CN114214441B - CDA primer group and kit for detecting Listeria and application of CDA primer group and kit - Google Patents
CDA primer group and kit for detecting Listeria and application of CDA primer group and kit Download PDFInfo
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Abstract
The invention discloses a CDA primer group for detecting Listeria, a kit and application thereof. The CDA primer group is a primer group for the amplification of a conserved region fragment of the listeria and is a primer pair LM-F2/LM-R2 and/or a primer pair LM-MF/LM-MR; wherein, the nucleotide sequence of LM-F2 is shown in SEQ ID NO.1, and the nucleotide sequence of LM-R2 is shown in SEQ ID NO. 2; the nucleotide sequence of LM-MF is shown in SEQ ID NO.3, and the nucleotide sequence of LM-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 listeria 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 rapid and accurate detection of Listeria.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a CDA primer group for detecting Listeria, a kit and application thereof.
Background
Listeria (Listeria monocytogenes) is a gram-positive, facultative, anaerobic, Bacillus-free bacterium that is parasitic intracellularly. It mainly uses food as the infection medium, and is one of the most deadly food-borne pathogenic bacteria. The susceptible population of Listeria is mainly pregnant women, neonates, the elderly and people with immunodeficiency. It can enter into body through eyes, damaged skin and mucosa to cause infection, and can pass through three barriers of intestinal tract, blood brain and placenta of human body after infection to cause gastroenteritis, septicemia, meningitis and other diseases, and can also cause abortion in pregnancy of female, and the death rate is up to 30%. Listeria is widespread and can be found in raw milk, dairy products, frozen meat, poultry, and fruits and vegetables, among others. Foreign investigations have shown that the listeria in fresh meat, meat products and deli meat products has the highest proportion among the products. Domestic investigation finds that the meat product has the highest proportion of listeria contamination. The listeria can grow and reproduce in the environment of 4 ℃, so the listeria becomes one of the main pathogenic bacteria threatening human health in refrigerated food, and also becomes an important detection target in sanitary microorganism detection.
The commonly used detection methods for listeria at present mainly include biochemical culture identification, serological detection and the like. The traditional detection method for the listeria generally requires 5-10 days in the whole process, has extremely low detection limit, wastes time and labor, cannot detect pathogenic bacteria in food timely and sensitively, and therefore cannot be used for treating food emergencies. The PCR method has excellent sensitivity compared to other Listeria detection methods, but must be selectively enriched to dilute certain components that may interfere with the detection reaction, and a complete PCR-based Listeria detection method requires 2 d. The establishment of a rapid, sensitive and specific Listeria detection method is imminent.
A Closed loop mediated Isothermal Amplification (CDA) based nucleic acid Amplification technology 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 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 Listeria. 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 Listeria, 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 Listeria, which is not the purpose of disease diagnosis, wherein the CDA primer group is one primer or two primers in the following primer pairs,
the primer pair LM-F2/LM-R2, wherein the nucleotide sequence of LM-F2 is shown as SEQ ID NO.1, and the nucleotide sequence of LM-R2 is shown as SEQ ID NO. 2;
and the primer pair LM-MF/LM-MR, wherein the nucleotide sequence of LM-MF is shown as SEQ ID No.3, and the nucleotide sequence of LM-MR is shown as SEQ ID No. 4.
The invention also provides a kit for detecting Listeria, which comprises the CDA primer group.
In a preferred embodiment, in the CDA primer group, the concentration of the primers LM-F2 and LM-R2 is 0.2-0.4 mu M, and the concentration of the primers LM-MF and LM-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 ) 2 SO 4 、MgSO 4 And 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(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
100-150 mu mol/L color developing agent
0.2-0.4 mu M primer LM-F2
0.2-0.4 mu M primer LM-R2
1-2 mu M primer LM-MF
1-2 mu M of primer LM-MR;
the reaction solvent is ultrapure water.
The invention also provides a method for detecting the listeria by using the kit, which comprises the following steps:
and 2, reacting the prepared amplification reaction liquid at 60-65 ℃ for 20-80 min, and judging whether the sample contains the listeria according to a color development result.
The invention also provides application of the CDA primer group in detection of Listeria for non-disease diagnosis.
The invention also provides the application of the kit in the detection of Listeria for non-disease diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
the CDA primer group provided by the invention is directed at a specific conserved region of Listeria, and consists of 4 primers, including an inner primer pair (LM-MF/LM-MR) and an outer primer pair (LM-F2/LM-R2) in sequence. LM-F2/LM-R2 are respectively the upstream and downstream external primers, 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 Listeria 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 listeria 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 rapid and accurate detection of Listeria.
