A SLAMP primer set and kit for detecting Vibrio parahaemolyticus

Abstract

The present application proposes a SLAMP primer set and a kit for detecting Vibrio parahaemolyticus, and relates to the field of biological detection technology. The SLAMP primer set is a primer set for amplifying the conserved region fragment of Vibrio parahaemolyticus, which is a primer pair VP‑SF / VP‑SR; wherein the nucleotide sequence of VP‑SF is shown in SEQ ID NO.1, and the nucleotide sequence of VP‑SR is shown in SEQ ID NO.2. The kit and the detection process provided in the present application can complete the rapid and accurate detection of Vibrio parahaemolyticus without expensive instruments or complicated operations, and therefore, are suitable for on-site rapid detection in airports, customs, ports, communities and other places with low professionalism. The visualization kit provided by the present invention will provide great convenience for on-site detection, and can realize rapid and accurate detection of Vibrio parahaemolyticus.

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 Vibrio parahaemolyticus. Background Art

[0002] Vibrio parahaemolyticus is a halophilic, Gram-negative, polymorphic coccobacillus that is widely found in the marine environment. It is an important foodborne pathogen that mainly originates from seafood such as fish, shrimp, crab, shellfish and seaweed. When people eat food contaminated by Vibrio parahaemolyticus, they are very likely to develop acute gastroenteritis with abdominal pain, diarrhea, nausea, vomiting, fever and other symptoms as the main symptoms. One of its characteristics is that the feces of the poisoned person are mostly watery and often mixed with mucus or pus and blood. In addition, severe patients may also experience symptoms of dehydration, shock, coma, and even death, so Vibrio parahaemolyticus is also known as Vibrio enteritidis. Cases of food poisoning caused by this bacterium have been reported all over the world, so it is very important to conduct qualitative or quantitative detection of Vibrio parahaemolyticus in food.

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

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

[0005] Another object of the present application is to provide a kit for detecting Vibrio parahaemolyticus, which has the advantages of high detection accuracy, fast speed and convenient 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 Vibrio parahaemolyticus, wherein the SLAMP primer set is a VP-SF / VP-SR primer pair, wherein the nucleotide sequence of primer VP-SF is shown in SEQ ID NO.1, and the nucleotide sequence of primer VP-SR is shown in SEQ ID NO.2.

[0008] On the other hand, the present application provides a kit for detecting Vibrio parahaemolyticus, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0009] 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) that was 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. 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.

[0010] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0011] 1. The present application designs multiple primer sets by using the specific conserved region of Vibrio parahaemolyticus (GenBank: CP051112.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of two primers, including an intrasequence primer pair (VP-SF / VP-SR) as upstream and downstream external primers, respectively, consisting of the F2 region, and complementary to the F2c region of the target gene. The primer set provided by the present invention has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Vibrio parahaemolyticus contained in the sample to be tested.

[0012] 2. The primer set provided in this application has extremely high specificity, and the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation;

[0013] 3. The kit and detection process provided by the present application can complete the rapid and accurate detection of Vibrio parahaemolyticus without expensive instruments or complicated operations, and therefore, is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided by the present invention will greatly facilitate on-site detection and can achieve rapid and accurate detection of Vibrio parahaemolyticus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] Figure 1 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 1 of the present invention.

[0016] Figure 2 This is a curve diagram showing the change of fluorescence intensity with reaction time in Example 2 of the present invention.

[0017] Figure 3 This is an endpoint monitoring diagram based on color change of the reaction using HNB in ​​Example 3 of the present invention. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods, and can be specifically referred to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook, or carried out according to the kit and product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0019] 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.

[0020] A SLAMP primer set for detecting Vibrio parahaemolyticus, wherein the SLAMP primer set is a VP-SF / VP-SR primer pair, wherein the nucleotide sequence of primer VP-SF is shown in SEQ ID NO.1, and the nucleotide sequence of primer VP-SR is shown in SEQ ID NO.2. The specific sequences are shown in Table 1:

[0021] Table 1 Primer sequences

[0022]

[0023]

[0024] A kit for detecting Vibrio parahaemolyticus, wherein the reaction system of the kit comprises the above-mentioned SLAMP primer set.

[0025] In some embodiments of the present application, in the above SLAMP primer set, the concentrations of primer VP-SF and primer VP-SR are both 1-2 μM.

[0026] In some embodiments of the present application, the components of the above-mentioned kit also include Bst polymerase, SLAMP reaction buffer, reaction solvent and color developer.

[0027] 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.

[0028] In some embodiments of the present application, the SLAMP reaction buffer comprises: Tris-HCl, KCl, (NH 4 ) 2 SO 4 MgSO 4 and Triton X-100.

[0029] In some embodiments of the present application, the components of the reaction system of the above-mentioned kit are: 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 VP-SF and 1-2μM primer VP-SR; the reaction solvent is ultrapure water.

[0030] The present application also provides a method for detecting Vibrio parahaemolyticus using the kit, comprising the following steps:

[0031] Step 1: Mix the nucleic acid sample to be detected and the reaction system of the kit to prepare an amplification reaction solution;

[0032] Step 2: The amplification reaction solution prepared above is reacted at 60-65° C. for 20-80 min, and whether the sample contains Vibrio parahaemolyticus is determined based on the color development result.

