CN111286551A - Primer and kit for rapidly detecting mycobacterium tuberculosis and using method thereof - Google Patents
Primer and kit for rapidly detecting mycobacterium tuberculosis and using method thereof Download PDFInfo
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Abstract
The kit comprises a nucleotide sequence shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, or a pharmaceutically acceptable salt thereof. Based on the principle of nested PCR, specific gene design high-specificity primers which are not matched with other species are obtained through logic screening. PCR is carried out to verify the specificity of the primers, and the specificity detection which can be used for identifying the mycobacterium tuberculosis is determined. The PCR amplification product is detected by agarose electrophoresis, and the simple, economical and rapid detection of the mycobacterium tuberculosis is realized according to whether the target fragment can be amplified or not. The method has the characteristics of rapidness, simplicity, economy, accuracy and sensitivity, has low requirements on instruments, and can be widely applied to hospitals at or above the county level. The method can help clinicians to quickly and accurately diagnose tuberculosis patients and take medicines in time, thereby achieving the purposes of early diagnosis and early treatment.
Description
Technical Field
The invention relates to the field of a rapid detection kit for mycobacterium tuberculosis, in particular to a rapid detection primer and a rapid detection kit for mycobacterium tuberculosis and a use method of the rapid detection primer and the rapid detection kit.
Background
Tuberculosis is a highly contagious disease caused by infection with mycobacterium tuberculosis, which is transmitted mainly through the respiratory system, and thus the most affected organs of most patients are the lungs, commonly referred to as tuberculosis. Tuberculosis infection of other organs or tissues of a body is relatively rare compared with that of lung tissues, but tuberculosis infection occurs frequently, and the common infection parts of extrapulmonary tuberculosis include brain, bones, lymph nodes and the like. Tuberculosis is one of the infectious diseases with extremely high morbidity and mortality at present, the main susceptible population is young and young, the health of human beings is seriously harmed, a heavy burden is brought to infected persons, families and the society, and the tuberculosis becomes a serious public health problem in the global range. In the early 19 th century, about one fifth of the european population died of tuberculosis due to the lack of effective preventive and therapeutic measures; until the early and middle of the 20 th century, with the advent of bcg and tuberculosis therapeutics, the morbidity and mortality of tuberculosis has been effectively controlled. However, due to abuse of antibiotics, severe environmental pollution, epidemic of AIDS and the like, the morbidity, drug resistance and mortality of the disease only shortly after the application of the antituberculous drugs tend to rise. Global infectious disease death data show that deaths due to mycobacterium tuberculosis infection exceed aids deaths and are only lower than deaths due to diarrhea and malaria. China is one of 30 tuberculosis high-load countries in the world, and the sick people live in the world second and only second to India.
The molecular biology detection technology is a detection method developed in recent years and has the characteristics of high speed, specificity, high sensitivity and the like. The basic principle of molecular detection technology is applied to carry out sequence comparison on the mycobacterium tuberculosis and other similar mycobacteria, thereby designing a primer capable of specifically detecting the mycobacterium tuberculosis and rapidly detecting whether the mycobacterium tuberculosis exists in a sputum sample of a patient.
Through research, the sputum of a clinical tuberculosis patient is used as an experimental material, two pairs of primers for specific amplification of mycobacterium tuberculosis are designed, and the mycobacterium tuberculosis molecular detection method which is good in specificity, high in sensitivity, rapid and accurate is established. The method can help scientific research workers and clinical laboratory to diagnose tuberculosis sample quickly and accurately, so as to achieve the purpose of quick and accurate diagnosis.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a primer and a kit for rapidly detecting mycobacterium tuberculosis and a using method thereof, which are used for rapidly detecting mycobacterium tuberculosis in sputum or other body fluid samples.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the primer for rapidly detecting the mycobacterium tuberculosis has the following sequence: as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
A kit for rapidly detecting mycobacterium tuberculosis comprises a detection reagent of the kit, a DNA extraction reagent, a PCR amplification reagent, a specific primer and an agarose gel preparation reagent.
Further, the kit comprises a nucleotide sequence shown as SEQ ID NO: 1, SEQ id no: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and (b) specific primers shown in the specification.