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 graph showing color change based end point monitoring of the reaction using HNB in example 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 validation of amplification reaction on Listeria 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.
Reaction solution combinationAs follows (the remainder being ddH 2 O to 25 μ L):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X Eva Green(Biotum)
Primer:
200nM LM-F2 (shown in SEQ ID NO. l)
200nM LM-R2(SEQ ID NO. 2)
1600nM LM-MF (SEQ ID NO.3 shows)
1600nM LM-MR (SEQ ID NO.4 shows)
Target Listeria-genomic DNA dsDNA
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 listeria, the fluorescence detection is applied to the primer group, the purpose of real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.
Example 2 the same amplification reaction method as in example 1 was used to verify the extraction of listeria genome using Eva Green.
The reaction solutions were combined as follows (the remainder was added ddH) 2 O to 25 μ L):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X Eva Green(Biotum)
Primer:
200nM LM-F2/SEQ ID NO.l
200nM LM-R2/SEQ ID NO.2
1600nM LM-MF/SEQ ID NO.3
1600nM LM-MR/SEQ ID NO.4
extraction of genomic DNA from Listeria
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 listeria extracted genome, and the fluorescence detection is applied to the listeria extracted genome, so that the purpose of real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.
Example 3 validation of amplification reaction of Listeria CDA against 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) 2 O to 25 μ L):
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X Eva Green(Biotum)
Primer:
200nM LM-F2/SEQ ID NO.l
200nM LM-R2/SEQ ID NO.2
1600nM LM-MF/SEQ ID NO.3
1600nM LM-MR/SEQ ID NO.4
target nucleic acid Listeria plasmid DNA, set as a positive control
Target nucleic acid 2 extraction of genomic DNA from Listeria (nucleic acid from bacteria of different concentrations)
Target nucleic acid 3: extracting genome DNA from Staphylococcus aureus, and setting as negative control
Target nucleic acid 4: extracting genome DNA from Shigella, and setting as negative control
Target nucleic acid 5: extracting genome DNA from salmonella, and setting as negative control
Target nucleic acid 6: 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 distinguish Listeria, staphylococcus aureus, Shigella, salmonella and escherichia coli, and further illustrates the specificity of the CDA primer group provided by the invention.
Example 4 Listeria CDA amplification reaction endpoint monitoring Using Hydroxynaphtholate (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 listeria CDA amplification using hydroxynaphthol blue (HNB) is shown below.
The reaction solutions were combined as follows, and ddH was added to the rest 2 O to 25. mu.L
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
120μM HNB
Primer:
200nM LM-F2/SEQ ID NO.l
200nM LM-R2/SEQ ID NO.2
1600nM LM-MF/SEQ ID NO.3
1600nM LM-MR/SEQ ID NO.4
target nucleic acid Listeria 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 Listeria CDA amplification reaction endpoint monitoring for multiple bacteria extracted genomes 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 listeria CDA amplification using hydroxynaphthol blue (HNB) is shown below.
The reaction solutions were combined as follows, and ddH was added to the rest 2 O to 25. mu.L
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH 4 ) 2 SO 4
14mM MgSO 4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
120μM HNB
Primer:
200nM LM-F2/SEQ ID NO.l
200nM LM-R2/SEQ ID NO.2
1600nM LM-MF/SEQ ID NO.3
1600nM LM-MR/SEQ ID NO.4
target nucleic acid Listeria Synthesis DNA, set as a Positive control
Target nucleic acid 2 extraction of genomic DNA from Listeria (nucleic acid from bacteria of different concentrations)
Target nucleic acid 3: extracting genome DNA from Shigella, 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: extracting genome DNA from Staphylococcus aureus, and setting as negative control
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 Listeria, Shigella, salmonella, Escherichia coli and staphylococcus aureus, the reaction result can be judged by color 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 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 Listeria, kit and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> primer (primer)
<400> 1
ttgacgctgc cgtaagtgg 19
<210> 2
<211> 19
<212> DNA
<213> primer (primer)
<400> 2
gcaatgggaa ctcctggtg 19
<210> 3
<211> 30
<212> DNA
<213> primer (primer)
