CN115927674A - CDA primer group and kit for detecting staphylococcus aureus and application of CDA primer group and kit - Google Patents

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

A CDA primer set, kit and application for detecting Staphylococcus aureus

technical field

The invention belongs to the technical field of biological detection, and in particular relates to a CDA primer set, a kit and an application thereof for detecting Staphylococcus aureus.

Background technique

Staphylococcus aureus (SAU) is a common food-borne pathogenic microorganism that widely exists in the natural environment. Staphylococcus aureus can produce enterotoxin and cause food poisoning under appropriate conditions. Reports of food poisoning caused by Staphylococcus aureus emerge in endlessly. Food poisoning caused by Staphylococcus aureus accounts for about 25% of food-borne microbial food poisoning incidents. Microbial pathogens.

The classic detection method of Staphylococcus aureus is isolation and culture, specifically: inoculate the neutralized bacteria-collecting material on solid medium, culture at 37°C with a rubber stopper on the mouth of the vessel, and observe once a week. However, Staphylococcus aureus grows slowly, and generally takes 2 to 4 weeks to grow into colonies visible to the naked eye. Other relatively rapid and accurate methods, including RT-PCR-based detection of the amplification signal of Staphylococcus aureus DNA, only need to contain a few bacteria per milliliter to detect positive results, and the results can be obtained within 1-2 days result. However, the RT-PCR method requires the use of a sophisticated real-time fluorescent PCR instrument in a standard biochemical analysis laboratory, and requires three separate test areas for reagent preparation, specimen preparation, and PCR amplification detection, and is not suitable for on-site rapid screening. In addition, the test strips made by immunoassay technology have the advantage of being simple and quick, but this method has a high false detection rate and low sensitivity, making it difficult to detect SAU carriers in the incubation period, which may cause the spread of the epidemic. Therefore, the development of rapid, sensitive, and highly specific nucleic acid marker on-site detection technology will provide a rapid and powerful treatment for diseases caused by Staphylococcus aureus infection.

Closed Dumbbell mediated Isothermal Amplification of nucleic acids (CDA) is a new method developed by Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences (China Patent No.: ZL202110473121.8), which can An alternative to the Japanese LAMP nucleic acid amplification method. This method mainly uses two different specific primers to identify specific regions of the target gene, and performs amplification reactions under isothermal conditions. Compared with conventional genetic detection methods (such as PCR, etc.), the CDA reaction can be completed in a constant temperature water bath, which requires less equipment and is easy to operate. It can also be completed accurately by non-professionals. It is suitable for primary medical institutions and local inspection and quarantine departments. Moreover, CDA can also shorten the operation time, improve the detection efficiency, and reduce the probability of sample contamination, which is suitable for the rapid diagnosis of Staphylococcus aureus. In addition, the key looping primer used by CDA is about 30bp, which is shorter than that of LAMP (40bp), which will save the detection cost.

Contents of the invention

The purpose of the present invention is to provide a CDA primer set, kit and application for detecting Staphylococcus aureus.

The technical scheme that the present invention adopts for realizing the above object is as follows:

In the present invention, by screening the 4 pairs of primer sets described in Table 1, the primer set with the best detection effect on Staphylococcus aureus is SAU-MF-3/SAU-MR-3.

Table 1

serial number Primer name Nucleotide sequence (5'-3') SEQ ID NO.1 SAU-MF-1 CAGCTTGCTTACTAATCAATCACTGGACCG SEQ ID NO.2 SAU-MR-1 CAATGACCTCGTTTTTCACGCAAACTGTTG 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 present invention also provides a kit for detecting Staphylococcus aureus, which includes a CDA primer set SAU-MF-3/SAU-MR-3.

As a preferred embodiment, the concentrations of the primers SAU-MF and SAU-MR in the CDA primer set in the reaction system are both 1-2 μM.

As a preferred embodiment, the kit also includes Bst polymerase, CDA reaction buffer, ultrapure water, and chromogen.

As a preferred embodiment, the color developer is selected from Sybr green I, Eva green, hydroxynaphthol blue or chrome black T.

As a preferred embodiment, the CDA reaction buffer includes Tris-HCl, KCl, (NH ₄ ) ₂ SO ₄ , MgSO ₄ and Triton X-100.

As a preferred embodiment, the composition of the kit reaction system is:

2～50mM Tris-HClpH8.8

2～20mM KCl

2～20mM(NH ₄ ) ₂ SO ₄

2～20mM _MgSO4

0.1～0.5%TritonX-100

0.2～1M betaine

1～1.6mM dNTP

5～10U Bst DNA polymerase

100～150μmol/L developer

1～2μM Primer SAU-MF-3

1～2μM Primer SAU-MR-3

The reaction solvent is ultrapure water.

