CN113322338B - CDA primer group and kit for detecting Shigella and application of CDA primer group and kit - Google Patents
CDA primer group and kit for detecting Shigella and application of CDA primer group and kit Download PDFInfo
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Abstract
The invention discloses a CDA primer group for detecting Shigella, a kit and application thereof. The CDA primer group is a primer group amplified aiming at a conserved region fragment of Shigella and is a primer pair Shi-F2/Shi-R2 and/or a primer pair Shi-MF/Shi-MR; wherein the nucleotide sequence of Shi-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of Shi-R2 is shown as SEQ ID NO. 2; the nucleotide sequence of Shi-MF is shown in SEQ ID NO.3, and the nucleotide sequence of Shi-MR is shown in SEQ ID NO. 4. The method or the kit provided by the invention can complete the rapid and accurate detection of the shigella without expensive instruments and 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 rapid and accurate detection of Shigella.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a CDA primer group for detecting Shigella, a kit and application thereof.
Background
Shigella (Shigella) is a gram-negative bacterium that was first discovered by Shigella, a japanese bacteriologist, 1898. The susceptibility of humans to shigella (shigella) is high and its only host, mainly through ingestion (fecal-oral contamination) of food infections, the most common symptoms being diarrhea (watery diarrhea), fever, nausea, vomiting, gastric cramps, flatulence and constipation. According to different crowds and ages, only about 100 pathogenic bacteria are required to be taken to cause infection. Shigella is the most common pathogenic bacterium of human bacillary dysentery and is collectively called dysentery bacillus. The genus bacteria are classified into four groups of shigella dysenteriae, shigella flexneri, and baumannii according to biochemical reactions and serological tests. Bacillary dysentery caused by Shigella flexneri and Shigella sonnei is the most common in China. Bacillary dysentery is a common disease, and is mainly prevalent in developing countries, and the number of annual cases worldwide exceeds 2 hundred million, 500 thousands of cases need hospitalization, and 65 thousands of annual death cases exist.
The conventional biochemical identification method for Shigella requires proliferation culture, separation and purification, biochemical test, serological test and the like, has the disadvantages of complicated detection steps, long time consumption, low accuracy and fewer samples after one-time detection. According to the food safety national standard Shigella test for food microbiology inspection (GB4789.5-2012), the conventional biochemical identification method adopted by the current standard in China is used for detecting Shigella, the whole process needs 4-5 days, and the requirement of rapid and accurate inspection in the market cannot be met, so that the research and development of the on-site rapid, sensitive and high-specificity nucleic acid marker detection technology can provide a powerful guarantee for the prevention and control of Shigella. However, the RT-PCR method requires a precise real-time fluorescence PCR instrument in a standard biochemical analysis laboratory, three independent experimental areas of reagent preparation, specimen preparation and PCR amplification detection, and is not suitable for on-site rapid screening. Therefore, the rapid, sensitive and high-specificity nucleic acid marker detection technology oriented to the field is to provide a powerful guarantee for shigella prevention and control.
A Closed loop mediated Isothermal Amplification (CDA) based nucleic acid Amplification technology is a method developed by Ningbo Life and health industry research institute of Chinese academy of sciences, which replaces Japanese LAMP nucleic acid Amplification (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 isothermal 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 tank, the requirements on instruments and equipment are low, the operation is simpler than that of the traditional PCR and culture method, the CDA reaction can be finished accurately without professionals, and the CDA reaction is suitable for being applied to 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 Shigella. In addition, the key loop forming primer used by CDA is about 30bp, which is shorter than 40bp of loop forming primer in LAMP, so that the cost is greatly saved.
Disclosure of Invention
The invention aims to provide a CDA primer group for detecting Shigella, a kit and application thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows:
a CDA primer group for detecting Shigella, which is not used for disease diagnosis, is one or two of the following primer pairs,
the primer pair Shi-F2/Shi-R2, wherein the nucleotide sequence of Shi-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of Shi-R2 is shown as SEQ ID NO. 2;
the primer pair Shi-MF/Shi-MR, wherein the nucleotide sequence of Shi-MF is shown in SEQ ID NO.3, and the nucleotide sequence of Shi-MR is shown in SEQ ID NO. 4.
The invention also provides a kit for detecting Shigella, which comprises the CDA primer group.
In a preferred embodiment, in the CDA primer set, the concentration of the primers Shi-F2 and Shi-R2 is 0.2-0.4. mu.M, and the concentration of the primers Shi-MF and Shi-MR is 1-2. mu.M.
As a preferred embodiment, the kit further comprises Bst polymerase, CDA reaction buffer, ultrapure water, and a color developing agent.
As a preferred embodiment, the color-developing agent is selected from one of Sybr green I, Eva green, hydroxynaphthol blue, 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 Shi-F2
0.2-0.4. mu.M primer Shi-R2
1-2. mu.M primer Shi-M
1-2 μ M primer Shi-MR;
the reaction solvent is ultrapure water.
