CN114350827A - CDA primer group and kit for detecting vibrio parahaemolyticus and application of CDA primer group and kit - Google Patents
CDA primer group and kit for detecting vibrio parahaemolyticus and application of CDA primer group and kit Download PDFInfo
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Abstract
The invention relates to a rapid detection technology of vibrio parahaemolyticus based on Closed loop mediated Isothermal Amplification of nucleic acid (CDA). The invention discloses a CDA primer group for detecting vibrio parahaemolyticus, a kit and application thereof. The CDA primer group is a primer group for amplifying a conserved region fragment of vibrio parahaemolyticus and comprises a primer pair VP-F2/VP-R2 and a primer pair VP-MF/VP-MR; wherein, the nucleotide sequence of VP-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of VP-R2 is shown as SEQ ID NO. 2; the nucleotide sequence of VP-MF is shown in SEQ ID NO.3, and the nucleotide sequence of VP-MR is shown in SEQ ID NO. 4. The method or the visual kit provided by the invention can complete the rapid and accurate detection of the vibrio parahaemolyticus without expensive instruments or complex operations, so that the method or the visual kit is suitable for the rapid field detection of airports, customs, ports, communities and the like with low professional degree.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a CDA primer group for detecting vibrio parahaemolyticus, a kit and application thereof.
Background
Vibrio parahaemolyticus is a halophilic growing gram-negative polymorphic bacterium, widely present in marine environments. The thallus is arc-shaped, rod-shaped or filamentous, and has no spore and no capsule. The bacterium contains cytochrome oxidase, does not decompose sucrose, but can decompose glucose to produce acid without producing gas or hydrogen sulfide. Since Vibrio parahaemolyticus is a halophilic bacterium, it must grow in an environment containing salt of 0.5% to 8%, and the salt content is most preferable in the case of 2% to 4%. The vibrio parahaemolyticus can grow at the temperature of 5-44 ℃, but the optimal temperature is 30-35 ℃, and the growth is stopped when the temperature is 40 ℃ lower; the pH value suitable for growth is 7.5-8.5, the optimum pH value is 7.7, and the growth is not good under the acidic condition that the pH value is lower than 6.
Vibrio parahaemolyticus is an important food-borne pathogenic bacterium and mainly comes from marine products such as fish, shrimp, crab, shellfish and seaweed. People eating food contaminated by vibrio parahaemolyticus are likely to cause acute gastroenteritis with abdominal pain, diarrhea, nausea, vomiting, fever and the like as main symptoms. One of the characteristics is that the feces of the poisoned person is mostly watery and often mixed with mucus or purulent blood. In addition, severe patients can also have symptoms of dehydration, shock coma and even death, so Vibrio parahaemolyticus is also called Vibrio enteritis. The cases of food poisoning caused by this bacterium are reported all over the world, and therefore, it is very important to perform qualitative or quantitative detection of vibrio parahaemolyticus in food.
The existing detection methods mainly comprise PCR, DNA probe technology and the like. Although these techniques can rapidly detect the amount of total or pathogenic Vibrio parahaemolyticus in a sample, these methods rely on special and expensive equipment and are complicated and time-consuming in their manipulation (including DNA isolation, hybridization, colorimetry, and probe and primer synthesis), and only those skilled in their manipulation skills can perform the tests. Therefore, the above method is not suitable for rapid detection in the field, and the establishment of a rapid, sensitive and specific vibrio parahemolyticus detection method is imminent.
A Closed loop mediated Isothermal Amplification of nucleic acids (CDA) is a method for replacing Japanese LAMP nucleic acid Amplification developed by national Ningbo Life and health industry research institute (Chinese patent application No. 202110473121.8). The method mainly utilizes 2 different specific primers to identify a specific region of a target gene and carries out amplification reaction under a constant temperature condition. Compared with the conventional gene detection means (such as PCR and the like), the CDA reaction can be finished in a constant-temperature water bath box, the requirement on instruments and equipment is low, the operation is simpler than that of the traditional PCR and culture method, the CDA reaction can be finished accurately without professional personnel, and the amplification result can be combined with the metal ion indicator Hydroxy Naphthol Blue (HNB), so that the result can be judged by observing the color change by naked eyes, and the CDA reaction is suitable for basic medical institutions and local inspection and quarantine departments. In addition, the CDA can greatly shorten the operation time and reduce the sample pollution chance, and is suitable for the rapid diagnosis of the vibrio parahaemolyticus. In addition, the key loop forming primer used by the CDA is about 30bp, which is shorter than the loop forming primer in LAMP by 40bp, so that the cost is greatly saved.