[0033] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0034] Example 1

[0035] This application uses Eva Green to verify the amplification reaction of the extracted genome containing the conservative sequence DNA fragment of Vibrio parahaemolyticus

[0036] 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.

[0037] The reaction solution was composed as follows (add ddH 2 0 to 25 μL):

[0038] 20mM Tris-HCl pH8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% Triton X-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), and primers: 1600nM VP-SF (SEQ ID NO.1) and 1600nM VP-SR (SEQ ID NO.2)

[0039] Target: Extracted genome containing DNA fragments of conservative sequences of Vibrio parahaemolyticus (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 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 achieve rapid amplification of the specific conserved region of Vibrio parahaemolyticus, 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.

[0042] Example 2

[0043] This application uses Eva Green to verify the amplification reaction of Vibrio parahaemolyticus SLAMP on genomes extracted from various bacteria:

[0044] The method is the same as in Example 1.

[0045] The reaction solution was composed as follows (add ddH 2 0 to 25 μL):

[0046] 20mM Tris-HCl pH8.8, 10mM KCl, 10mM (NH 4 ) 2 SO 4 、14 mM MgSO 4 , 0.1% Triton X-100, 1 M betaine, 1.25 mM dNTP, 8 U Bst DNA polymerase (NEW ENGLAND Biolabs), 1× Eva Green (Biotum), and primers: 1600 nM VP-SF (SEQ ID NO. 1) and 1600 nM VP-SR (SEQ ID NO. 2).

[0047] Target nucleic acid 2: genomic DNA extracted from Vibrio parahaemolyticus, set as a positive control (the sequence contained is as SEQ ID NO.3);

[0048] Target nucleic acid 3: Genomic DNA extracted from Staphylococcus aureus, set as negative control;

[0049] Target nucleic acid 4: Salmonella extracted genomic DNA, set as negative control;

[0050] Target nucleic acid 5: genomic DNA extracted from Escherichia coli, set as negative control;

[0051] A non-target control group was also set up.

[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 effectively distinguish Vibrio parahaemolyticus from Staphylococcus aureus, Salmonella, and Escherichia coli, further illustrating the specificity of the SLAMP primer set provided by the present invention.

[0053] Example 3

[0054] This application uses hydroxynaphthol blue (HNB) to monitor the endpoint of the SLAMP amplification reaction of Vibrio parahaemolyticus:

[0055] 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.

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

[0057] The reaction solution was composed as follows, and ddH 2 0 to 25 μL:

[0058] 20mM Tris-HCl pH8.8, 10mM KCl, 10mM (NH4)2SO4, 14mM MgSO4, 0.1% Triton X-100, 1M betaine, 1.25mM dNTP, 8U Bst DNA polymerase (NEW ENGLAND Biolabs), 120μM HNB and primers: 1600nM VP-SF (SEQ ID NO.1) and 1600nM VP-SR (SEQ ID NO.2)

[0059] Target nucleic acid: genomic DNA extracted from Vibrio parahaemolyticus (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 63°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 Vibrio parahaemolyticus according to the embodiments of the present application have the following advantages:

[0062] 1. The present application designs multiple primer sets by using the specific conserved region of Vibrio parahaemolyticus (GenBank: CP051112.1), and screens out the primer set with the highest amplification efficiency. The primer set consists of two primers, including an intrasequence primer pair (VP-SF / VP-SR) as upstream and downstream external primers, respectively, consisting of the F2 region, and complementary to the F2c region of the target gene. The primer set provided by the present invention has high sensitivity and strong specificity, and the kit made therefrom can quickly and accurately detect Vibrio parahaemolyticus contained in the sample to be tested.

[0063] 2. The primer set provided in this application has extremely high specificity, and the time required for SLAMP amplification is short, which further shortens the detection time and simplifies the operation;

[0064] 3. The kit and detection process provided by the present application can complete the rapid and accurate detection of Vibrio parahaemolyticus without expensive instruments or complicated operations, and therefore, is suitable for on-site rapid detection in airports, customs, ports, communities, etc. where the professional level is not high. The visualization kit provided by the present invention will greatly facilitate on-site detection and can achieve rapid and accurate detection of Vibrio parahaemolyticus.

[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 Vibrio parahaemolyticus, characterized in that: The SLAMP primer set is a VP-SF / VP-SR primer pair, wherein the nucleotide sequence of the primer VP-SF is shown in SEQ ID NO.1, and the nucleotide sequence of the primer VP-SR is shown in SEQ ID NO.

2.

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

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

4. A kit for detecting Vibrio parahaemolyticus 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 Vibrio parahaemolyticus 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 Vibrio parahaemolyticus 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 Vibrio parahaemolyticus according to claim 2, characterized in that: The components of the reaction system of the kit are: 2-50mM Tris-HCl pH8.8, 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 VP-SF and 1-2μM primer VP-SR; the reaction solvent is ultrapure water.

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