A use method of a rapid detection kit for mycobacterium tuberculosis comprises the following steps:
(1) collecting sputum samples of tuberculosis patients, and extracting the whole genome DNA of the mycobacterium tuberculosis;
(2) using the extracted DNA as a template, and amplifying the extracted DNA by using the two pairs of specific primers of claim 1 to obtain corresponding PCR amplification products;
(3) carrying out agarose gel electrophoresis on the obtained PCR product;
(4) carrying out sensitivity detection on the kit, and setting 6 gradients for detecting that the copy number of the mycobacterium tuberculosis DNA with the lowest level which can be detected is 10 copies;
(5) and (3) carrying out kit specificity detection, randomly selecting 5 clinical common pathogenic microorganisms, carrying out nucleic acid extraction, carrying out amplification by using the two pairs of primers, and detecting whether a PCR product can be amplified.
Further, in the step (4), the 6 gradients set for the sensitivity detection specifically include: 106、105、104、103、102And 10 copies.
Compared with the prior art, the invention has the beneficial effects that:
compared with other prior art, the kit has the characteristics of rapidness, simplicity, economy, accuracy and sensitivity, the requirements of instruments and equipment are not high, the reagents and experimental conditions are easy to achieve, only DNA extraction reagents, PCR amplification reagents and the two most important pairs of high-specificity primers are needed, PCR products are quickly detected by an agarose electrophoresis method, and the kit is suitable for popularization and application in large-scale sample screening. The invention has important significance for the rapid detection research of the mycobacterium tuberculosis.
Drawings
FIG. 1 shows a part of the detection results of the method of the present invention.
FIG. 2 is a graph of a sensitivity detection electropherogram of the method of the invention.
FIG. 3 is a specificity experimental electropherogram of the method of the present invention.
Detailed Description
The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:
a primer and a kit for rapidly detecting mycobacterium tuberculosis and a using method thereof develop an accurate, rapid, simple and economic detection kit aiming at the mycobacterium tuberculosis in a sputum sample of a patient. The whole genome sequence of the standard strain of the mycobacterium tuberculosis and the sequences of other isolated strains are downloaded 208 pieces from NCBI (http:// www.ncbi.nlm.nih.gov/genome /), the genome sequences of other mycobacteria are downloaded 173 pieces, sequence analysis and manual proofreading screening are carried out, and a highly conserved and specific sequence section of the mycobacterium tuberculosis is obtained by screening, wherein the sequence of the section has no obvious similarity with the sequences of other mycobacteria. Based on the principle of nested PCR, a primer which is highly specific to the mycobacterium tuberculosis is designed according to the sequence obtained in the section. The availability of the primers is detected through PCR amplification, the optimal working conditions of the primers are searched, the specificity of the designed primers is verified, and the specificity of the primers, which can be used for identifying the mycobacterium tuberculosis, is determined. At present, a kit and an instrument capable of carrying out rapid molecular detection on mycobacterium tuberculosis are X-pert detection, but the instrument and the reagent are expensive, the requirements on hospitals and inspection departments are high, the detection cost is high, and not all patients can bear the detection cost. The developed kit contains a highly specific mycobacterium tuberculosis detection primer obtained by repeated experimental groping, and after the primer is subjected to PCR amplification, a product can be detected through agarose electrophoresis, so that the simple, economical, rapid and accurate detection of mycobacterium tuberculosis can be realized according to whether a target fragment can be amplified or not.
In order to determine the specificity and sensitivity of detecting the mycobacterium tuberculosis by the nested PCR method, 5 common pathogenic microorganisms are selected for primer specificity detection. Positive plasmids are constructed by using the nested PCR one-step product, and positive plasmids with different copy numbers are amplified to determine the sensitivity and provide the technical parameters of the lowest level of DNA copy number detected by the technology.
The invention utilizes a nested PCR method to detect mycobacterium tuberculosis, which comprises one or a combination of several reagents as follows:
(1) a reagent for extracting DNA of Mycobacterium tuberculosis from sputum.
(2) Primers and related PCR reagents for one-step and two-step amplification of specific sequences of Mycobacterium tuberculosis;
(3) and preparing the reagent of the agarose gel.