<400> 3
acggctttga aggatctgtc tcaggtgatg 30
<210> 4
<211> 29
<212> DNA
<213> primer (primer)
<400> 4
aatttacggt ggtatctcgt aagtctccg 29
<210> 5
<211> 1077
<212> DNA
<213> Listeria monocytogenes
<400> 5
atgtatgatc gattacaggc ggtggaagac cgttatgatg aactaaatga actactaagt 60
gatccagatg tagtatctga tccaaagcga ctacgcgacc tttctaaaga acaatcaggt 120
attacagcga cagtggaaac ataccgcgaa tacaaaaatg tgaacgaaca aattaacgaa 180
acaaaagaac tcctaggaga aaaactagac gatgaaatgc gcgaaatggc caaagaagaa 240
ttcgcggaac ttcaaaaaga aaaaacagac ttggaagaac gactaaaact attactcgtt 300
ccaaaagatc ctaatgatga caaaaacgtt attttagaaa ttcgcggagc agctggtgga 360
gacgaagcag ctttatttgc cggcgattta ttccgtatgt acagcaaata tgcggaatca 420
cgcggctgga aagtagaaat tatggacgca aatccaactg gtattggcgg ttacaaagaa 480
attattgcga tgatgaacgg aaatgatgct ttttctcgaa tgaaatatga aaacggggcg 540
caccgtgtac aacgtgttcc tgaaacagag tcaggcggtc gaattcatac atcaacagcg 600
actgtagcaa ttttgccaga agcggaagaa gtggaaatcg agttgcatga caaagatatc 660
cgtacagata cattcgcatc cactggtgcc ggtggacaaa gtgtcaatac aacgatgtca 720
gctgtacgtt taacgcatat tccgaccgga attgtcgttt cgatgcaaga tgagcgttct 780
cagttgaaaa acaaagataa agcgatgaaa gtattgcgcg cacgtgttta tgataaattt 840
gagcgtgagg ctcgtgagga atacgatgcc aaccgtaagt cagctgttgg aacgggtgac 900
cgctccgaac gtatccggac ttataactat ccgcaaaacc gcgtaaccga ccaccgtatc 960
ggcttaacca ttcaaaagct agatcaaatc atggaaggca aactcgatga aatcattgac 1020
gcgctcatcc tagaagatca aacaagtaaa ctggagcatt taaatgacgc aaattag 1077
Claims (9)
1. A CDA primer set for detecting Listeria, which is not for disease diagnosis, is characterized in that: the CDA primer group comprises two pairs of primers,
the primer pair LM-F2/LM-R2, wherein the nucleotide sequence of LM-F2 is shown as SEQ ID number 1, and the nucleotide sequence of LM-R2 is shown as SEQ ID number 2;
and the primer pair LM-MF/LM-MR, wherein the nucleotide sequence of the LM-MF is shown as SEQ ID number 3, and the nucleotide sequence of the LM-MR is shown as SEQ ID number 4.
2. A kit for detecting Listeria, which is characterized in that: the kit comprises the CDA primer set of claim 1.
3. The listeria detection kit of claim 2, wherein: in the CDA primer group, the concentrations of the primers LM-F2 and LM-R2 are both 0.2-0.4 mu M, and the concentrations of the primers LM-MF and LM-MR are both 1-2 mu M.
4. The listeria detection kit of claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and color developing agent; the CDA reaction buffer solution comprises Tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 And Triton X-100.
5. The Listeria detection kit 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 listeria detection kit of 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 (NH 4 ) 2 SO 4
2～20 mM MgSO 4
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 LM-F2
0.2-0.4 mu M primer LM-R2
1-2 mu M primer LM-MF
1-2 mu M of primer LM-MR;
the reaction solvent is ultrapure water.
7. The kit of claim 6 for use in a method of detecting Listeria 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 the listeria according to a color development result.
8. Use of the CDA primer set of claim 1 for the detection of listeria that is not of disease diagnostic interest.
9. Use of the kit of any one of claims 2-6 for the detection of listeria for non-disease diagnostic purposes.
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| CN113322354A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院大学宁波生命与健康产业研究院 | CDA primer group for detecting 2019-nCoV, kit and application thereof |
| CN113416799A (en) * | 2021-08-04 | 2021-09-21 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit |
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| JP2007117022A (en) * | 2005-10-28 | 2007-05-17 | Kirin Brewery Co Ltd | Primer set for detection of listeria monocytogenes and detection method |
| CN104313173B (en) * | 2014-11-11 | 2016-05-04 | 舟山市质量技术监督检测研究院 | The real-time turbidity LAMP of Listeria Monocytogenes detection method |
| CN113201583B (en) * | 2021-04-29 | 2022-02-08 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
| CN113322338B (en) * | 2021-07-19 | 2022-03-22 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit |
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| CN113322354A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院大学宁波生命与健康产业研究院 | CDA primer group for detecting 2019-nCoV, kit and application thereof |
| CN113416799A (en) * | 2021-08-04 | 2021-09-21 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting African swine fever virus and application of CDA primer group and kit |
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