CN111793703A - Kit for enzyme digestion probe constant temperature detection of mycobacterium tuberculosis nucleic acid - Google Patents
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Abstract
The invention provides a kit for detecting mycobacterium tuberculosis nucleic acid by using an enzyme digestion probe at constant temperature, which comprises an isothermal amplification primer for detecting mycobacterium tuberculosis IS6110 gene and a corresponding probe capable of being digested by RNaseH; isothermal amplification primers for detecting a mycobacterium tuberculosis Pab gene and a corresponding probe which can be digested by RNaseH enzyme. The primers and the probes which are efficient and can be amplified at constant temperature are utilized to carry out constant-temperature multiple detection on the mycobacterium tuberculosis, and the kit has high sensitivity and high specificity.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a kit for detecting mycobacterium tuberculosis nucleic acid by using an enzyme digestion probe at a constant temperature.
Background
Tuberculosis is a chronic infectious disease caused by infection with mycobacterium tuberculosis. Mycobacterium tuberculosis invades various organs of the body, but is mainly infected with lung, and is called tuberculosis. Tuberculosis, also known as tuberculosis and 'white plague', is one of the longest diseases accompanied with human history. At present, about 1/3 people worldwide infect Mycobacterium tuberculosis, with 900 ten thousand new cases appearing each year, 80% of which are concentrated in developing countries. The tuberculosis epidemic situation of China ranks second in 22 tuberculosis-high-burden countries in the world, and people dying from tuberculosis every year are as many as 25 thousands. Therefore, the early and accurate diagnosis and active tuberculosis monitoring are the basis and the premise for treating and controlling the respiratory infectious disease, and the search for a rapid and accurate diagnosis method is always the first research field of the tuberculosis science.
Currently, the main diagnostic methods for mycobacterium tuberculosis are: pathogen isolation methods, immunological methods, PCR detection methods based on nucleic acid levels. The pathogen separation method is a gold standard for clinical diagnosis, but has the defects of long time consumption, complex operation and the like; immunological methods have not been widely used because of their non-specific amplification and low sensitivity; the PCR detection method has high specificity and sensitivity, but has the defects of expensive experimental instruments, stable experimental environment, high consumable cost and the like, and is limited by the operating technology of detection personnel.
In order to overcome the disadvantages of the PCR amplification technology, a number of isothermal nucleic acid amplification technologies have been developed in recent years. The method does not require template denaturation, long temperature cycling, i.e., a process of adjusting temperature, and thus, does not require specific expensive equipment. Compared with the traditional nucleic acid level detection method, the method has the characteristics of simplicity, convenience, rapidness, accuracy, low price, easy detection and the like. The kit is widely applied to pathogen detection at present, and has great significance in the development of clinical diagnosis technology by using a constant-temperature nucleic acid amplification technology to detect the mycobacterium tuberculosis. However, most of the currently used isothermal nucleic acid amplification techniques still have some defects, such as that most of the techniques cannot perform multiplex detection in a single tube, and some isothermal fluorescence detection methods are nonspecific fluorescence detection during interpretation, which affect the practicability of detection in the isothermal nucleic acid amplification method.
Disclosure of Invention
In order to solve the problems, the invention provides a kit for detecting mycobacterium tuberculosis nucleic acid by using an enzyme digestion probe at constant temperature, which comprises: detecting isothermal amplification primers of the mycobacterium tuberculosis IS6110 gene and a corresponding probe which can be digested by RNaseH; isothermal amplification primers for detecting a mycobacterium tuberculosis Pab gene and a corresponding probe which can be digested by RNaseH enzyme.