The kit for molecular detection of mycobacterium tuberculosis uses two pairs of PCR primers
(1)
myco--F1：5’-ATGACGGCAATCTCGTGCTCA-3’
myco-R1:5’-TTAGCTGGCCGCCAGCTGCTCG-3’
(2)
myco-F2:5’-GCAAGACCGTCGAGGTCACC-3’
myco-R2:5’-CCAGGACGTTGTTGAGCAGCA-3’
The kit for molecular detection of mycobacterium tuberculosis comprises a DNA extraction reagent, a PCR amplification reagent, a specific primer and an agarose gel preparation reagent.
The specific operation of the kit for detecting the mycobacterium tuberculosis is as follows:
(1) collecting sputum samples of tuberculosis patients, and extracting the whole genome DNA of the mycobacterium tuberculosis;
(2) using the extracted DNA as a template, and applying the two pairs of specific primers designed by the invention to amplify the DNA to obtain corresponding PCR amplification products;
(3) carrying out agarose gel electrophoresis on the obtained PCR product, and detecting whether the strip exists or not and whether the size is correct or not;
(4) the kit sensitivity detection is carried out, and 6 gradients (namely 10 gradients) are set6、105、104、103、102And 10 copies) to detect the lowest level of mutant DNA detectable by the present technology.
(5) And (3) carrying out kit specificity detection, randomly selecting 5 clinical common pathogenic microorganisms, carrying out nucleic acid extraction, carrying out amplification by using the two pairs of primers, and detecting whether a PCR product can be amplified.
Experimental example: as shown in fig. 1.
1. Primer design
Based on the principle of nested PCR, primers with high specificity of specific gene design which are not matched with other species are obtained by screening sequences of various mycobacteria through Blast analysis, the sequences of the primers are shown in a sequence table, and the size of the products is 375 bp.
2. Sample collection
Clinical samples required for the experiment were provided by a hospital in Kunming.
3. Sample genomic DNA extraction
Extracting the genomic DNA of the mycobacterium tuberculosis from the sputum sample by using a kit method.
4. Nested PCR amplification
Nested PCR reaction system:
(1) total volume of inner PCR was 20. mu.L, containing 30ng of genomic DNA, 10. mu.L
2×TSINGKETMMaster Mix, 3. mu.M each of peripheral forward and reverse primers, ddH for the remainder2And (4) supplementing and finishing.
(2) The total volume of peripheral PCR was 20. mu.L, containing 1.5. mu.L of inner PCR product DNA,
PCR reaction procedure:
(1) inner wall PCR: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 57.5 ℃ for 30s, and extension at 72 ℃ for 10s, and repeating for 20 cycles; extension at 72 ℃ for 5 min.
(2) Peripheral PCR: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 10s, and repeating for 25 cycles; extension at 72 ℃ for 5 min.
5. PCR product detection
And (3) detecting a PCR product: taking 3 mu L of PCR product to carry out electrophoresis on 2% agarose gel, wherein the electrophoresis buffer is 1 XTAE, carrying out constant-pressure electrophoresis at 150V for 23min, and observing and photographing under a gel imaging system. The results of the experiment are shown (see FIG. 1). In FIG. 1, lane M is a 2000bp nucleic acid molecular standard, and lanes 1-10 are nucleic acid PCR products of sputum specimens of tuberculosis patients. Lane 11 is a negative control. Lane 12 is a positive control. The detection result of the detection method is consistent with the clinical diagnosis result. The result shows that the specific primer designed by the inventor can efficiently detect the mycobacterium tuberculosis existing in clinical samples.
6. Sensitivity detection
Using successfully constructed positive plasmids as templates, 6 gradients (i.e., 10) were set6、105、104、103、102And 10 copies) to detect the lowest level of mutant DNA detectable by the present technology. Following the same PCR reaction as in steps 4 and 5The system and procedure should be used for amplification and detection.