The invention also provides a method for detecting Shigella by using the kit, which comprises the following steps:
step 1, mixing a nucleic acid sample to be detected with a reaction system of a kit to prepare an amplification reaction solution;
and 2, reacting the prepared amplification reaction liquid at 60-65 ℃ for 20-80 min, and judging whether the sample contains shigella according to a color development result.
The invention also provides application of the CDA primer group in detection of Shigella viruses for non-disease diagnosis.
The invention also provides application of the kit in detection of Shigella viruses for non-disease diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
1. the CDA primer group provided by the invention aims at a specific conserved region (GenBank: CP055125.1) of Shigella, consists of 4 primers and comprises an inner sequence primer pair (Shi-MF/Shi-MR) and an outer sequence primer pair (Shi-F2/Shi-R2). Shi-F2/Shi-R2 are upstream and downstream outer primers, respectively, composed of an 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 shigella contained in a sample to be detected.
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 shigella without expensive instruments and 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 rapid and accurate detection of Shigella.
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 chart based on color change for reaction performed by HNB in embodiment 4 of the present invention.
Fig. 5 is a terminal monitoring chart 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 conventional unless otherwise specified, and may be specifically carried out by the methods specified in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to 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 on Shigella genomic DNA fragments using Eva Green
Eva Green is similar to SYBR Green I, is a dye with Green excitation wavelength and combined with all double helix minor groove regions of dsDNA, and has far less inhibition on nucleic acid amplification reactions such as PCR and the like. 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 correlated with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from 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)
1X Eva Green(Biotum)
Primer:
200nM Shi-F2 (shown in SEQ ID NO. l)
200nM Shi-R2 (shown in SEQ ID NO. 2)
1600nM Shi-MF (SEQ ID NO. 3)
1600nM Shi-MR (SEQ ID NO.4 shows)
Target Shigella-genomic DNA dsDNA (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 Shigella, 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 through a real-time amplification curve.
Example 2 the amplification reaction method for extracting genome from shigella using Eva Green was the same 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)
1X Eva Green(Biotum)
Primer:
200nM Shi-F2/SEQ ID NO.l
200nM Shi-R2/SEQ ID NO.2
1600nM Shi-MF/SEQ ID NO.3
1600nM Shi-MR/SEQ ID NO.4
shigella as target for extracting genomic DNA
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 shigella extracted genome, 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 through a real-time amplification curve.
Example 3 verification of Shigella CDA amplification reaction on 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)
1X Eva Green(Biotum)
Primer:
200nM Shi-F2/SEQ ID NO.l
200nM Shi-R2/SEQ ID NO.2
1600nM Shi-MF/SEQ ID NO.3
1600nM Shi-MR/SEQ ID NO.4
the target nucleic acid 1 is genome DNA (shown as SEQ ID NO. 5) extracted from Shigella and set as a positive control
Target nucleic acid 2: extraction of genomic DNA from Escherichia coli
Target nucleic acid 3: extraction of genomic DNA from Listeria monocytogenes
Target nucleic acid 4: extraction of genomic DNA from Salmonella
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 distinguish Shigella, Escherichia coli, Listeria monocytogenes and Salmonella, and further illustrates the specificity of the CDA primer group provided by the invention.
Example 4 end-point monitoring of Shigella CDA amplification 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 shigella CDA amplification 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 Shi-F2/SEQ ID NO.l
200nM Shi-R2/SEQ ID NO.2
1600nM Shi-MF/SEQ ID NO.3
1600nM Shi-MR/SEQ ID NO.4
the target nucleic acid, Shigella, extracts genomic DNA. The amplification reaction was set up with 8 positive controls and 8 negative controls. The reaction temperature of the constant temperature water bath kettle is set to be 63 ℃, 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 can be applied to the system to judge the reaction result through color without the assistance of an instrument.
Example 5 verification of Shigella CDA for end-point monitoring of amplification reactions on extracted genomes of various bacteria Using Hydroxynaphtholan (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 shigella CDA amplification 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 Shi-F2/SEQ ID NO.l
200nM Shi-R2/SEQ ID NO.2
1600nM Shi-MF/SEQ ID NO.3
1600nM Shi-MR/SEQ ID NO.4
target nucleic acid 1 Shigella synthetic DNA
Target nucleic acid 2: extraction of genomic DNA from Escherichia coli
Target nucleic acid 3: extraction of genomic DNA from Listeria monocytogenes
Target nucleic acid 4: extraction of genomic DNA from Salmonella
Amplification reactions were set up 8 each. The reaction temperature of the constant temperature water bath kettle is set to be 63 ℃, 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 Shigella, Escherichia coli, Listeria monocytogenes and salmonella, and the application of HNB system can realize the judgment of reaction result through color without the assistance of instrument, thus further explaining the specificity of the CDA primer group.