In one embodiment, the isothermal amplification primer for detecting the Mycobacterium tuberculosis IS6110 gene SEQ ID NO.1-SEQ ID NO.6 and the corresponding RNaseH restriction enzyme probe are SEQ ID NO.7, the probe comprises an RNA base, the base shown by italic underlining IS the RNA base,
in one embodiment, the isothermal amplification primer SEQ ID NO.8-SEQ ID NO.13 for detecting the Pab gene of Mycobacterium tuberculosis and the corresponding RNaseH-cleavable probe are SEQ ID NO.7, the probe comprises an RNA base, the base shown by italic underlining is the RNA base,
in one embodiment, the kit further comprises: detecting a section of artificially synthesized internal reference gene isothermal amplification primer SEQ ID NO.15-SEQ ID NO.20 which is not homologous with mycobacterium tuberculosis nucleic acid and human nucleic acid and a corresponding RNaseH enzyme digestion probe SEQ ID NO.21, wherein the probe comprises an RNA base, and the base displayed by italic underlining is the RNA base
In one embodiment, the ratio OF IF: IB: LF: LB: OF: OR in each OF the primers for detecting IS6110 gene, Pab gene and reference gene OF Mycobacterium tuberculosis IS 8:8:4:4:1:1, preferably, the concentrations OF IF and IB primers OF the three genes for detection are 0.8 μ M, the concentrations OF LF and LB primers are 0.4 μ M, and the concentrations OF OF and OR primers are 0.1 μ M.
In one embodiment, the probes for detecting IS6110 gene and Pab gene in the kit are SEQ ID NO: 7 and SEQ ID NO: 14 and a probe SEQ ID NO: 21 labeled with different fluorophores.
The invention has the advantages that: the primers and the probes which are efficient and can be amplified at constant temperature are utilized to carry out constant-temperature multiple detection on the mycobacterium tuberculosis, and the kit has high sensitivity and high specificity. Multiple target genes can be detected in one reaction tube simultaneously, quality control is carried out by using internal reference genes, detection is fast, and the whole detection time is within 30 min.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a fluorescence amplification curve diagram of three templates of the special primer probe system of the invention, namely L1, L2 and L3, which are subjected to fluorescence isothermal amplification;
FIG. 2 is a fluorescence amplification curve diagram of three tuberculosis positive clinical samples of the special primer probe system of the invention, namely S1, S2 and S3, which are subjected to fluorescence isothermal amplification;
FIG. 3 is a fluorescent amplification curve diagram of the specific primer probe system in the invention for fluorescent isothermal amplification of Mycobacterium tuberculosis genome DNA, human DNA and other various pathogen genome nucleic acids; and
FIG. 4 is a graph showing the amplification curve of 5 tuberculosis positive samples detected by the kit of the present invention.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, but not all of the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In the following examples, unless otherwise specified, all methods are conventional in the art. In the following examples, the materials used, unless otherwise specified, were purchased from biochemicals companies in the field.
In the following examples, unless otherwise specified, all methods are conventional in the art. Isothermal amplification buffer, MgSO4, BstDNA polymerase and AMV reverse transcriptase were purchased from New England Biolabs, N.Y., dNTP was purchased from Bao bioengineering, Inc., and RNaseH was purchased from IDT, Inc., all of which were synthesized by Shanghai Bioengineering services, Inc. The plasmid mini-extraction kit, the bacterial genome DNA and the RNA extraction kit are purchased from Beijing Tiangen Biotechnology GmbH. The mycobacterium tuberculosis nucleic acid detection kit (PCR-fluorescent probe method) is purchased from Beijing Hua Dagibbi love biotechnology limited.
The first embodiment is as follows: optimization of the primer Probe in the present invention
Downloading standard sequences of mycobacterium tuberculosis IS6110 and Pab genes from NCBI, artificially synthesizing a gene sequence which IS not homologous with mycobacterium tuberculosis nucleic acid and human nucleic acid as a standard sequence of an exogenous internal reference gene, and designing a constant-temperature amplification primer and an RNaseH enzyme digestion probe for detecting the IS6110, Pab and the exogenous internal reference gene.
