CN112126695A - Primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and reaction system - Google Patents
Primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and reaction system Download PDFInfo
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Abstract
The invention provides a primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and a reaction system, and relates to the technical field of biological detection. The primer composition comprises at least 3 pairs of primers designed and formed for a target gene based on a common insertion sequence IS6110 in a mycobacterium tuberculosis complex, wherein the at least 3 pairs of primers comprise an external primer F3/B3 with nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2, an internal primer FIP/BIP with nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 and a loop primer LF/LB with nucleotide sequences shown as SEQ ID NO.5 and SEQ ID NO. 6. The invention adopts the loop-mediated isothermal amplification technology and the primer composition to detect the mycobacterium tuberculosis quickly, conveniently, efficiently, specifically and highly sensitively, and the invention breaks away from the dependence on large instruments and even small and medium-sized laboratory instruments and realizes the quick detection on the spot. Moreover, the reaction system adopts domestic Bst DNA polymerase, so that the cost of the reagent is greatly reduced, and the detection cost is greatly reduced.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a primer composition for detecting Mycobacterium tuberculosis complex loop-mediated isothermal amplification and a reaction system.
Background
According to the '2019 global tuberculosis report' issued by the World Health Organization (WHO), tuberculosis is still one of 10 global causes of death, and is always the first cause of death of a single infectious disease since 2007. The number of tuberculosis deaths worldwide in 2018 is about 124 ten thousand, and the death rate is 16/10 ten thousand. China is one of 30 tuberculosis high-load countries in the world, and the number of the tuberculosis patients is second to Indian and second in the world. About 86 thousands of new infectious patients are sent in 2018 in China. It is estimated that about 48.4 million people worldwide are new forms of rifampicin resistant tuberculosis (RR-TB), and 78% of these people are multiple drug resistant tuberculosis (MDR-TB). Of the MDR-TB patients, 6.2% were estimated to be broadly drug resistant tuberculosis (XDR-TB). Among them, about 6.8 million cases of MDR/RR-TB in China are known. From the epidemic situation of tuberculosis in China, rural areas are higher than towns, economically undeveloped areas are higher than economically developed areas, tuberculosis is a poverty-stricken disease, and the income of most tuberculosis patients is below the average level. Tuberculosis treatment cycle is long, cost is high, most patients are poor population, and difficulty is increased for preventing and treating tuberculosis. According to statistics, at present, more than 80% of tuberculosis patients in China are in rural areas, the prevalence rate of the tuberculosis patients in the rural areas is more than twice that of the tuberculosis patients in cities, and the age of the tuberculosis patients is mainly concentrated on 20-45 years; the floating population is also a high-incidence population of tuberculosis, 40% and 50% of tuberculosis patients reported in Beijing and Shanghai respectively originate from the floating population, and Shenzhen is as high as 80%. Due to the high fluidity, the diagnosis rate of suspected tuberculosis patients in the floating population is very low, wherein the treatment success rate is lower, and both Beijing and Shanghai are only 20%.
The real difficulty of tuberculosis prevention and treatment is that the early symptoms are variable and are not easy to be perceived by patients, and the other main reason is that the current diagnosis means is not in place. The bacteria-positive rate of sputum is low, and if the patient is diagnosed with the symptom, more than 40% of patients do not have any symptom. Many patients cannot be found by X-ray chest radiographs, and still need to go through more expensive imaging examination items such as bronchoscopes and CT. However, some smaller tuberculosis lesions are easily confused with pneumonia and lung cancer, and cannot be diagnosed by only imaging examination. However, the Point-of-Care testing (POCT) diagnostic products approved by CFDA still mainly detect antigen and antibody, and molecular diagnostic devices and reagents are rare. The POCT molecular detection method with high sensitivity and specificity is still the key point of research and development.
In recent years, Loop-mediated isothermal amplification (Loop-mediated isothermal amplification) has attracted much attention for its unique advantages in the development of POCT molecular diagnostic methods, and its application in the detection of infectious disease pathogen nucleic acids is designed to 8 pairs of specific primers for target gene sequences, as shown in fig. 1, wherein F1c and F2 constitute an upstream Inner primer fip (forward Inner primer), B1c and B2 constitute a downstream Inner primer bip (backward Inner primer), an upstream Loop primer lf (Loop forward) and a downstream Loop primer lb (Loop backward) constitute Loop primers. The upstream outer primer F3 and the downstream outer primer B3 constitute the outer primers. Under the action of DNA polymerase with replication activity and high strand displacement activity, the 3 pairs of primers such as F3, B3, FIP, BIP, loop primer and the like can realize rapid specific amplification of 6 regions of a target gene sequence at a constant temperature of 60-65 ℃.
