CN115927674A - CDA primer group and kit for detecting staphylococcus aureus and application of CDA primer group and kit - Google Patents
CDA primer group and kit for detecting staphylococcus aureus and application of CDA primer group and kit Download PDFInfo
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a CDA primer group for detecting Staphylococcus Aureus (SAU), a kit and application thereof. The CDA primer group is a primer group for amplifying a conserved region fragment of staphylococcus aureus, namely SAU-MF-3/SAU-MR-3; wherein, the nucleotide sequence of SAU-MF-3 is shown as SEQ ID NO.5, and the nucleotide sequence of SAU-MR-3 is shown as SEQ ID NO. 6. 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 whether a sample to be detected contains staphylococcus aureus. In addition, the visual kit provided by the invention can provide great convenience for field detection of customs, port, remote areas 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 staphylococcus aureus, a kit and application thereof.
Background
Staphylococcus Aureus (SAU) is a common food-borne pathogenic microorganism, which is widely present in the natural environment. Under appropriate conditions, staphylococcus aureus can produce enterotoxin, causing food poisoning. The reports of food poisoning caused by staphylococcus aureus are infinite, the food poisoning caused by staphylococcus aureus accounts for about 25% of food-borne microbial food poisoning events, and staphylococcus aureus becomes the third largest microbial pathogenic bacterium second only to salmonella and haemolyticus parahaemolyticus.
The classical detection method of staphylococcus aureus is separation culture, which specifically comprises the following steps: the neutralized bacterial material was inoculated on a solid medium, cultured at 37 ℃ with a rubber plug at the mouth of the vessel, and observed 1 time per week. However, staphylococcus aureus grows slowly and generally takes 2-4 weeks to grow into colonies visible to the naked eye. Other methods, which are relatively rapid and accurate, include RT-PCR-based detection of amplification signals for Staphylococcus aureus DNA, which only requires several bacteria per ml to detect positivity, and results are obtained in 1-2 days. However, the RT-PCR method requires a precise real-time fluorescence PCR instrument in a standard biochemical analysis laboratory, requires three independent test areas for reagent preparation, specimen preparation and PCR amplification detection, and is not suitable for rapid on-site screening. In addition, the test strip prepared by the immunoassay technology has the advantages of simplicity, convenience and rapidness, but the method has high false detection rate and low sensitivity, is difficult to detect SAU carriers in a latent period, and can cause spreading of epidemic situations. Therefore, the research and development of the rapid, sensitive and high-specificity nucleic acid marker on-site detection technology can provide a rapid and powerful treatment means for diseases caused by staphylococcus aureus infection.
A Closed loop mediated Isothermal Amplification technology (CDA) based on nucleic acid is a novel method developed by Ningbo life and health industry research institute of Chinese academy of sciences (Chinese patent No. ZL 202110473121.8), and can replace the Japanese LAMP nucleic acid Amplification method. 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 completed in a constant-temperature water bath box, the requirement on instruments and equipment is lower, the operation is simple, non-professionals can accurately complete the reaction, and the method is suitable for basic medical institutions and local inspection and quarantine departments. In addition, the CDA can also shorten the operation time, improve the detection efficiency, reduce the probability of sample pollution and be suitable for the rapid diagnosis of staphylococcus aureus. In addition, the key loop primers used in CDA are about 30bp shorter than LAMP (40 bp), which saves the detection cost.
Disclosure of Invention
The invention aims to provide a CDA primer group for detecting staphylococcus aureus, a kit and application thereof.
The technical scheme adopted by the invention for realizing the purpose is as follows:
according to the invention, 4 pairs of primer groups described in Table 1 are screened, and the primer group with the optimal detection effect on staphylococcus aureus is SAU-MF-3/SAU-MR-3.
TABLE 1
| Serial number | Name of primer | Nucleotide sequence (5 '-3') |
| SEQ ID NO.1 | SAU-MF-1 | CAGCTTGCTTACTAATCAATCACTGGACCG |
| SEQ ID NO.2 | SAU-MR-1 | CAATGACCTCGTTTTCACGCAAACTGTTG |
| SEQ ID NO.3 | SAU-MF-2 | GCGATGACTACTGTTGTCGCATTCATCGTC |
| SEQ ID NO.4 | SAU-MR-2 | AACTAACGATATGGATACTGGTGTCTACGGCTT |
| SEQ ID NO.5 | SAU-MF-3 | AGTTGTCGCAGGAGCTACCAATGACAATGCCAT |
| SEQ ID NO.6 | SAU-MR-3 | GGCACATTGGCTTACTGTGACGATGAATGCGA |
| SEQ ID NO.7 | SAU-MF-4 | GGCAAGTGCAACGAATGTTCTGGTCCAATAC |
| SEQ ID NO.8 | SAU-MR-4 | CATCTTCATCTTGGGCGATGATACTGATAAAC |
The invention also provides a kit for detecting staphylococcus aureus, which comprises a CDA primer group SAU-MF-3/SAU-MR-3.