Selection of IS6110 Gene reaction conditions
Screening of primer probe for IS6110 gene
TABLE 1 primers and probes used in IS6110 Gene screening Process
2. The template used was: two dilutions of 5 and 10 copies/. mu.L of IS6110 gene standard plasmid, high-concentration genomic nucleic acid of human genomic DNA and 10 other common pathogens of lung infection (such as streptococcus pneumoniae, legionella pneumophila, haemophilus influenzae, klebsiella pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, acinetobacter baumannii, influenza virus, neocoronavirus and adenovirus) (the concentrations of the nucleic acids are equal to each other>104copies/μL)。
3. Enzyme digestion probe constant temperature amplification system
TABLE 2 isothermal amplification system of IS6110 gene restriction enzyme probe
4. The amplification procedure was: 30cycles:63 ℃ for 1min (Collection of fluorescence)
5. The screening results are shown in the following table, and from the aspect of sensitivity, the second set of primer matched probe 1 or matched probe 2, and the fourth set of primer matched probe 1 can be amplified to 5 copies/mu L; from the aspect of specificity, the three combinations only have the fourth set of primer matched probe 1 without amplification signals when other pathogens are amplified, namely, cross reaction cannot be generated; therefore, the fourth set of primers and probe 1 IS selected as the special primer probe of IS6110 gene.
TABLE 3 IS6110 Gene differential primer pairs and Probe screening results
(II) selection of Pab Gene reaction conditions
Screening of primer probe for Pab Gene
TABLE 4 primers and probes used in the Pab Gene screening Process
2. The template used was: two dilutions of 5 and 10 copies/. mu.L of Pab gene standard plasmid, human genomic DNA, and high genomic nucleic acid concentration of 10 other common pathogens of lung infection such as Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, influenza virus, neocoronavirus, and adenovirus (all nucleic acid concentrations are equal to each other)>104copies/μL)。
3. Enzyme digestion probe constant temperature amplification body
TABLE 5 isothermal amplification System for Pab Gene restriction enzyme
| Reaction reagent | Quantitative concentration of end substance or end enzyme activity unit |
| 10 × |
1× |
| MgSO4 | 6mM |
| dNTP | 1.4mM |
| Bst DNA polymerase | 8U |
| AMV reverse transcriptase (amplified DNA is not added, amplified RNA is added) | 10U (when adding) |
| Betaine solution, PCR grade (5M) | 0.24M |
| RNaseH2 | 15mU |
| Primer mix | 1.3μM |
| Probe (20P) | 0.2μM |
| Water (W) | Make up to 30 mu L |
4. The amplification procedure was: 30cycles:63 ℃ for 1min (Collection of fluorescence)
5. The screening results are shown in the following table, and only the third set of primers can amplify to 5 copies/mu L when matched with the probe 1 from the aspect of sensitivity; from the aspect of specificity, the third set of primer matched probe 1 has no amplification signal when amplifying other pathogens, namely, no cross reaction is generated; therefore, the third set of primers and probe 1 is selected as the special primer probe of Pab gene.
TABLE 6 screening results of different primer pairs and probes for Pab genes
(III) selection of reaction conditions of reference genes
TABLE 7 primers and probes used in the screening Process of reference genes
2. The template used was: two dilutions of 5 and 10 copies/. mu.L of the internal reference gene standard plasmid, human genomic DNA, Mycobacterium tuberculosis genomic DNA, and high concentration genomic nucleic acid (nucleic acid concentrations are all equal) of 10 other common pathogens in pulmonary infection such as Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, influenza virus, neocoronavirus, and adenovirus>104copies/μL)。
3. Constant-temperature amplification system of internal reference gene enzyme digestion probe
TABLE 8 isothermal amplification System of reference Gene restriction enzyme
4. The amplification procedure was: 30cycles:63 ℃ for 1min (Collection of fluorescence)
5. The screening results are shown in the table below, and only the second set of primers with probe 2 amplified to 5 copies/. mu.L from the sensitivity aspect; from the aspect of specificity, the second set of primer matched probes 2 has no amplification signal when amplifying human DNA, Mycobacterium tuberculosis genome DNA and other pathogens, namely, cross reaction can not be generated; therefore, the second set of primers and probe 2 are selected as the special primer probes for the reference gene.
TABLE 9 results of screening different primer pairs and probes for reference genes
(IV) combination of optimal primer probes for detecting mycobacterium tuberculosis targets and internal references
The multiplex primers and probes used for detecting the mycobacterium tuberculosis nucleic acid are obtained by screening through a large amount of repeated screening and optimization and are shown in the table 10.
