CN116790770A - CDA primer set and kit for detecting mycobacterium tuberculosis and application of CDA primer set and kit - Google Patents
CDA primer set and kit for detecting mycobacterium tuberculosis and application of CDA primer set and kit Download PDFInfo
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Abstract
The invention discloses a CDA primer set for detecting Mycobacterium Tuberculosis (MTB), a kit and application thereof. The CDA primer set is a primer set for amplifying a conserved region fragment of the mycobacterium tuberculosis, namely MTB-MF-3/MTB-MR-3; the nucleotide sequence of the MTB-MF-3 is shown as SEQ ID NO.5, and the nucleotide sequence of the MTB-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 by the primer group can rapidly and accurately detect whether the sample to be detected contains mycobacterium tuberculosis. In addition, the visual kit provided by the invention can provide great convenience for on-site detection of customs, ports, 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 set for detecting mycobacterium tuberculosis, a kit and application thereof.
Background
Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB) is a pathogen causing tuberculosis, which can invade organs throughout the body, but is most common in causing tuberculosis. Tuberculosis is an ancient disease, widely distributed worldwide, and is the first cause of mortality from bacterial infectious diseases.
The classical detection method of the mycobacterium tuberculosis is separation culture, specifically, the neutralized bacteria-collecting material is inoculated in a solid culture medium, a rubber plug is added at the mouth of a vessel for culture at 37 ℃, and the bacteria are observed for 1 time per week. But the mycobacterium tuberculosis grows slowly, and generally needs to grow into macroscopic falling bacteria for 2-4 weeks. Other relatively rapid and accurate methods, including RT-PCR based detection of amplified signals for Mycobacterium tuberculosis DNA, require only a few bacteria per milliliter to detect positives, and give results in 1-2 days. However, the RT-PCR method requires the use of a precision real-time fluorescent PCR instrument in a standard biochemical analysis laboratory, and three separate test areas for reagent preparation, sample preparation and PCR amplification detection, and is not suitable for rapid screening on site. In addition, the test strip manufactured by the immunodetection technology has the advantages of simplicity, convenience and rapidness, but the method has high false detection rate and low sensitivity, and is difficult to detect MTB carriers in a latent period, so that epidemic situation can be possibly spread. Thus, the development of rapid, sensitive, highly specific nucleic acid marker on-site detection techniques would provide a rapid and powerful treatment for diseases caused by mycobacterium tuberculosis infection.
The closed neck ring-mediated isothermal amplification technology (Closed Dumbbell mediated Isothermal Amplification of nucleic acids, CDA) is a new method developed by the university of Chinese academy of sciences Ningbo life and health industry institute (Chinese patent number: ZL 202110473121.8) and can replace the Japanese LAMP nucleic acid amplification method. The method mainly utilizes 2 different specific primers to identify specific regions of target genes, and carries out amplification reaction under isothermal conditions. Compared with the conventional gene detection means (such as PCR, etc.), the CDA reaction can be completed in a constant-temperature water bath box, has lower requirements on instruments and equipment, is simple to operate, can be accurately completed by non-professionals, and 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 is suitable for rapid diagnosis of mycobacterium tuberculosis. In addition, the key loop-forming primer used by CDA is about 30bp, which is shorter than LAMP (40 bp), thus saving detection cost.
Disclosure of Invention
The invention aims to provide a CDA primer set for detecting mycobacterium tuberculosis, a kit and application thereof.
The technical scheme adopted by the invention for achieving the purpose is as follows:
the invention obtains the primer group with optimal detection effect on the mycobacterium tuberculosis as MTB-MF-3/MTB-MR-3 by screening the 4 pairs of primer groups in the table 1.
TABLE 1
| Sequence number | Primer name | Nucleotide sequence (5 '-3') |
| SEQ ID NO.1 | MTB-MF-1 | ATCAGCCGCGTCCAGGGTTAGCCACACTTTGCG |
| SEQ ID NO.2 | MTB-MR-1 | GTGCTCCTTGAGCCTACTACGACCACATCAACCG |
| SEQ ID NO.3 | MTB-MF-2 | ATCAGCCGCGTCCCACGGTTCAGGGTTAGC |
| SEQ ID NO.4 | MTB-MR-2 | GTGCTCCTTGAGCCTACTACGACCACATCAACCGG |
| SEQ ID NO.5 | MTB-MF-3 | ATCAGCCGCGTCCGTTAGCCACACTTTGCG |
| SEQ ID NO.6 | MTB-MR-3 | GTGCTCCTTGAGCCTACACCACATCAACCGGGAG |
| SEQ ID NO.7 | MTB-MF-4 | ATCAGCCGCGTCCAGGGTTAGCCACACTTT |
| SEQ ID NO.8 | MTB-MR-4 | GTGCTCCTTGAGCCTACTCGACCTACTACGACCACA |
The invention also provides a kit for detecting the mycobacterium tuberculosis, which comprises a CDA primer set MTB-MF-3/MTB-MR-3.