Disclosure of Invention
The invention aims to provide a CDA primer group for detecting vibrio parahaemolyticus, a kit and application thereof. The technical problems of complex operation and high cost in the prior art are solved.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a CDA primer group for detecting vibrio parahaemolyticus for non-disease diagnosis purposes, wherein the CDA primer group comprises the following two pairs of primers:
a primer pair VP-F2/VP-R2, wherein the nucleotide sequence of VP-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of VP-R2 is shown as SEQ ID NO. 2;
the primer pair VP-MF/VP-MR, wherein the nucleotide sequence of VP-MF is shown in SEQ ID NO.3, and the nucleotide sequence of VP-MR is shown in SEQ ID NO. 4.
The invention also provides a kit for detecting the vibrio parahaemolyticus, and the kit comprises the CDA primer group.
In a preferred embodiment, in the CDA primer group, the concentration of the primers VP-F2 and VP-R2 is 0.2-0.4. mu.M, and the concentration of the primers VP-MF and VP-MR is 1-2. mu.M.
As a preferred embodiment, the kit further comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent.
In a preferred embodiment, the color-developing agent is one selected from SYBR green I, Eva green, hydroxynaphthol blue and chrome black T.
As a preferred embodiment, the CDA reaction buffer comprises Tris-HCl, KCl, (NH)4)2SO4、MgSO4And Triton X-100.
As a preferred embodiment, the reaction system of the kit comprises the following components:
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
100-150 mu mol/L color developing agent
0.2-0.4 mu M primer VP-F2
0.2-0.4. mu.M primer VP-R2
1-2 mu M primer VP-MF
1-2 mu M primer VP-MR;
the reaction solvent is ultrapure water.
The invention also provides a method for detecting the vibrio parahaemolyticus by using the kit for non-disease diagnosis, which comprises the following steps:
and 2, reacting the prepared amplification reaction liquid at the temperature of 60-65 ℃ for 20-80 min, and judging whether the sample contains vibrio parahaemolyticus according to a color development result.
The invention also provides application of the CDA primer group in detection of vibrio parahaemolyticus for non-disease diagnosis.
The invention also provides application of the kit in detection of vibrio parahaemolyticus for non-disease diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
1. in the invention, a plurality of primer groups are designed for the specific conserved region (GenBank: CP051112.1) of the vibrio parahaemolyticus, and the primer group with the highest amplification efficiency is screened out. The primer group consists of 4 primers, and comprises a sequence inner primer pair (VP-MF/VP-MR) and an outer primer pair (VP-F2/VP-R2). VP-F2/VP-R2 are upstream and downstream outer primers, respectively, consisting of the F2 region, and are complementary to the F2c region of the target gene. The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared from the primer group can quickly and accurately detect the vibrio parahaemolyticus contained in a sample to be detected. The specific primer nucleotide sequences are shown in table 1:
TABLE 1 primer sequences
| Serial number | Primer name | Nucleotide sequence |
| SEQ ID NO.1 | VP-F2 | TTTGCCCATTCCCAATC |
| SEQ ID NO.2 | VP-R2 | TTACTCCCGCTTGCTTC |
| SEQ ID NO.3 | VP-MF | ATCAGCACGCAATGCGATACCAACAGCGA |
| SEQ ID NO.4 | VP-MR | CACTTCAGACGCGCCGAAGAGCCAACCTTAT |
2. The primer group provided by the invention has extremely high specificity, so that the time required by CDA amplification is short, the detection time is further shortened, and the operation is simplified.