TABLE 10 sequence information of the primer probes specific for detecting Mycobacterium tuberculosis nucleic acid in the present invention
Note: the detection probes of IS6110 and Pab are labeled with FAM, and the detection probe of the reference gene IS labeled with Cy 5.
The primer probes selected from each gene were combined, and the combined multiplex reaction solution was verified, and the reaction system of the multiplex reaction solution is shown in Table 11.
TABLE 11 reaction solution system after combination of multiple primer probes for detecting Mycobacterium tuberculosis in the present invention
(V) preparation of plasmid template for verifying multiple reaction solutions
IS6110 and Pab are mixed with standard plasmids of three genes of an internal reference and diluted to form template diluents with three concentrations: the ingredients and concentrations of L1, L2, L3, L1, L2 and L3 are shown in table 12.
TABLE 12 ingredient of template diluent and description of the concentration of each ingredient
(VI) sensitivity verification
The reaction systems in table 11 are used to verify the DNA of three templates L1, L2 and L3 in table 12 (the results are shown in fig. 1) and the DNA of 3 tuberculosis positive clinical samples S1, S2 and S3 with different pathogen capacities (the results are shown in fig. 2), wherein the Ct values of the clinical samples S1, S2 and S3 detected by the fluorescence PCR kit are: 25.98, 32.92, 36.47. According to the interpretation principle, the interpretation of the above-mentioned template is detected as shown in the following table:
| sample name | Target channel | Internal reference channel | Interpretation of results |
| L1 | With amplification | With amplification | Positive for tuberculosis |
| L2 | With amplification | With amplification | Positive for tuberculosis |
| L3 | With amplification | With amplification | Positive for tuberculosis |
| S1 | With amplification | / | Positive for tuberculosis |
| S2 | With amplification | / | Positive for tuberculosis |
| S3 | With amplification | / | Positive for tuberculosis |
From the above table, the amplification sensitivity of the multiplex reaction solution can still reach the concentration of 5copies/μ L, and the low-load positive samples with Ct value >36 can also be detected, which has higher sensitivity.
(VII) specificity verification
The reaction systems shown in Table 11 above were used to verify the genomic DNA of Mycobacterium tuberculosis, human DNA, and 10 other common pathogens of pulmonary infection, such as Streptococcus pneumoniae, Legionella pneumophila, Haemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, influenza virus, neocoronavirus, and adenovirus, respectively. As shown in FIG. 3, according to the interpretation principle, when the nucleic acid of the above-mentioned pathogen is detected, only amplification signals are present when the genomic nucleic acid of Mycobacterium tuberculosis is amplified, and amplification signals are absent when the nucleic acid of human DNA and other pathogens are detected, so that the specificity of the reaction system for detecting Mycobacterium tuberculosis is high, and the specific detection of Mycobacterium tuberculosis can be achieved.
Example two: preparation and use examples of Mycobacterium tuberculosis detection kit in the present invention
1. Preparation of the kit
Packaging the nucleic acid reaction solution, AMV reverse transcriptase, a blank reference substance, a positive reference substance and an internal reference substance together, and matching with an accessory product use instruction (recording detection modes for detecting DNA and RNA) to obtain the kit for detecting the mycobacterium tuberculosis nucleic acid by the enzyme digestion probe at the constant temperature.
