SLAMP primer group and kit for detecting salmonella

Abstract

The invention provides an SLAMP primer group and a kit for detecting salmonella, and relates to the technical field of biological detection. The SLAMP primer group is a primer group aiming at the amplification of a conserved region fragment of salmonella, and is a primer pair SAL-SF / SAL-SR; wherein the nucleotide sequence of the SAL-SF is as shown in SEQ ID NO. 1, and the nucleotide sequence of the SAL-SR is as shown in SEQ ID NO. 2. According to the method or the kit provided by the invention, the salmonella can be rapidly and accurately detected without expensive instruments or complicated operation, so that the method or the kit is suitable for on-site rapid detection of ports, communities and the like with low professional degree. According to the visual kit provided by the invention, great convenience is provided for field detection, and rapid and accurate non-diagnostic purpose detection of salmonella can be realized.

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 Salmonella. Background Art

[0002] Salmonella, a gram-negative enteric bacillus belonging to the family Enterobacteriaceae, is a zoonotic intestinal pathogen that is ubiquitous in nature and can cause a variety of diseases such as typhoid, paratyphoid and food poisoning, which has a direct impact on the development of industry, agriculture and animal husbandry. The endotoxins released after the death of Salmonella can induce a fever and a decrease in the number of white blood cells in the host. Once the toxin dose is too large, it can also cause poisoning reactions or even shock. Therefore, it is very important to conduct qualitative or quantitative detection of Salmonella in food.

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

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

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

[0006] Another object of the present application is to provide a method for detecting Salmonella, which is simple to operate.

[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 Salmonella, wherein the SLAMP primer set is a primer pair SAL-SF / SAL-SR;

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

[0010] In a second aspect, the present application provides a kit, wherein the reaction system of the kit includes the above-mentioned SLAMP primer set.

[0011] In a third aspect, the present application provides a method for detecting Salmonella using the above-mentioned kit, comprising the following steps:

[0012] Step 1: Mix the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0013] Step 2: react the amplification reaction solution prepared above at 60-65° C. for 20-80 min, and determine whether the sample contains Salmonella based on the color development result.

[0014] 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. The developed SLAMP 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, 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 change can be observed by naked eyes 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 Salmonella. 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.

[0015] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0016] 1. The present application designs multiple primer sets based on the specific conserved region of Salmonella (GenBank: CP163542.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of two primers, including an intrasequence primer pair (SAL-SF / SAL-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 the Salmonella contained in the sample to be tested.

[0017] 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.

[0018] 3. The method or kit provided in this application can complete the rapid and accurate detection of Salmonella 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 Salmonella. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] 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.

[0020] Figure 1 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 1.

[0021] Figure 2 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 2.

[0022] Figure 3 This is an endpoint monitoring chart based on color change of the reaction using HNB in ​​Example 3. DETAILED DESCRIPTION

[0023] 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.

[0024] 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.

[0025] A SLAMP primer set for detecting Salmonella, wherein the SLAMP primer set is a primer pair SAL-SF / SAL-SR;

[0026] Among them, the nucleotide sequence of SAL-SF is shown in SEQ ID NO.1, and the nucleotide sequence of SAL-SR is shown in SEQ ID NO.2.

[0027] A kit for detecting Salmonella, 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 CA-SF and primer CA-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 SAL-SF and 1-2μM primer SAL-SR; the reaction solvent is ultrapure water

[0033] A method for detecting Salmonella using a kit, comprising 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 Salmonella 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 Salmonella conserved sequence DNA fragment.

[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-1001M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), and primers: 1600 nM SAL-SF (shown in SEQ ID NO.1) and 1600 nM SAL-SR (shown in SEQ ID NO.2)

[0041] Target: Salmonella extracted genomic DNA (target sequence is shown in SEQ ID NO.3);

[0042] A non-target control group was also set up.

[0043] 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 extracted genome of Salmonella, 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 Salmonella 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), primers: 1600 nM SAL-SF (shown in SEQ ID NO. 1) and 1600 nM SAL-SR (shown in SEQ ID NO. 2);

[0047] Target nucleic acid 1: Salmonella extracted genomic DNA (target sequence as shown in SEQ ID NO.3), set as positive control;

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

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

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

[0051] Target nucleic acid 5: Listeria monocytogenes, extract genomic DNA, 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 Salmonella from Staphylococcus aureus, Shigella, Escherichia coli, and Listeria monocytogenes, 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 Salmonella SLAMP amplification reaction.

[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 Salmonella 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 SAL-SF (shown in SEQ ID NO. 1) and 1600 nM SAL-SR (shown in SEQ ID NO. 2);

[0059] Target nucleic acid: genomic DNA extracted from Salmonella (the target sequence 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 Salmonella 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 Salmonella (GenBank: CP163542.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of 2 primers, including an intra-sequence primer pair (SAL-SF / SAL-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 the Salmonella 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 Salmonella 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 Salmonella.

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

2.

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

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

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

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