As a preferred embodiment, the concentration of the primers MTB-MF and MTB-MR in the CDA primer set in the reaction system is 1-2 mu M.
As a preferred embodiment, the kit further comprises Bst polymerase, CDA reaction buffer, ultrapure water, and a color developer.
As a preferred embodiment, the color-developing agent 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 composition of the kit reaction system is:
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 MTB-MF-3
1-2 mu M primer MTB-MR-3
The reaction solvent is ultrapure water.
The invention also provides a method for detecting mycobacterium tuberculosis 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 liquid obtained in the step 1 at 60-65 ℃ for 20-80 min, and judging whether the sample contains mycobacterium tuberculosis according to a color development result.
The invention also provides application of the CDA primer set in detection of mycobacterium tuberculosis for non-disease diagnosis.
The invention also provides application of the kit in detection of mycobacterium tuberculosis for the purpose of non-disease diagnosis.
Compared with the prior art, the invention has the beneficial effects that:
1, the CDA primer set provided by the invention is aimed at a specific conserved region Mycobacterium tuberculosis strain SGF0702019 chromoname (GenBank: CP 095023.1) of mycobacterium tuberculosis, consists of 2 primers, and is an intrasequence primer pair (MTB-MF-3/MTB-MR-3). The primer group provided by the invention has high sensitivity and strong specificity, and the kit prepared by the primer group can rapidly and accurately detect whether the sample to be detected contains mycobacterium tuberculosis.
2, because 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.
The method or the kit provided by the invention can complete related detection without expensive instruments or complicated operations, so that the visual kit provided by the invention can provide great convenience for on-site detection of airports, customs, communities and the like with low professionals, and can realize rapid and accurate detection of mycobacterium tuberculosis.
Drawings
FIG. 1 is a graph showing the change of fluorescence intensity with time of the amplification reaction of four pairs of primer sets in example 1 of the present invention.
FIG. 2 is a graph showing the change in fluorescence intensity with time of the amplification reaction of the 3 rd primer set in example 2 of the present invention.
Fig. 3 is a graph showing endpoint monitoring based on color change for reactions using HNB in example 3 of the present invention.
Detailed Description
The following describes the technical scheme of the present invention in detail by referring to examples. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and can be carried out with reference to the specific methods in the "molecular cloning Experimental guidelines" (third edition) J.Sam Brookfield, or according to the kit and product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 application of Eva Green to respective verification of amplification reaction of Mycobacterium tuberculosis Gene DNA fragments by four primer sets
Eva Green, like SYBR Green I, is a dye with Green excitation wavelength that binds to all dsDNA double helix minor groove regions, and inhibits nucleic acid amplification reactions such as PCR far less than the latter. The fluorescent signal intensity of Eva Green correlates with the number of double stranded DNA. In the free state, eva Green emits weak fluorescence, but after binding to double-stranded DNA, its fluorescence is greatly enhanced. Thus, 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 (ddH was added to the rest 2 O to 25 μl):
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)
Single tube use primers:
1600nM MTB-MF-1 (shown in SEQ ID NO. 1)
1600nM MTB-MR-1 (shown in SEQ ID NO. 2);
1600nM MTB-MF-2 (shown in SEQ ID NO. 3)
1600nM MTB-MR-2 (shown in SEQ ID NO. 4);
1600nM MTB-MF-3 (shown in SEQ ID NO. 5)
1600nM MTB-MR-3 (shown in SEQ ID NO. 6);
1600nM MTB-MF-4 (shown in SEQ ID NO. 7)
1600nM MTB-MR-4 (shown in SEQ ID NO. 8).
And (3) target: mycobacterium tuberculosis genomic DNA dsDNA (shown in SEQ ID NO. 9).
SEQ ID NO.9：
gatcagcgatcgtggtcctgcgggctttgccgcgggtggtcccggacaggccgagtttggtcatcagccgttcgacggtgcatctggccacctcgatgccctcacggttcagggttagccacactttgcgggcaccgtaaacaccgtagttggcggcgtggacgcggctgatgtgctccttgagttcgccatcgcgcagctcgcggcggctgggctcccggttgatgtggtcgtagtaggtcgatggggcgatcggcacacccagctcggtcagctgtgtgcagatcgactcgacaccccaccgcaaaccatcggggcc
Meanwhile, four pairs of primers are respectively provided with a control group without targets.
The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min. The detection result is shown in fig. 1, and can be found by fig. 1: amplification occurs in positive groups of the four primer sets, false positives occur in the primer set 2, and the amplification Ct value is fastest in the primer set 3. The results show that: the 3 rd primer group provided by the invention can realize rapid amplification of the mycobacterium tuberculosis genes, no false positive appears in a negative control group, the fluorescent detection is applied to the primer group, the real-time monitoring can be carried out, and the result can be judged in advance through a real-time amplification curve.
Example 2 verification of amplification reaction of Mycobacterium tuberculosis Gene DNA fragments Using Eva Green 3 rd primer set (repeated experiments)
The procedure is as in example 1.