2. 5 clinical samples were tested using the kit prepared in the present invention
The kit of the invention is used for extracting and detecting DNA of 5 tuberculosis positive sputum clinical samples S4-S8 according to the kit specification, the detection result is shown in figure 4, and the interpretation is shown in the following table according to the interpretation principle:
| sample name | Target channel | Internal reference channel | Interpretation of results |
| S4 | With amplification | With amplification | Positive for tuberculosis |
| S5 | With amplification | With amplification | Positive for tuberculosis |
| S6 | With amplification | No amplification | Positive for tuberculosis |
| S7 | With amplification | With amplification | Positive for tuberculosis |
| S8 | With amplification | With amplification | Positive for tuberculosis |
The detection Ct values of the 5 clinical samples detected by the fluorescence PCR detection kit are respectively as follows: 32.07, 31.44, 28.48, 36.86, 33.89. Therefore, the detection results of the kit and the fluorescence PCR kit in the invention for detecting tuberculosis positive clinical samples are consistent, and the samples with Ct values of more than 36 (such as S7) detected by the fluorescence PCR kit can still be detected by using the kit in the invention, which shows high sensitivity, while the detection of the kit in the invention only needs 30min, the whole detection time is shorter, and the operation is simple and convenient.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
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BEIJING MACRO & MICRO TEST BIO TECH Co.,Ltd.
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Claims (6)
1. A kit for detecting mycobacterium tuberculosis nucleic acid by using an enzyme digestion probe at constant temperature is characterized by comprising: detecting isothermal amplification primers of the mycobacterium tuberculosis IS6110 gene and a corresponding probe which can be digested by RNaseH; isothermal amplification primers for detecting a mycobacterium tuberculosis Pab gene and a corresponding probe which can be digested by RNaseH enzyme.
2. The kit according to claim 1, wherein the isothermal amplification primer for detecting Mycobacterium tuberculosis IS6110 gene SEQ ID NO.1-SEQ ID NO.6 and the corresponding RNaseH restriction enzyme probe are SEQ ID NO.7, the probe comprises an RNA base, the base shown by italic underlining IS the RNA base,
3. the kit of claim 1, wherein the isothermal amplification primer for detecting the Pab gene of Mycobacterium tuberculosis is SEQ ID NO.8-SEQ ID NO.13 and the corresponding RNaseH restriction enzyme probe is SEQ ID NO.14, the probe comprises an RNA base, the base shown by italic underlining is the RNA base,
4. the kit of claim 1, further comprising: detecting a section of artificially synthesized internal reference gene isothermal amplification primer SEQ ID NO.15-SEQ ID NO.20 which is not homologous with mycobacterium tuberculosis nucleic acid and human nucleic acid and a corresponding RNaseH enzyme digestion probe SEQ ID NO.21, wherein the probe comprises an RNA base, the base displayed by italic underlining is the RNA base,
5. the kit according to claim 4, wherein the ratio OF IF, IB, LF, LB, OF, OR in the primers for detecting the IS6110 gene OF Mycobacterium tuberculosis, the Pab gene OF Mycobacterium tuberculosis and the foreign reference gene IS 8:8:4:4:1:1, preferably, the concentrations OF IF and IB primers OF the three genes for detection are 0.8 μ M, the concentrations OF LF and LB primers are 0.4 μ M, and the concentrations OF OF and OR primers are 0.1 μ M.
6. The kit according to claim 5, wherein the probe for detecting IS6110 gene and Pab gene in the kit IS SEQ ID NO: 7 and SEQ ID NO: 14 and a probe SEQ ID NO: 21 labeled with different fluorophores.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112779344A (en) * | 2021-01-20 | 2021-05-11 | 中国人民解放军总医院 | Kit for detecting respiratory tract infection bacterial pathogen by enzyme digestion probe at constant temperature |
| CN113073152A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | LAMP primer, probe and kit for detecting influenza B virus |
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| US20170044594A1 (en) * | 2015-07-14 | 2017-02-16 | Abbott Molecular Inc. | Compositions and methods for identifying drug resistant tuberculosis |
| CN109750091A (en) * | 2019-03-13 | 2019-05-14 | 江苏宏微特斯医药科技有限公司 | Single tube detects the method and its kit of one or more object to be measured nucleic acid sequences |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112779344A (en) * | 2021-01-20 | 2021-05-11 | 中国人民解放军总医院 | Kit for detecting respiratory tract infection bacterial pathogen by enzyme digestion probe at constant temperature |
| CN113073152A (en) * | 2021-05-12 | 2021-07-06 | 广州普世利华科技有限公司 | LAMP primer, probe and kit for detecting influenza B virus |
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| CN111793703B (en) | 2023-09-22 |
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