The results of electrophoresis of the detection sensitivity test for Mycobacterium tuberculosis in sputum samples were carried out by the method of the present invention (see FIG. 2). In FIG. 2, lane M shows a 2000bp nucleic acid molecule standard, and lanes 1-6 show plasmid copy numbers of 10 and 102、103、104、105、106. Lane 7 is a negative control. When the copy number of the template is from low to high, the change of the strip from weak to bright can be obviously seen, and when the plasmid with the copy number of 10 is used as the template, the brightness of the strip is higher than that of Marker compared with the Marker, so that the template still can effectively work under the condition of the copy number of 10. Therefore, the two pairs of primers for detecting the mycobacterium tuberculosis have higher sensitivity, namely, a specific fragment can be amplified when 10 copies exist, and the existence of the mycobacterium tuberculosis can be detected.
7. Experiment of specificity
In order to verify the specificity of the nested PCR primer, 5 common pathogenic bacteria are selected for nucleic acid extraction, and the nested PCR primer is tested. The template was amplified and detected according to the same PCR reaction system and procedure as in steps 4 and 5.
The electrophoresis results of the test for the specificity of detection of Mycobacterium tuberculosis in sputum specimens were carried out by the method of the present invention (see FIG. 3). In FIG. 3, lane M shows a 2000bp nucleic acid molecular standard, and lanes 1 to 5 show the nucleic acid amplification products of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus haemolyticus, and Acinetobacter baumannii, respectively. Lane 6 is the product of amplification of mycobacterium tuberculosis, and lane 7 is a negative control. In FIG. 3, only the target band was amplified from the genomic DNA of M.tuberculosis, but the amplification results of the genomic DNA of other bacteria were negative. Therefore, the two pairs of primers for detecting the mycobacterium tuberculosis have good specificity, and the mycobacterium tuberculosis can be effectively detected in common clinical respiratory pathogenic bacteria.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Sequence listing
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Claims (5)
1. The primer for rapidly detecting the mycobacterium tuberculosis is characterized by comprising the following sequences: as shown in SEQ ID NO: 1, SEQ id no: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
2. A kit for rapidly detecting mycobacterium tuberculosis is characterized in that detection reagents of the kit comprise a DNA extraction reagent, a PCR amplification reagent, a specific primer and an agarose gel preparation reagent.
3. The kit of claim 2, wherein: the kit comprises a nucleotide sequence shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, and (b) specific primers shown in the specification.
4. The use method of the kit for rapid detection of Mycobacterium tuberculosis as claimed in any one of claims 2 to 3, wherein: the method comprises the following steps:
(1) collecting sputum samples of tuberculosis patients, and extracting the whole genome DNA of the mycobacterium tuberculosis;
(2) using the extracted DNA as a template, and amplifying the extracted DNA by using the two pairs of specific primers of claim 1 to obtain corresponding PCR amplification products;
(3) carrying out agarose gel electrophoresis on the obtained PCR product;
(4) carrying out sensitivity detection on the kit, and setting 6 gradients for detecting that the copy number of the mycobacterium tuberculosis DNA with the lowest level which can be detected is 10 copies;
(5) and (3) carrying out kit specificity detection, randomly selecting 5 clinical common pathogenic microorganisms, carrying out nucleic acid extraction, carrying out amplification by using the two pairs of primers, and detecting whether a PCR product can be amplified.
5. The method according to claim 4, characterized in that in step (4), the 6 gradients of the sensitivity detection settings are in particular: 106、105、104、103、102And 10 copies.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114990240A (en) * | 2022-06-01 | 2022-09-02 | 昆明理工大学 | Multiple qPCR detection reagent for detecting gynecological disease exogenous pathogens |
| CN115992272A (en) * | 2022-10-17 | 2023-04-21 | 杭州遂真生物技术有限公司 | Composition for detecting mycobacterium tuberculosis and drug-resistant gene thereof and integrated kit |
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| CN114990240B (en) * | 2022-06-01 | 2024-05-10 | 昆明理工大学 | Multiple qPCR detection reagent for detecting gynecological disease exogenous pathogens |
| CN115992272A (en) * | 2022-10-17 | 2023-04-21 | 杭州遂真生物技术有限公司 | Composition for detecting mycobacterium tuberculosis and drug-resistant gene thereof and integrated kit |
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Application publication date: 20200616 |