As a preferable embodiment, the concentration of the primers SAU-MF and SAU-MR in the CDA primer group in the reaction system is 1 to 2 μ 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 colour developer is selected from Sybr green I, eva green, hydroxynaphthol blue or 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-HClpH8.8
2～20mM KCl
2～20mM(NH 4 ) 2 SO 4
2～20mM MgSO 4
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
1-2 mu M primer SAU-MF-3
1-2 mu M primer SAU-MR-3
The reaction solvent is ultrapure water.
The invention also provides a method for detecting staphylococcus aureus by using the kit for non-disease diagnosis, 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 amplification reaction solution obtained in the step 1 at the temperature of 60-65 ℃ for 20-80 min, and judging whether the sample contains staphylococcus aureus according to the color development result.
The invention also provides application of the CDA primer group in staphylococcus aureus detection with non-disease diagnosis as a purpose.
The invention also provides application of the kit in detection of staphylococcus aureus 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 Staphylococcus aureus strain 332chromosome (GenBank: CP 077893.1), consists of 2 primers and is a primer pair in a sequence (SAU-MF-3/SAU-MR-3). 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 whether a sample to be detected contains staphylococcus aureus.
2, the primer group provided by the invention has extremely high specificity, the time required by CDA amplification is short, the detection time is further shortened, and the operation is simple and easy.
3, the method or the kit provided by the invention can complete related detection without expensive instruments and complex operation, so that the visual kit provided by the invention can provide great convenience for field detection of airports, customs, communities and the like with low specialty and can realize rapid and accurate detection of the staphylococcus aureus.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with reaction time in the amplification reaction of four primer sets in example 1 of the present invention.
FIG. 2 is a graph showing the change of fluorescence intensity with reaction time in the amplification reaction of the primer set 3 in example 2 of the present invention.
FIG. 3 is a graph showing the change of fluorescence intensity with reaction time in the amplification reaction of the primer set 3 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.
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 can be carried out according to the methods specified in molecular cloning, a laboratory manual (third edition) J. SammBruke, 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 of four primer sets to DNA fragment of Staphylococcus aureus Gene 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. The fluorescence signal intensity of Eva Green is related to the amount of double stranded DNA. In the free state, eva Green emits weak fluorescence, but the fluorescence is greatly enhanced after the Eva Green is combined with double-stranded DNA. Therefore, the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from the fluorescent signal.
The single-tube reaction solutions were combined as follows (the remainder was ddH added) 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)
Primers used for single tubes:
1600nM SAU-MF-1 (SEQ ID NO. 1)
1600nM SAU-MR-1 (shown in SEQ ID NO. 2);
1600nM SAU-MF-2 (SEQ ID NO. 3)
1600nM SAU-MR-2 (shown in SEQ ID NO. 4);
1600nM SAU-MF-3 (SEQ ID NO. 5)
1600nM SAU-MR-3 (shown in SEQ ID NO. 6);
1600nM SAU-MF-4 (SEQ ID NO. 7)
1600nM SAU-MR-4 (shown in SEQ ID NO. 8).
Target: staphylococcus aureus genomic DNA dsDNA (shown in SEQ ID NO. 9).