3. The method or the kit provided by the invention can complete the rapid and accurate detection of the vibrio parahemolyticus without expensive instruments or complex operation, so that the method or the kit is suitable for the rapid field detection of airports, customs, ports, communities and the like with low professional degree. The visual kit provided by the invention provides great convenience for field detection and can realize quick and accurate detection of vibrio parahaemolyticus.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with reaction time in example 1 of the present invention.
FIG. 2 is a graph showing the change of fluorescence intensity with reaction time in example 2 of the present invention.
FIG. 3 is a graph showing the change of fluorescence intensity with reaction time in example 3 of the present invention.
Fig. 4 is a terminal monitoring diagram based on color change for reaction performed by HNB in embodiment 4 of the present invention.
Fig. 5 is a terminal monitoring diagram based on color change for reaction performed by HNB in embodiment 5 of the present invention.
Detailed Description
The technical solution of the present invention will be described in detail with reference to examples. The experimental procedures used in the following examples are, unless otherwise specified, conventional and may be carried out in accordance with the procedures specified in molecular cloning, a laboratory manual (third edition) J. SammBruke, or in accordance with kits and product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 verification of amplification reaction of Vibrio parahaemolyticus conserved sequence DNA fragment by Eva Green
Similar to SYBR Green I, Eva Green is a dye which is combined with all DNA double helix minor groove regions and has Green excitation wavelength, and the inhibition effect on nucleic acid amplification reactions such as PCR and the like is far smaller than that of the dye. In the free state, Eva Green emits weak fluorescence, but once bound to double-stranded DNA, 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 existing in the nucleic acid amplification system can be detected according to the intensity of the fluorescence signal.
The reaction solutions were combined as follows (the remainder was added ddH)2O to 25 μ L):
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)
1×Eva Green(Biotum)
Primer:
200nM VP-F2 (shown in SEQ ID NO. l)
200nM VP-R2 (shown in SEQ ID NO. 2)
1600nM VP-MF (SEQ ID NO.3 shows)
1600nM VP-MR (shown in SEQ ID NO. 4)
Target is a conserved sequence DNA fragment of vibrio parahaemolyticus (shown in SEQ ID NO. 5)
A control group without target was also set.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 1. The results in FIG. 1 show that: the primer group provided by the invention can realize rapid amplification of the specific conserved region of the vibrio parahaemolyticus, the purpose of real-time monitoring can be realized by applying fluorescence detection to the primer group, and the result can be judged in advance by observing a real-time amplification curve.
Example 2 verification of amplification reaction of extracted genome of Vibrio parahaemolyticus Using Eva Green
The procedure is as in example 1.
The reaction solutions were combined as follows (the remainder was added ddH)2O to 25 μ L):
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)
1×Eva Green(Biotum)
Primer:
200nM VP-F2 (shown in SEQ ID NO. l)
200nM VP-R2 (shown in SEQ ID NO. 2)
1600nM VP-MF (SEQ ID NO.3 shows)
1600nM VP-MR (shown in SEQ ID NO. 4)
Genomic DNA extracted from Vibrio parahaemolyticus as target
A control group without target was also set.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 2. The results in FIG. 2 show that: the primer group provided by the invention can realize rapid amplification of the extracted genome of vibrio parahaemolyticus, the purpose of real-time monitoring can be realized by applying fluorescence detection to the primer group, and the result can be judged in advance by observing a real-time amplification curve.
Example 3 verification of the amplification reaction of Vibrio parahaemolyticus CDA on the extracted genomes of various bacteria Using Eva Green
The procedure is as in example 1.
The reaction solutions were combined as follows (the remainder was added ddH)2O to 25 μ L):
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)
1×Eva Green(Biotum)
Primer:
200nM VP-F2 (shown in SEQ ID NO. l)
200nM VP-R2 (shown in SEQ ID NO. 2)
1600nM VP-MF (SEQ ID NO.3 shows)
1600nM VP-MR (shown in SEQ ID NO. 4)
Target nucleic acid 1, Vibrio parahaemolyticus conserved sequence DNA fragment (sequence shown in SEQ ID NO. 5)
Target nucleic acid 2 extraction of genomic DNA of Vibrio parahaemolyticus, set as positive control
Target nucleic acid 3: extracting genome DNA from Staphylococcus aureus, and setting as negative control
Target nucleic acid 4: extracting genome DNA from salmonella, and setting as negative control
Target nucleic acid 5: extracting genome DNA from Escherichia coli, and setting as negative control
A control group without target was also set.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 3. The results in FIG. 3 show that: the primer group provided by the invention can effectively distinguish vibrio parahaemolyticus, staphylococcus aureus, salmonella and escherichia coli, and further illustrates the specificity of the CDA primer group provided by the invention.
