑SR is shown in SEQ ID NO.2. The kit provided in the present application and the use of the kit can complete the rapid and accurate detection of Haemophilus influenzae without the need for expensive instruments or complicated operations. The visualization kit provided by the present invention can sensitively and specifically detect Haemophilus influenzae infection, is easy to operate, and can be used as primary health prevention and pathogen screening at sites with limited conditions.

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 Haemophilus influenzae. Background Art

[0002] Haemophilus influenzae (HI) is a Gram-negative bacillus and one of the main pathogens of respiratory tract infection in children. In addition to causing respiratory tract lesions, it can also cause meningitis, sepsis, conjunctivitis, female genital tract infection, cellulitis, etc. It is not only a common pathogen of infection in infants and young children, but also one of the pathogens of infection in adults.

[0003] Existing detection methods mainly include PCR, DNA probe technology, etc. Although these technologies can quickly detect the total or pathogenic Haemophilus influenzae 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 Haemophilus influenzae detection method. Summary of the invention

[0004] The purpose of the present application is to provide a SLAMP primer set for detecting Haemophilus influenzae, which has the advantages of high sensitivity and strong specificity.

[0005] Another object of the present application is to provide a kit for detecting Haemophilus influenzae, which has fast detection speed, high accuracy and is easy to use.

[0006] In order to solve the above technical problems, the technical solution adopted in this application is:

[0007] In one aspect, the present application provides a SLAMP primer set for detecting Haemophilus influenzae, wherein the SLAMP primer set is a primer pair HI-SF / R-SR;

[0008] Among them, the nucleotide sequence of HI-SF is shown in SEQ ID NO.1, and the nucleotide sequence of HI-SR is shown in SEQ ID NO.2.

[0009] On the other hand, the present application provides a kit for detecting Haemophilus influenzae, wherein the reaction system of the kit includes the above-mentioned SLAMP primer set.

[0010] Short loop mediated isothermal amplification of nucleic acids (SLAMP) is a newly developed nucleic acid amplification technology under constant temperature conditions (Chinese patent application number: 202411655233.5) based on the CDA technology (Chinese patent authorization number: ZL202110473121.8) and the Japanese LAMP nucleic acid amplification method developed by the inventor team in the early stage. This method mainly uses two different specific primers to identify the specific region 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 and much simpler operation than traditional PCR and culture methods. It can be accurately completed without professionals, and the amplification results can be combined with the metal ion indicator hydroxynaphthol blue (HNB) to observe the color change by naked eye to realize the judgment of the results, making it suitable for primary medical institutions and local inspection and quarantine departments. Moreover, SLAMP can greatly shorten the operation time and reduce the chance of sample contamination, making it suitable for rapid diagnosis of Haemophilus influenzae. In addition, the key looping primer used in SLAMP is about 30 bp, which is shorter than the 40 bp looping primer in LAMP. The design of the related primers is extremely simple, requiring only simple rearrangement, which will greatly save design time and development costs.

[0011] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0012] 1. The present application designs multiple primer sets based on the specific conserved region of Haemophilus influenzae (GenBank: CP043770.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of two primers, including an intrasequence primer pair (HI-SF / HI-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 Haemophilus influenzae contained in the sample to be tested.

[0013] 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.

[0014] 3. The method or kit provided in this application can complete the rapid and accurate detection of Haemophilus influenzae 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 Haemophilus influenzae. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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.

[0016] Figure 1 This is a graph showing the change in fluorescence intensity versus reaction time in Example 1 of the present application.

[0017] Figure 2 This is a graph showing the change in fluorescence intensity versus reaction time in Example 2 of the present application.

[0018] 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

[0019] 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.

[0020] 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.

[0021] A SLAMP primer set for detecting Haemophilus influenzae, wherein the SLAMP primer set is a primer pair HI-SF / R-SR;

[0022] The nucleotide sequence of HI-SF is shown in SEQ ID NO.1, and the nucleotide sequence of HI-SR is shown in SEQ ID NO.2. The specific primer nucleotide sequences are shown in Table 1:

[0023] Table 1 Primer sequences

[0024]

[0025] A kit for detecting Haemophilus influenzae, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0026] In some embodiments of the present application, in the above SLAMP primer set, the concentrations of primers HI-SF and HI-SR are both 1-2 μM.

[0027] 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.

[0028] 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.