SEQ ID NO.9：
ctgacgtatcttccataaatgatctaaaaattggtagttcttcttcagataaaaatcttactttaacaccattctttttaacttttttcgtgtttctttttctaagtccatccatatttttaatgatgtcatctgctgttttatcttttaaatctaacactgagtgataacgaatttgtagcacaggatcaaatcctttatggaatccagtatgttcaaatcctaagttactcattttatcaaagaaccaatcattaccagcattacctgtaatctcgccatcatgattcaagtattgatatggtaaatatggatcgatatgtaggtatagacaacgatgttttttaacatattttgataattcattaaagaaaaagtgtacgagttcttgattttcataatcaatcactggaccgcgatttgaataaaaatacttgaacactttcataacaggtacagcagtaagtaagcaagctgcaatgacctcgttattattgttttttattcccactaaatgtgtttcataaccttcagcaagctttaactcatagtggccaacagtttgcgtgaaatgactgtatggcatgctatctgtaaaggcaccaaactctttagctgttaaatttgtaaacttcattatcattactcctatttgtctctcgttaattaatttcatttccgtatttgcagtttttctatttcccctctgcaaatgtcaaaaataataaatctaatctaaataagtatacaatagttaatgttaaaactaaaacataaacgctttaattgcgtatacttttatagtaatatttagatttttgaatacaatttcaaaaaaagtaatatgaacgtttgggtttgctcatattacttttgtatcatttctattcaattttataattcaccgtttttcactttttcaaacagtattcgcctaatttttttaaatcaagtaaacttaattattcaatgtttgttggatagattgtaaatatttaatgatttcctcacgcgtgttagatttaaatcgcttaacgatttcgctaccaatgacaatgccatctgcaacctcttttatatctgcaacatgttgtggtgttcttataccaaatcctgcgacaactggcacattggctatcgctttaattgactcaatttttcgttttaattctggatgaaacgcaccgttttgccctgttgtcgcattcatcgtcacagtataaataaagccttccgcatgggatacgatatcttttatacgtttgtcatcagtagtcatcgcaactaacgatatgattttgacgccatagtgactaaattgttgttttaaacgctgcgataattcatatggtaaatcaggaataattaagccgtagacaccagtatctcgacatttttcaaaaaacgcttgttctccataatgacaaataatattataatacgtcattaatacatagttacacttaatttgatcaccatgtttttctaattgattgaaaatataatctatcgtgatgccttgtttaatcgcttgttgacctgcttccatgataactggaccatcagcaaccggatcagagaaaggtactccaatttcaattatatctgcaccattttcactcaacaatgttgcattttcaatcaaatctttattgcccataatataaggtataaataatttagtcatttgcaagacctcgctctaccatatattgtctaattgtttccatatctttatcgccacgtccagaaatagttactacaataatatcttctttcgacatcgtaggcgctagtctttcaacataactcagtgcatgtgcactttcaattgcaggtataataccttcatgttttgtaaagttgattaaagcattcattgcttgtgtatcactagcattttcaaaagttactctaccaatgtcgtggtaataagaatgttctggtccaataccaggataatcaagtcc
Meanwhile, four pairs of primer groups are respectively provided with a control group without targets.
The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60min. The detection result is shown in fig. 1, and it can be found from fig. 1 that: only the positive group of the two groups of primer groups has amplification, the amplification rate of the 2 nd group is not high, and the Ct value of the 3 rd group of primer groups has the fastest amplification value. The results show that: the 3 rd group primer group provided by the invention can realize rapid amplification of staphylococcus aureus genes, and a negative control group has no false positive, and the result can be judged in advance by applying fluorescence detection to the primer group for real-time monitoring and a real-time amplification curve.
Example 2 validation of amplification reaction of group 3 primer set on DNA fragment of Staphylococcus aureus Gene by Eva Green (repeat experiment)
The procedure is as in example 1.
The single-tube reaction solutions were combined as follows (the remainder was ddH added) 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)
Primers used for single tubes:
1600nM SAU-MF-3 (SEQ ID NO. 5)
1600nM SAU-MR-3 (shown in SEQ ID NO. 6);
the reaction solution was combined in the same manner as in example 1, with 8 target and no target control groups being provided. Target: staphylococcus aureus genomic DNA dsDNA (SEQ ID NO. 9).
The SLAN 96real time PCR reaction temperature is set to be constant at 63 ℃ and the reaction time is set to be 60min. 8 negative repeated experiments are carried out on the primer group 3, no false positive appears, the repeatability is good, 8 positive samples are subjected to control experiments, and the curve of the change of the fluorescence intensity along with the reaction time is shown in figure 2. The results in FIG. 2 show that: the 3 rd group primer group provided by the invention can realize accurate detection of staphylococcus aureus genes, real-time monitoring can be realized through fluorescence detection, and results can be judged in advance through a real-time amplification curve.
Example 3 validation of amplification reaction of group 3 primers on extraction of genomes from 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)
Group 3 primers:
1600nM SAU-MF-3 (SEQ ID NO. 5)
1600nM SAU-MR-3 (shown in SEQ ID NO. 6)
Target nucleic acid 1: staphylococcus aureus positive quality control
Target nucleic acid 2: staphylococcus aureus genome
Target nucleic acid 3: shigella genome
Target nucleic acid 4: escherichia coli genome
Target nucleic acid 5: salmonella genome
Target nucleic acid 6: listeria genome
Target nucleic acid 7: vibrio parahaemolyticus genome
A control group without target was also set as a negative control.