Example 4 application of Hydroxynaphthol blue (HNB) to Vibrio parahaemolyticus CDA amplification reaction endpoint monitoring
Hydroxynaphthol blue (HNB) belongs to a metal ion indicator, and aims at the change of the amount of magnesium ions or manganese ions combined with a byproduct pyrophosphate in the reaction, so that different indicating colors are presented to judge the result.
The combination of the reaction solutions for the amplification of Vibrio parahaemolyticus CDA using hydroxynaphthol blue (HNB) is shown below.
The reaction solutions were combined as follows, and ddH was added to the rest2O to 25. mu.L
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
Primer:
200nM VP-F2 (shown in SEQ ID NO. l)
200nM VP-R2 (shown in SEQ ID NO. 2)
1600nM VP-MF (SEQ ID NO.3 shows)
1600nM VP-MR (shown in SEQ ID NO. 4)
Extracting genome DNA from the target nucleic acid, vibrio parahaemolyticus. The amplification reaction was set up with 8 positive controls and 8 negative controls. The reaction temperature of the water bath kettle is set to be 63 ℃ constantly, and the reaction time is 60 min. The results of the negative-positive reaction end points are shown in FIG. 4, in which violet indicates negative and sky blue indicates positive. The experimental results show that: the HNB is applied to the reaction product, so that the reaction result can be judged by observing color change through naked eyes.
Example 5 verification of the end-point monitoring of the amplification reaction of Vibrio parahaemolyticus CDA on the extracted genomes of various bacteria Using hydroxynaphthol blue (HNB)
Hydroxynaphthol blue (HNB) belongs to a metal ion indicator, and aims at the change of the amount of magnesium ions or manganese ions combined with a byproduct pyrophosphate in the reaction, so that different indicating colors are presented to judge the result.
The combination of the reaction solutions for the amplification of Vibrio parahaemolyticus CDA using hydroxynaphthol blue (HNB) is shown below.
The reaction solutions were combined as follows, and ddH was added to the rest2O to 25. mu.L
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
Primer:
200nM VP-F2 (shown in SEQ ID NO. l)
200nM VP-R2 (shown in SEQ ID NO. 2)
1600nM VP-MF (SEQ ID NO.3 shows)
1600nM VP-MR (shown in SEQ ID NO. 4)
Target nucleic acid 1: Vibrio parahaemolyticus synthetic DNA (sequence shown in SEQ ID NO. 5)
Target nucleic acid 2 extraction of genomic DNA of Vibrio parahaemolyticus, set as positive control
Target nucleic acid 3: extracting genome DNA from Staphylococcus aureus, and setting as negative control
Target nucleic acid 4: extracting genome DNA from salmonella, and setting as negative control
Target nucleic acid 5: extracting genome DNA from Escherichia coli, and setting as negative control
Target nucleic acid 6: set as negative control without adding any genomic DNA
Amplification reactions were set up 8 each. The reaction temperature of the water bath kettle is set to be 63 ℃ constantly, and the reaction time is 60 min. The results of the negative-positive reaction end points are shown in FIG. 5, in which violet indicates negative and sky blue indicates positive. The experimental results show that: the CAD primer group provided by the invention can distinguish vibrio parahaemolyticus, staphylococcus aureus, salmonella and escherichia coli, the reaction result can be judged through color change by applying an HNB system, and the identification can be carried out without the assistance of an instrument, so that the specificity of the CDA primer group provided by the invention is further explained.
The above description is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made within the spirit of the invention, and any changes and modifications made are within the scope of the invention.