The present invention also provides the method for detecting Staphylococcus aureus that the kit is used for non-disease diagnosis purpose, comprising the following steps:

Step 1, mixing the nucleic acid sample to be detected with the reaction system of the kit to prepare an amplification reaction solution;

Step 2: react the amplification reaction solution obtained in step 1 at 60-65° C. for 20-80 minutes, and judge whether the sample contains Staphylococcus aureus according to the color development result.

The present invention also provides the application of the CDA primer set in the detection of Staphylococcus aureus for the purpose of non-disease diagnosis.

The present invention also provides the application of the above kit in the detection of Staphylococcus aureus for the purpose of non-disease diagnosis.

Compared with prior art, the beneficial effect of the present invention is:

1. The CDA primer set provided by the present invention is directed at the specific conserved region Staphylococcusaureus strain 332chromosome (GenBank: CP077893.1) of Staphylococcus aureus, and consists of 2 primers, which are primer pairs within the sequence (SAU-MF-3/SAU-MR -3). The primer set provided by the invention has high sensitivity and strong specificity, and the kit made from it can quickly and accurately detect whether the sample to be tested contains Staphylococcus aureus.

2. Since the primer set provided by the present invention has extremely high specificity, the time required for CDA amplification is short, the detection time is further shortened, and the operation is simple.

3. The method or kit provided by the present invention can complete the relevant detection without expensive instruments and complicated operations. Therefore, the visualization kit provided by the present invention will be used in airports, customs, communities and other places with low professionalism. It provides great convenience for on-site detection and can realize rapid and accurate detection of Staphylococcus aureus.

Description of drawings

Fig. 1 is a graph showing the variation of the fluorescence intensity of the amplification reaction of the four pairs of primer sets as a function of the reaction time in Example 1 of the present invention.

Fig. 2 is a graph showing the fluorescence intensity of the amplification reaction of the third group of primers as a function of reaction time in Example 2 of the present invention.

Fig. 3 is a graph showing the fluorescence intensity of the amplification reaction of the third group of primers as a function of reaction time in Example 3 of the present invention.

Fig. 4 is an end point monitoring diagram based on color change in the reaction using HNB in Example 4 of the present invention.

Detailed ways

The technical solution of the present invention will be described in detail below in conjunction with the embodiments. Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and can be carried out with reference to the specific methods in the book "Molecular Cloning Experiment Guide" (Third Edition) J. Sambrook, or according to the kit and product manual. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1 uses Eva Green to verify respectively the amplification reaction of four pairs of primer sets to Staphylococcus aureus gene DNA fragment

Similar to SYBR Green I, Eva Green is a dye with a green excitation wavelength that binds to the minor groove region of all dsDNA double helices, and its inhibition on nucleic acid amplification reactions such as PCR is much smaller than that of the latter. The fluorescence signal intensity of Eva Green correlates with the amount of double-stranded DNA. In the free state, Eva Green emits weak fluorescence, but when combined with double-stranded DNA, its fluorescence is greatly enhanced. Therefore, the amount of double-stranded DNA present in the nucleic acid amplification system can be detected based on the fluorescent signal.

The single-tube reaction solution was combined as follows (add ddH ₂ O to 25 μL for the rest):

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 dNTPs

8U Bst DNA Polymerase (NEW ENGLAND Biolabs)

1X Eva Green(Biotum)

Primers used in a single tube:

1600nM SAU-MF-1 (shown in SEQ ID NO.1)

1600nM SAU-MR-1 (shown in SEQ ID NO.2);

1600nM SAU-MF-2 (shown in SEQ ID NO.3)

1600nM SAU-MR-2 (shown in SEQ ID NO.4);

1600nM SAU-MF-3 (shown in SEQ ID NO.5)

1600nM SAU-MR-3 (shown in SEQ ID NO.6);

1600nM SAU-MF-4 (shown in 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:

