SLAMP primer group and kit for detecting klebsiella pneumoniae

Abstract

The invention provides an SLAMP primer group and a kit for detecting klebsiella pneumoniae, and relates to the technical field of biological detection. The SLAMP primer group is a primer group for amplifying a conserved region fragment of klebsiella pneumoniae, namely KP-SF / KP-SR; wherein the nucleotide sequence of the KP-SF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the KP-SR is as shown in SEQ ID NO. 2. The primer group provided by the invention is high in sensitivity and strong in specificity, and the kit prepared from the primer group can quickly and accurately detect whether a sample to be detected contains klebsiella pneumoniae or not. In addition, the visual kit provided by the invention can provide great convenience for on-site detection of places such as customs, ports, community vegetable markets and the like with low professional degrees.

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 Klebsiella pneumoniae. Background Art

[0002] Klebsiella pneumoniae (KP) belongs to Klebsiella in the Enterobacteriaceae family and is a G(-) facultative anaerobic bacterium. Klebsiella pneumoniae has two main colonization channels in human hosts: the upper respiratory tract and the intestine. According to statistics, Klebsiella pneumoniae is the main pathogen of the human respiratory system that causes pneumonia.

[0003] Existing methods for detecting KP include RT-PCR-based detection of amplification signals of specific nucleic acid sequences of the viral genome, but the RT-PCR method requires the use of a sophisticated real-time fluorescence PCR instrument in a standard biochemical analysis laboratory, and requires three independent test areas for reagent preparation, specimen preparation, and PCR amplification detection, and is not suitable for rapid on-site screening. In addition, the test strips made by immunoassay technology have the advantages of being simple and fast, but this method has a high false positive rate and low sensitivity, making it difficult to detect KP carriers in the incubation period, which may cause the spread of the epidemic. Therefore, the development of rapid, sensitive, and highly specific nucleic acid marker on-site detection technology will provide a rapid and powerful treatment for diseases caused by Klebsiella pneumoniae infection. Summary of the invention

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

[0005] Another object of the present application is to provide a kit for detecting Klebsiella pneumoniae, 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] On the one hand, the present application provides a SLAMP primer set for detecting Klebsiella pneumoniae, wherein the SLAMP primer set is a primer pair KP-SF / KP-SR;

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

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

[0010] Short loop mediated isothermal amplification of nucleic acids (SLAMP) is a newly developed and improved 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 Vibrio parahaemolyticus. In addition, the key circular primer used in SLAMP is about 30 bp, which is shorter than the 40 bp circular primer in LAMP. The design of the relevant 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 SLAMP primer set provided in this application is targeted at the gene-specific conserved region of Klebsiella pneumoniae rcsA (GenBank: AY059955.1), and is composed of two primers, which are an intra-sequence primer pair (KP-SF / KP-SR). The primer set provided in this application has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect whether the sample to be tested contains Klebsiella pneumoniae.

[0013] 2. The primer set provided in this application has extremely high specificity, so the time required for SLAMP amplification is short, further shortening the detection time and making the operation simple.

[0014] 3. The method or kit provided in this application can complete the relevant detection without expensive instruments or complicated operations. Therefore, the visualization kit provided in this application will provide great convenience for on-site detection in airports, customs, communities and other places with low professionalism, and can achieve rapid and accurate detection of Klebsiella pneumoniae. 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 curve chart showing the change in fluorescence intensity of the Klebsiella pneumoniae SLAMP primer set amplification reaction versus reaction time in Example 1 of the present application.

[0017] Figure 2 This is a curve chart showing the change in fluorescence intensity versus reaction time of the amplification reaction of the Klebsiella pneumoniae SLAMP primer set on the genomes of various bacteria 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 performed with reference to the specific methods 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 channels.

[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 Klebsiella pneumoniae, wherein the SLAMP primer set is a primer pair KP-SF / KP-SR;

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

[0023] Table 1 Primer sequences

[0024] Serial Number Primer name Nucleotide sequence (5'-3') SEQ ID NO.1 KP-SF GCAGCATTGTTGA-ACAGGAAATCGTTGAGG SEQ ID NO.2 KP-SR GTGCATGATGAT-ATAATGCCATTACTTTC

[0025] A kit for detecting Klebsiella pneumoniae, 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 KP-SF and KP-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 DNA polymerase, SLAMP reaction buffer, reaction solvent and color developer.

[0028] In some embodiments of the present application, the 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, (NH4)2SO4, MgSO4 and Triton X-100.

[0030] 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-1000.2-1M betaine, 1-1.6mM dNTP, 5-10U Bst DNA polymerase, 100-150μmol / L color developer, 1-2μM primer KP-SF and 1-2μM primer KP-SR; the reaction solvent is ultrapure water.

[0031] A method for detecting Klebsiella pneumoniae using the above kit comprises the following steps:

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

[0033] Step 2, reacting the amplification reaction solution prepared in step 1 at 60-65° C. for 20-80 minutes, and judging whether the sample contains Klebsiella pneumoniae 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 primer sets to the Klebsiella pneumoniae gene DNA fragment.