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> Ningbo Living and health industry research institute of university of Chinese academy of sciences
NINGBO HUAMEI HOSPITAL University OF CHINESE ACADEMY OF SCIENCES
<120> CDA primer group for detecting Shigella, kit and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> primer (primer)
<400> 1
aacacctttt ccgcgttc 18
<210> 2
<211> 17
<212> DNA
<213> primer (primer)
<400> 2
gagattgttc catgtga 17
<210> 3
<211> 31
<212> DNA
<213> primer (primer)
<400> 3
ttttccagcc atgcctttcc gataccgtct c 31
<210> 4
<211> 31
<212> DNA
<213> primer (primer)
<400> 4
aactcagtgc ctctctcagt ggcatcagca g 31
<210> 5
<211> 500
<212> DNA
<213> Shigella (Shigella)
<400> 5
accacggccc acggatttac ttctccatga gtgacggaca acagaataca ctccatcgcc 60
cctggctgat gccgtgacag catggttccc ggaaaacaaa caatctgatg tatcacagat 120
atggcatgct tttgaacatg aagagcatgc caacaccttt tccgcgttcc ttgaccgcct 180
ttccgatacc gtctctgcac gcaatacctc cggattccgt gaacaggtcg ctgcatggct 240
ggaaaaactc agtgcctctg cggagcttcg acagcagtct ttcgctgttg ctgctgatgc 300
cactgagagc tgtgaggacc gtgtcgcgct cacatggaac aatctccgga aaaccctcct 360
ggtccatcag gcatcagaag gccttttcga taatgatacc ggcgctctgc tctccctggg 420
cagggaaatg ttccgcctcg aaattctgga ggacattgcc cgggataaag tcagaactct 480
ccattttgtg gacgagatag 500
Claims (10)
1. A CDA primer group for detecting Shigella, which is not used for disease diagnosis, is characterized in that: the CDA primer group comprises the following two pairs of primers,
the primer pair Shi-F2/Shi-R2, wherein the nucleotide sequence of Shi-F2 is shown as SEQ ID number 1, and the nucleotide sequence of Shi-R2 is shown as SEQ ID number 2;
the primer pair Shi-MF/Shi-MR, wherein the nucleotide sequence of Shi-MF is shown as SEQ ID number 3, and the nucleotide sequence of Shi-MR is shown as SEQ ID number 4.
2. A kit for detecting Shigella, comprising: the kit comprises the CDA primer set of claim 1.
3. The kit for detecting shigella according to claim 2, wherein: in the CDA primer group, the concentration of the primers Shi-F2 and Shi-R2 is 0.2-0.4 mu M, and the concentration of the primers Shi-MF and Shi-MR is 1-2 mu M.
4. The kit for detecting shigella according to claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent.
5. The kit for detecting Shigella according to 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. As claimed inClaim 4 discloses a kit for detecting shigella, which is characterized in that: the CDA reaction buffer solution comprises Tris-HCl, KCl and (NH)4)2SO4、MgSO4And Triton X-100.
7. The kit for detecting shigella according to claim 2, wherein: the reaction system of the kit consists of a reaction system,
2-50 mM Tris-HCl pH8.8
2-20 mM KCl
2-20 mM (NH4)2SO4
2～20 mM MgSO4
0.1～0.5 % Triton X-100
0.2-1M betaine
1～1.6 mM dNTP
5-10U Bst DNA polymerase
100-150 mu mol/L color developing agent
0.2-0.4. mu.M primer Shi-F2
0.2-0.4. mu.M primer Shi-R2
1-2. mu.M primer Shi-MF
1-2 μ M primer Shi-MR;
the reaction solvent is ultrapure water.
8. The method for detecting shigella bacteria 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 a kit to prepare an amplification reaction solution;
and 2, reacting the prepared amplification reaction liquid at 60-65 ℃ for 20-80 min, and judging whether the sample contains shigella according to a color development result.
9. Use of the CDA primer set of claim 1 in shigella detection for non-disease diagnostic purposes.
10. Use of a kit according to any one of claims 2 to 7 in shigella detection for non-disease diagnostic purposes.
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| CN114182001B (en) * | 2021-12-16 | 2022-07-26 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under asymmetric loop-mediated isothermal condition, kit and application |
| CN114214441B (en) * | 2021-12-17 | 2022-08-12 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting Listeria and application of CDA primer group and kit |
| CN114250311A (en) * | 2022-02-09 | 2022-03-29 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting spotted fever group rickettsia and application of CDA primer group and kit |
| CN114381554A (en) * | 2022-02-10 | 2022-04-22 | 国科宁波生命与健康产业研究院 | CDA primer pair and kit for detecting porcine circovirus type II and application of CDA primer pair and kit |
| CN114965764A (en) * | 2022-05-18 | 2022-08-30 | 陕西安宁云生生物技术有限公司 | Diagnosis and treatment of constipation |
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