8 replicates were set up for each set of amplification reactions. The constant SLAN 96real time PCR reaction temperature is set to 63 ℃, and the reaction time is set to 60min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 3. The results in FIG. 3 show that: the 3 rd group primer group provided by the invention can only amplify the positive quality control of the staphylococcus aureus of the positive sample group and the genomic DNA of the staphylococcus aureus, and can not amplify other bacteria; therefore, the primer group can distinguish staphylococcus aureus, shigella, escherichia coli, salmonella, listeria and vibrio parahaemolyticus. The experimental results further illustrate the specificity of the CDA primer group for detecting staphylococcus aureus provided by the invention.
Example 4 end-point monitoring of Staphylococcus aureus CDA amplification 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 following combinations of reaction solutions for amplification of staphylococcus aureus CDA using hydroxynaphthol blue (HNB) were used.
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)
120μM HNB
Primer set 3:
1600nM SAU-MF-3 (SEQ ID NO. 5)
1600nM SAU-MR-3 (shown in SEQ ID NO. 6)
Target nucleic acid 1: staphylococcus aureus positive quality control
Target nucleic acid 2: staphylococcus aureus genome
Target nucleic acid 3: shigella genome
Target nucleic acid 4: escherichia coli genome
Target nucleic acid 5: salmonella genome
Target nucleic acid 6: listeria genome
Target nucleic acid 7: vibrio parahaemolyticus genome
A control group without target was also set as a negative control.
8 replicates were set up for each set of amplification reactions. The reaction temperature of the constant temperature water bath kettle is set to be 63 ℃, and the reaction time is 60min. 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 used for intuitively judging the reaction result through the color without the assistance of an instrument.
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.
Claims (10)
1. A CDA primer set for the detection of staphylococcus aureus for non-disease diagnostic purposes, comprising: the primer group is SAU-MF-3/SAU-MR-3, wherein the nucleotide sequence of SAU-MF-3 is shown as SEQ ID NO.5, and the nucleotide sequence of SAU-MR-3 is shown as SEQ ID NO. 6.
2. A kit for detecting staphylococcus aureus, which is characterized in that: the kit comprises the CDA primer set of claim 1.
3. The kit for detecting staphylococcus aureus according to claim 2, wherein: the concentration of the primers SAU-MF-3 and SAU-MR-3 in the CDA primer group in a reaction system is 1-2 mu M.
4. The kit for detecting staphylococcus aureus according to claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer solution, ultrapure water and a color developing agent.
5. The kit for detecting staphylococcus aureus 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. The kit for detecting staphylococcus aureus according to claim 4, wherein: the CDA reaction buffer solution comprises Tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 And Triton X-100.
7. The kit for detecting staphylococcus aureus according to claim 2, wherein: the reaction system of the kit comprises the following components:
2～50mM Tris-HClpH8.8
2～20mM KCl
2～20mM(NH 4 ) 2 SO 4
2～20mM MgSO 4
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
1-2 mu M primer SAU-MF-3
1-2 mu M primer SAU-MR-3
The reaction solvent is ultrapure water.
8. The kit of claim 7 for use in a method of detecting staphylococcus aureus 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 amplification reaction solution prepared in the step 1 at the temperature of 60-65 ℃ for 20-80 min, and judging whether the sample contains staphylococcus aureus according to the color development result.
9. Use of the CDA primer set of claim 1 for the detection of staphylococcus aureus for the purpose of non-disease diagnosis.
10. Use of a kit according to any one of claims 2 to 7 for the detection of staphylococcus aureus for the purpose of non-disease diagnosis.
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| CN202210969466.7A CN115927674A (en) | 2022-08-12 | 2022-08-12 | CDA primer group and kit for detecting staphylococcus aureus and application of CDA primer group and kit |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202210969466.7A CN115927674A (en) | 2022-08-12 | 2022-08-12 | CDA primer group and kit for detecting staphylococcus aureus and application of CDA primer group and kit |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090305975A1 (en) * | 2006-04-17 | 2009-12-10 | Guang Yang | Use of Trap Protein Per se as an Active Ingredient for the Manufacture of a Medicament for the Treatment of Staphylococcus Aureus Infection |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| CN113201583A (en) * | 2021-04-29 | 2021-08-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
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2022
- 2022-08-12 CN CN202210969466.7A patent/CN115927674A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090305975A1 (en) * | 2006-04-17 | 2009-12-10 | Guang Yang | Use of Trap Protein Per se as an Active Ingredient for the Manufacture of a Medicament for the Treatment of Staphylococcus Aureus Infection |
| CN106434917A (en) * | 2016-09-24 | 2017-02-22 | 中华人民共和国广州机场出入境检验检疫局 | LAMP primer group and detection kit for staphylococcus aureus and use method of detection kit |
| CN113201583A (en) * | 2021-04-29 | 2021-08-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
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