ctgacgtatcttccataaatgatctaaaaattggtagttcttcttcagataaaaatcttactttaacaccattctttttaactttttcgtgtttctttttctaagtccatccatatttttaatgatgtcatctgctgttttcttttaaatctaacactgagtgataacgaatttgtagcacaggat caaatcctttatggaatccagtatgttcaaatcctaagttatcatttttcaaagaaccaatcattaccagcattacctgtaatctcgccatcatgattcaagtattgatatggtaaatatggatcgatatgtaggtatagacaacgatgttttttaacatattttgataattcattaaagaaaaagtgtacgagttcttgattt tcataatcaatcactggaccgcgatttgaataaaaatacttgaacactttcataacaggtacagcagtaagtaagcaagctgcaatgacctcgttattattgttttttattccactaaatgtttcataaccttcagcaagctttaactcatagtggccaacagtttgcgtgaaatgactgtatggcatgctatctgtaa aggcaccaaactctttagctgttaaatttgtaaacttcattatcattactcctatttgtctctcgttaattaatttcattccgtatttgcagtttttctatttcccctctgcaaatgtcaaaaataataaatctaatctaaataagtatacaatagttaatgttaaaactaaaacataaacgctttaattgcgtatactt ttatagtaatatttagatttttgaatacaatttcaaaaaaagtaatatgaacgtttgggtttgctcatattacttttgtatcatttctattcaattttataattcaccgtttttcactttttcaaacagtattcgcctaatttttttaaatcaagtaaacttaattattcaatgtttgttggatagattgtaaatatttaatgatt tcctcacgcgtgttagatttaaatcgcttaacgatttcgctaccaatgacaatgccatctgcaacctcttttatctgcaacatgttgtggtgttcttataccaaatcctgcgacaactggcacattggctatcgctttaattgactcaatttttcgttttaattctggatgaaacgcaccgttttgccctgt tgtcgcattcatcgtcacagtataaataaagccttccgcatgggaatacgatatcttttatacgtttgtcatcagtagtcatcgcaactaacgatatgattttgacgccatagtgactaaattgttgttttaaacgctgcgataattcatatggtaaatcaggaataattaagccgtagacaccagtatctcgacatttt tcaaaaaacgcttgttctccataatgacaaataatattataatacgtcattaatacatagttacacttaatttgatcaccatgtttttctaattgattgaaaatataatctatcgtgatgccttgtttaatcgcttgttgacctgcttccatgataactggaccatcagcaaccggatcagagaaaggtactccaatttcaattatatct gcaccattttcactcaacaatgttgcattttcaatcaaatctttatgcccataatataaggtataaataatttagtcatttgcaagacctcgctctaccatatattgtctaattgtttccatatctttatcgccacgtccagaaatagttactacaataatcttctttcgacatcgtaggcgctagtctttcaacataactcag tgcatgtgcactttcaattgcaggtataataccttcatgttttgtaaagttgattaaagcattcattgcttgtgtatcactagcattttcaaaagttactctaccaatgtcgtggtaataagaatgttctggtccaataccaggataatcaagtcc

At the same time, the four pairs of primer groups were respectively set up as control groups without targets.

Set the SLAN 96 real time PCR reaction temperature to be constant at 63°C, and the reaction time to be 60min. The detection results are shown in Figure 1. From Figure 1, it can be found that only the positive groups of the two primer sets were amplified, the amplification rate of the second set was not high, and the amplification Ct value of the third set of primer sets was the fastest. The result shows: the 3rd group of primer sets provided by the present invention can realize the fast amplification to Staphylococcus aureus gene, and negative control group does not have false positive to occur, and fluorescence detection is applied wherein can carry out real-time monitoring, through real-time amplification curve The results can be judged in advance.

Embodiment 2 uses Eva Green to verify the amplification reaction of the 3rd group of primer sets to Staphylococcus aureus gene DNA fragment (repeat experiment)

Method is with embodiment 1.

The single-tube reaction solution was combined as follows (add ddH ₂ O to 25 μL for the rest):

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 dNTPs

8U Bst DNA Polymerase (NEW ENGLAND Biolabs)

1X Eva Green(Biotum)

Primers used in a single tube:

1600nM SAU-MF-3 (shown in SEQ ID NO.5)

1600nM SAU-MR-3 (shown in SEQ ID NO.6);

Simultaneously, 8 control groups with targets and without targets were set up, and the reaction solution combinations were the same as in Example 1. Target: Staphylococcus aureus genomic DNA dsDNA (shown in SEQ ID NO.9).

Set the SLAN 96 real time PCR reaction temperature to be constant at 63°C, and the reaction time to be 60min. 8 negative repeated experiments were carried out on primer group 3, no false positives occurred, and the repeatability was good, and a control experiment of 8 positive samples was carried out. The curve of the fluorescence intensity changing with the reaction time is shown in Figure 2. The results in Fig. 2 show that: the third set of primers provided by the present invention can realize the accurate detection of Staphylococcus aureus gene, the fluorescence detection can realize real-time monitoring, and the result can be judged in advance through the real-time amplification curve.

Example 3 Application of Eva Green to verify the amplification reaction of the third group of primer sets to the extraction genome of various bacteria

Method is with embodiment 1.

The reaction solution was combined as follows (add ddH ₂ O to 25 μL for the rest):

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 dNTPs

8U Bst DNA Polymerase (NEW ENGLAND Biolabs)

1X Eva Green(Biotum)

Group 3 primers:

1600nM SAU-MF-3 (shown in 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: Genome of Staphylococcus aureus

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

At the same time, a target-free control group was set up as a negative control.