The single tube reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
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)
Single tube use primers:
1600nM MTB-MF-3 (shown in SEQ ID NO. 5)
1600nM MTB-MR-3 (shown in SEQ ID NO. 6);
the reaction solution was combined in the same manner as in example 1, except that 8 groups of the target and the no-target control group were simultaneously provided. And (3) target: mycobacterium tuberculosis genomic DNA dsDNA (shown in SEQ ID NO. 9).
The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min. The repeated experiments of 8 negatives, no false positive, better repeatability and the control experiments of 8 positives are carried out on the primer group 3, 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 primer group provided by the invention can realize accurate detection of the mycobacterium tuberculosis genes, can realize real-time monitoring through fluorescence detection, and can judge the result in advance through a real-time amplification curve.
EXAMPLE 3 Mycobacterium tuberculosis CDA amplification reaction end-point monitoring Using hydroxynaphthol blue (HNB)
Hydroxyl Naphthol Blue (HNB) belongs to a metal ion indicator, and aims at the change of magnesium ions or manganese ions combined with byproduct pyrophosphates in the reaction, so that different indication colors are displayed to judge the result.
The combinations of the reaction solutions for CDA amplification of Mycobacterium tuberculosis using hydroxynaphthol blue (HNB) are shown below.
The procedure is as in example 1.
The single tube reaction solution was combined as follows (ddH was added to the rest 2 O to 25 μl):
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 (NE W ENGLAND Biolabs)
120μM HNB
Single tube use primers:
1600nM MTB-MF-3 (shown in SEQ ID NO. 5)
1600nM MTB-MR-3 (shown in SEQ ID NO. 6);
the reaction solution was combined in the same manner as in example 1, except that 8 groups of the target and the no-target control group were simultaneously provided. And (3) target: mycobacterium tuberculosis genomic DNA dsDNA (shown in SEQ ID NO. 9).
The SLAN 96real time PCR reaction temperature was set to be constant at 63℃and the reaction time was set to be 60min.
The amplification reaction was set up with 8 negative controls and 8 positive controls. The reaction temperature of the constant temperature water bath kettle is set to be 63 ℃ and the reaction time is set to be 60 minutes. The end point result of the yin-yang reaction is shown in fig. 3, wherein the color of violet is negative, and the sky blue is positive. The experimental results show that: the HNB can intuitively judge the reaction result through the color, and can judge without the assistance of an instrument.
The foregoing 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 scope of the technical solution of the present invention, and any changes and modifications are within the scope of the present invention.
Claims (10)
1. A CDA primer set for detecting mycobacterium tuberculosis for non-disease diagnosis purposes, characterized in that: the primer group is MTB-MF-3/MTB-MR-3, wherein the nucleotide sequence of the MTB-MF-3 is shown as SEQ ID NO.5, and the nucleotide sequence of the MTB-MR-3 is shown as SEQ ID NO. 6.
2. A kit for detecting mycobacterium tuberculosis, characterized in that: the kit comprises the CDA primer set of claim 1.
3. A kit for detecting mycobacterium tuberculosis as described in claim 2, wherein: the concentration of the primer MTB-MF-3 and the primer MTB-MR-3 in the CDA primer group in the reaction system is 1-2 mu M.
4. A kit for detecting mycobacterium tuberculosis as described in claim 2, wherein: the kit also comprises Bst polymerase, CDA reaction buffer, ultrapure water and a color developing agent.
5. A kit for detecting mycobacterium tuberculosis according to claim 4, wherein: the color developing agent is selected from one of Sybrgreen I, eva green, hydroxynaphthol blue and chrome black T.
6. A kit for detecting mycobacterium tuberculosis according to claim 4, wherein: the CDA reaction buffer comprises Tris-HCl, KCl and (NH) 4 ) 2 SO 4 、MgSO 4 And TritonX-100.
7. A kit for detecting mycobacterium tuberculosis as described in claim 2, wherein: the kit reaction system 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 MTB-MF-3
1-2 mu M primer MTB-MR-3
The reaction solvent is ultrapure water.
8. A method of detecting mycobacterium tuberculosis for non-disease diagnostic purposes using the kit of claim 7, 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 60-65 ℃ for 20-80 min, and judging whether the sample contains mycobacterium tuberculosis according to a color development result.
9. Use of the CDA primer set according to claim 1 for detection of mycobacterium tuberculosis for non-disease diagnosis purposes.
10. Use of a kit according to any one of claims 2-7 for the detection of mycobacterium tuberculosis for the purpose of non-disease diagnosis.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210842037.3A CN116790770A (en) | 2022-07-18 | 2022-07-18 | CDA primer set and kit for detecting mycobacterium tuberculosis and application of CDA primer set and kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210842037.3A CN116790770A (en) | 2022-07-18 | 2022-07-18 | CDA primer set and kit for detecting mycobacterium tuberculosis and application of CDA primer set and kit |
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| CN116790770A true CN116790770A (en) | 2023-09-22 |
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