[0029] In some embodiments of the present application, the SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH 4 ) 2 SO 4 MgSO 4 and TritonX-100.

[0030] In some embodiments of the present application, the reaction system of the above kit is composed of: 2-50 mM Tris-HCl pH 8.8, 2-20 mM KCl, 2-20 mM (NH 4 ) 2 SO 4 , 2~20mM MgSO 4 , 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 HI-SF and 1-2μM primer HI-SR; the reaction solvent is ultrapure water.

[0031] In the present application, the method for detecting Haemophilus influenzae by using the kit comprises the following steps:

[0032] Step 1: Mix the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0033] Step 2: react the amplification reaction solution prepared above at 60-65°C for 20-80 minutes, and determine whether the sample contains Haemophilus influenzae based on the color development result.

[0034] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0035] Example 1

[0036] In this example, Eva Green was used to verify the amplification reaction of the conserved sequence DNA fragment of Haemophilus influenzae.

[0037] 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.

[0038] The reaction solution was composed as follows (add ddH 2 (0 to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), and primers: 1600nM HI-SF (shown in SEQ ID NO.1) and 1600nM HI-SR (shown in SEQ ID NO.2);

[0039] Target nucleic acid: genomic DNA extracted from Haemophilus influenzae (sequence shown in SEQ ID NO.3);

[0040] A non-target control group was also set up.

[0041] 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 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 Haemophilus influenzae, 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.

[0042] Example 2

[0043] This example uses Eva Green to verify the amplification reaction of Haemophilus influenzae SLAMP on genomes extracted from various bacteria. The method is the same as that of Example 1.

[0044] The reaction solution was composed as follows (add ddH 2 (0 to 25 μL): 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 1◇Eva Green (Biotum), and primers: 1600nM HI-SF (shown in SEQ ID NO.1), 1600nM HI-SR (shown in SEQ ID NO.2);

[0045] Target nucleic acid 1: genomic DNA extracted from Haemophilus influenzae (sequence shown in SEQ ID NO.3);

[0046] Target nucleic acid 2: Genomic DNA extracted from Haemophilus influenzae, set as a positive control;

[0047] Target nucleic acid 3: Genomic DNA extracted from Staphylococcus aureus, set as negative control;

[0048] Target nucleic acid 4: Genomic DNA extracted from Haemophilus influenzae, set as negative control;

[0049] Target nucleic acid 5: genomic DNA extracted from Escherichia coli, set as negative control;

[0050] A non-target control group was also set up.

[0051] 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 Haemophilus influenzae from Staphylococcus aureus, Haemophilus influenzae, and Escherichia coli, further illustrating the specificity of the SLAMP primer set provided by the present invention.

[0052] Example 3

[0053] In this example, hydroxynaphthol blue (HNB) was used to monitor the endpoint of the Haemophilus influenzae SLAMP amplification reaction.

[0054] 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.

[0055] The composition of the reaction solution for SLAMP amplification of Haemophilus influenzae using hydroxynaphthol blue (HNB) is shown below.

[0056] The reaction solution was composed as follows, and ddH 2 O to 25 μL: 20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% TritonX-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 120μM HNB and primers: 1600nM HI-SF (shown in SEQ ID NO.1), 1600nM HI-SR (shown in SEQ ID NO.2).

[0057] Target nucleic acid: Genomic DNA extracted from Haemophilus influenzae (sequence shown in SEQ ID NO.3).

[0058] 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.

[0059] In summary, the SLAMP primer set and kit for detecting Haemophilus influenzae 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 Haemophilus influenzae (GenBank: CP043770.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of 2 primers, including an intra-sequence primer pair (HI-SF / HI-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 Haemophilus influenzae contained in the sample to be tested.

[0060] 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.

[0061] 3. The method or kit provided in this application can complete the rapid and accurate detection of Haemophilus influenzae 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 Haemophilus influenzae.

[0062] 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 Haemophilus influenzae, characterized in that: The SLAMP primer set is a primer pair HI-SF / R-SR; The nucleotide sequence of HI-SF is shown in SEQ ID NO.1, and the nucleotide sequence of HI-SR is shown in SEQ ID NO.

2.

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

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

4. A kit for detecting Haemophilus influenzae 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 Haemophilus influenzae 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 Haemophilus influenzae 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 Haemophilus influenzae 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 HI-SF and 1-2 μM primer HI-SR; the reaction solvent is ultrapure water.

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