Eight replicates were set up for each set of amplification reactions. Set the SLAN 96 real time PCR reaction temperature to be constant at 63°C, and the reaction time to be 60min. The curve of the fluorescence intensity changing with the reaction time is shown in Fig. 3 . Fig. 3 result shows: the 3rd group of primer sets provided by the present invention can only amplify positive sample group Staphylococcus aureus positive quality control and Staphylococcus aureus genomic DNA, all can't amplify for other bacteria; Therefore this primer set Able to distinguish Staphylococcus aureus from Shigella, Escherichia coli genome, Salmonella genome, Listeria genome, Vibrio parahaemolyticus genome. The experimental results further illustrate the specificity of the CDA primer set provided by the present invention for detecting Staphylococcus aureus.

Example 4 Application of hydroxynaphthol blue (HNB) to monitor the end point of Staphylococcus aureus CDA amplification reaction

Hydroxynaphthol blue (HNB) is a metal ion indicator, which is aimed at the change of the amount of magnesium ions or manganese ions combined with the by-product pyrophosphate during the reaction, so as to present different indicator colors to realize the judgment of the result.

Combinations of reaction solutions for CDA amplification of Staphylococcus aureus using hydroxynaphthol blue (HNB) are shown below.

The reaction solution was combined as follows (add ddH ₂ O to 25 μL for the rest):

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 dNTPs

8U Bst DNA Polymerase (NEW ENGLAND Biolabs)

120 μM HNB

Group 3 primers:

1600nM SAU-MF-3 (shown in 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: Genome of Staphylococcus aureus

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

At the same time, a target-free control group was set up as a negative control.

Eight replicates were set up for each set of amplification reactions. Set the constant temperature water bath to keep the reaction temperature at 63°C and the reaction time at 60min. The results of negative and positive reaction endpoints are shown in Figure 4, where the color is violet for negative, and sky blue for positive. The experimental results show that: using HNB, the reaction result can be judged intuitively through the color, and the interpretation can be carried out without the aid of the instrument.

The above are only some preferred embodiments of the present invention, and the present invention is not limited to the content of the embodiments. For those skilled in the art, various changes and modifications can be made within the concept scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims (10)

1. A CDA primer set for detecting Staphylococcus aureus of non-disease diagnosis purpose, is characterized in that: described primer set is SAU-MF-3/SAU-MR-3, wherein the nucleotide of SAU-MF-3 The sequence is shown in SEQ ID NO.5, and the nucleotide sequence of SAU-MR-3 is shown in SEQ ID NO.6. 2. A test kit for detecting Staphylococcus aureus, characterized in that: said test kit comprises the CDA primer set according to claim 1. 3. a kind of test kit for detecting Staphylococcus aureus as claimed in claim 2, is characterized in that: the concentration of primer SAU-MF-3 and SAU-MR-3 in the reaction system in the described CDA primer set is equal to 1~2μM. 4. A test kit for detecting Staphylococcus aureus as claimed in claim 2, characterized in that: said test kit also comprises Bst polymerase, CDA reaction buffer, ultrapure water and chromogen. 5. a kind of test kit for detecting Staphylococcus aureus as claimed in claim 4, is characterized in that: described chromogen is selected from the one in Sybr green I, Eva green, hydroxynaphthol blue, chrome black T . 6. A kind of test kit for detecting Staphylococcus aureus as claimed in claim 4, is characterized in that: described CDA reaction buffer comprises Tris-HCl, KCl, (NH ₄ ) ₂ SO ₄ , MgSO ₄ and Triton X -100. 7. a kind of test kit for detecting Staphylococcus aureus as claimed in claim 2, is characterized in that: the composition of described kit reaction system is: 2～50mM Tris-HClpH8.8 2～20mM KCl 2～20mM(NH ₄ ) ₂ SO ₄ 2～20mM _MgSO4 0.1～0.5%TritonX-100 0.2～1M Betaine 1～1.6mM dNTP 5～10U Bst DNA polymerase 100～150μmol/L developer 1～2μM Primer SAU-MF-3 1～2μM Primer SAU-MR-3 The reaction solvent is ultrapure water. 8. the kit described in claim 7 is used for the method for the detection staphylococcus aureus of non-disease diagnosis purpose, comprises the following steps: Step 1, mixing the nucleic acid sample to be detected with the reaction system of the kit to prepare an amplification reaction solution; Step 2: react the amplification reaction solution prepared in step 1 at 60-65° C. for 20-80 minutes, and judge whether the sample contains Staphylococcus aureus according to the color development result. 9. The application of the CDA primer set according to claim 1 in the detection of Staphylococcus aureus for the purpose of non-disease diagnosis. 10. The application of the kit according to any one of claims 2-7 in the detection of Staphylococcus aureus for the purpose of non-disease diagnosis.

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