Sequence listing
<110> national institute of Ningbo Life and health industry
NINGBO HUAMEI HOSPITAL University OF CHINESE ACADEMY OF SCIENCES
<120> CDA primer group for detecting vibrio parahaemolyticus, kit and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213> primer (primer)
<400> 1
tttgcccatt cccaatc 17
<210> 2
<211> 17
<212> DNA
<213> primer (primer)
<400> 2
ttactcccgc ttgcttc 17
<210> 3
<211> 29
<212> DNA
<213> primer (primer)
<400> 3
atcagcacgc aatgcgatac caacagcga 29
<210> 4
<211> 31
<212> DNA
<213> primer (primer)
<400> 4
cacttcagac gcgccgaaga gccaacctta t 31
<210> 5
<211> 469
<212> DNA
<213> Vibrio parahaemolyticus (Vibrio parahaemolyticus)
<400> 5
gccattgtga tgcgttaaag atgttgcctg tatcagacaa gctgtcaccg agtgcaacca 60
ctttgttgat ttgatctggc tgcattgctg cgtcgttgct ccagatcgtg tggttgtatg 120
agaagcgatt gtcagcggcg aagaacgtaa tgtctgcgtt ctcgttcgcc aaatctaatg 180
ttgcttcaca acgctgacgg ataacgtttt gcgacgtgtt ggtgtagaac atgtttttaa 240
atgaaacgga gctccaccag tagccgtcaa tggtgaagta gctaccatct tcgttttttg 300
cccattccca atcggtcgcc ggatcatctt tcgagtagct ggtgcgatac caacagcgaa 360
cataggtata ggtttggttt tcttgcgtgc tgatcacttc agacgctgaa accatttctg 420
gtgataaggt tggctcttcg gcaactgcag aagcaagcgg gagtaatgc 469
Claims (10)
1. A CDA primer group for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes, characterized in that: the CDA primer group comprises the following two pairs of primers,
a primer pair VP-F2/VP-R2, wherein the nucleotide sequence of VP-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of VP-R2 is shown as SEQ ID NO. 2;
the primer pair VP-MF/VP-MR, wherein the nucleotide sequence of VP-MF is shown in SEQ ID NO.3, and the nucleotide sequence of VP-MR is shown in SEQ ID NO. 4.
2. A kit for detecting vibrio parahaemolyticus is characterized in that: the kit comprises the CDA primer set of claim 1.
3. The kit for detecting Vibrio parahaemolyticus of claim 2, wherein: in the CDA primer group, the concentrations of the primers VP-F2 and VP-R2 are both 0.2-0.4 mu M, and the concentrations of the primers VP-MF and VP-MR are both 1-2 mu M.
4. The kit for detecting Vibrio parahaemolyticus of claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent.
5. The kit for detecting Vibrio parahaemolyticus of claim 4, wherein: the color developing agent is selected from one of SYBR green I, Eva green, hydroxyl naphthol blue and chrome black T.
6. The kit for detecting Vibrio parahaemolyticus of claim 4, wherein: the CDA reaction buffer comprises: Tris-HCl, KCl, (NH)4)2SO4、MgSO4And Triton X-100.
7. The kit for detecting Vibrio parahaemolyticus of claim 2, wherein: the reaction system of the kit comprises the following components:
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
100-150 mu mol/L color developing agent
0.2-0.4 mu M primer VP-F2
0.2-0.4. mu.M primer VP-R2
1-2 mu M primer VP-MF
1-2 mu M primer VP-MR;
the reaction solvent is ultrapure water.
8. The method for detecting Vibrio parahaemolyticus of the kit of claim 7 for non-disease diagnostic purposes, comprising the steps of:
step 1: mixing a nucleic acid sample to be detected with a reaction system of the kit to prepare an amplification reaction solution;
step 2: and (3) reacting the amplification reaction solution prepared at the temperature of 60-65 ℃ for 20-80 min, and judging whether the sample contains vibrio parahaemolyticus according to a color development result.
9. Use of the CDA primer set of claim 1 in the detection of Vibrio parahaemolyticus for non-disease diagnostic purposes.
10. Use of the kit of any one of claims 2 to 7 for the detection of vibrio parahaemolyticus for non-disease diagnostic purposes.
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