[0037] Eva Green is similar to SYBR Green I. It is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helixes. Its inhibition on nucleic acid amplification reactions such as PCR is much less than that of the latter. The fluorescence signal intensity of Eva Green is related to the amount of double-stranded DNA. In the free state, Eva Green emits weak fluorescence, but after binding to double-stranded DNA, its fluorescence is greatly enhanced. Therefore, the amount of double-stranded DNA in the nucleic acid amplification system can be detected based on the fluorescence signal.

[0038] The single-tube reaction solution was composed as follows (add ddH2O to the rest 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), 1X Eva Green (Biotum), and primers: 1600 nM KP-SF (shown in SEQ ID NO.1) and 1600 nM KP-SR (shown in SEQ ID NO.2);

[0039] Target: Klebsiella pneumoniae genomic DNA. The amplified sequence is shown in SEQ ID NO.3.

[0040] At the same time, two groups of reactions were set up for the positive control and the negative control group without target.

[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 As shown, the amplification primer set can be applied to the real-time monitoring of Klebsiella pneumoniae, and the result can be judged in advance through the real-time amplification curve.

[0042] Example 2

[0043] This example uses the Eva Green validation primer set to amplify genomes extracted from various bacteria; the method is the same as in Example 1.

[0044] 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), 1X Eva Green (Biotum), and primers: 1600 nM LM-SF (shown in SEQ ID NO. 1) and 1600 nM LM-SR (shown in SEQ ID NO. 2);

[0045] Target nucleic acid 1: Klebsiella pneumoniae DNA (SEQ ID NO. 3);

[0046] Target nucleic acid 2: Escherichia coli DNA;

[0047] Target nucleic acid 3: Salmonella DNA;

[0048] Target nucleic acid 4: Shigella DNA;

[0049] Target nucleic acid 5: Staphylococcus aureus DNA;

[0050] Target nucleic acid 6: Vibrio parahaemolyticus DNA;

[0051] At the same time, a control group without target was set up as a negative control.

[0052] 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 only amplify the DNA of Klebsiella pneumoniae in the positive sample group, and cannot amplify other bacteria; therefore, it can distinguish Klebsiella pneumoniae from Escherichia coli, Salmonella, Shigella, Staphylococcus aureus, and Vibrio parahaemolyticus. The experimental results further illustrate the specificity of the SLAMP primer set provided by the present invention for detecting Klebsiella pneumoniae.

[0053] Example 3

[0054] In this example, hydroxynaphthol blue (HNB) was used to monitor the endpoint of the Klebsiella pneumoniae SLAMP amplification reaction.

[0055] Hydroxynaphthol blue (HNB) is a metal ion indicator that displays different indicator colors based on the changes in the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate during the reaction to determine the results.

[0056] The composition of the reaction solution for SLAMP amplification of Klebsiella pneumoniae using hydroxynaphthol blue (HNB) is shown below.

[0057] 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), 120 μM HNB, and primers: 1600 nM LM-SF (shown in SEQ ID NO.1) and 1600 nM LM-SR (shown in SEQ ID NO.2).

[0058] Target nucleic acid 1: Klebsiella pneumoniae DNA (SEQ ID NO. 3);

[0059] At the same time, a control group without target was set up as a negative control.

[0060] Each amplification reaction was repeated 8 times. The reaction temperature of the thermostatic water bath 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 reaction results can be judged intuitively by color using HNB, and can be interpreted without the assistance of instruments.

[0061] In summary, the SLAMP primer set and kit for detecting Klebsiella pneumoniae in the embodiment of the present application have the following advantages: 1. The SLAMP primer set provided by the present application is targeted at the gene-specific conserved region of rcsA of Klebsiella pneumoniae (GenBank: AY059955.1), and is composed of 2 primers, which are in-sequence primer pairs (KP-SF / KP-SR). The primer set provided by the present application has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect whether the sample to be tested contains Klebsiella pneumoniae.

[0062] 2. The primer set provided in this application has extremely high specificity, so the time required for SLAMP amplification is short, further shortening the detection time and making the operation simple.

[0063] 3. The method or kit provided in this application can complete relevant detection without expensive instruments or complicated operations. Therefore, the visualization kit provided in this application will provide great convenience for on-site detection in airports, customs, communities and other places with low professionalism, and can realize rapid and accurate detection of Klebsiella pneumoniae.

[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 Klebsiella pneumoniae, characterized in that: The SLAMP primer set is a primer pair KP-SF / KP-SR; Among them, the nucleotide sequence of KP-SF is shown in SEQ ID NO.1, and the nucleotide sequence of KP-SR is shown in SEQ ID NO.

2.

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

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

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

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

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

7. A kit for detecting Klebsiella pneumoniae 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% TritonX-1000.2-1 M betaine, 1-1.6 mM dNTP, 5-10 U Bst DNA polymerase, 100-150 μmol / L color developer, 1-2 μM primer KP-SF and 1-2 μM primer KP-SR; the reaction solvent is ultrapure water.

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