The applicant finds that at least the following technical problems exist in the prior art: the existing Mycobacterium tuberculosis complex detection kit can be divided into two types. The first category of PCR-based assays, however, require the purchase of expensive PCR analyzers equipped with sophisticated thermal cyclers, as well as the provision of sophisticated laboratory conditions and highly trained professionals according to relevant regulations. In addition, the DNA polymerase (such as Taq polymerase) used in the PCR method has higher sensitivity to the inhibitor in the complex sample than the single strand displacement DNA polymerase (such as Bst polymerase) used in the loop-mediated isothermal amplification, namely, the DNA polymerase has poorer tolerance to the inhibitor; the second type is detection methods based on isothermal amplification techniques, in particular loop-mediated isothermal amplification techniques. However, in the prior art, the detection method for the mycobacterium tuberculosis complex based on the loop-mediated isothermal amplification technology is relatively high in cost, and a detection system which can be used for the mycobacterium tuberculosis complex has been developed by the nippondemand chemical company based on the loop-mediated isothermal amplification technology, but the system needs to be provided with a special multifunctional integrated detection instrument (Loopamp @ LF-160), and a detection kit of the system is a proprietary product, and is high in price, the selling price of only a detection reagent is as high as 150 yuan per person, and the detection cost is more 230 yuan per person. Therefore, a field rapid detection method for the mycobacterium tuberculosis complex, which has the advantages of low detection cost, cheaper construction, and quicker and more accurate detection, is needed to be provided so as to help improve the current situations of the deficiency and the return to deficiency of tuberculosis caused by diseases in China.
Disclosure of Invention
The invention aims to provide a primer composition for detecting mycobacterium tuberculosis complex by loop-mediated isothermal amplification and a reaction system, and the invention designs and screens an optimal primer pair by taking a conserved sequence of an insertion sequence 6110 gene (IS6110) which IS specific to a mycobacterium tuberculosis complex (comprising mycobacterium tuberculosis, mycobacterium africanum, mycobacterium bovis, mycobacterium microtus and mycobacterium capricolum) as a target sequence based on a loop-mediated isothermal amplification technology, and simultaneously replaces foreign high-cost imported enzymes with domestic Bst DNA polymerase to completely construct a novel on-site rapid detection method for the mycobacterium tuberculosis. The primer composition has good sensitivity and specificity for detecting the mycobacterium tuberculosis complex, the cost of the reagent can be controlled within 20 yuan/person, and the cost is far lower than that of similar products, so that the primer composition has a good application prospect.
In order to achieve the purpose, the invention provides the following technical scheme:
the primer composition comprises at least 3 pairs of primers designed and formed by taking an insertion sequence IS6110 shared by a mycobacterium tuberculosis complex as a target gene, wherein the at least 3 pairs of primers comprise an external primer F3/B3 with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2, an internal primer FIP/BIP with nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4 and a loop primer LF/LB with nucleotide sequences shown as SEQ ID No.5 and SEQ ID No. 6.
According to a preferred embodiment, the concentration of the inner primer is 0.6-2.4. mu.M; the concentration of the external primer is 0.1-0.2 mu M; the concentration of the loop primer is 0.4-1.2. mu.M. Preferably, the concentration of the outer primer is 0.1. mu.M or 0.2. mu.M.
According to a preferred embodiment, the concentrations of the inner primer, the outer primer and the loop primer are 1.2. mu.M, 0.2. mu.M and 0.6. mu.M, respectively.
The invention also provides a reaction system comprising the primer composition for the Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection, which is characterized in that the concentration dosage of the primer composition in the reaction system is as follows: the inner primer FIP/BIP is 0.8-2.4 mu M; the outer primers F3/B were 30.1-0.2. mu.M and the loop primer LF/LB was 0.4-1.2. mu.M.
According to a preferred embodiment, the reaction system further comprises Bst DNA polymerase, the volume dosage of the Bst DNA polymerase is 1 μ L, and the Bst DNA polymerase is selected from polymerase prepared in China.
According to a preferred embodiment, the reaction system further comprises manganese chloride and a fluorescent dye, and the concentration of the fluorescent dye is 0.2-1.0 mM; the concentration dosage of the manganese chloride is 5-20 mM.
According to a preferred embodiment, the reaction system further comprises the following components in the following concentrations: Tris-HCl 20-40 mM; KCl 5-15 mM; (NH)4)2SO410-30 mM; triton X-1000.2-1.5%; betaine 0.5-1.0M; MgSO (MgSO)45-10mM and dNTP mix 1-2 mM.
The invention also provides a kit for detecting the Mycobacterium tuberculosis complex group loop-mediated isothermal amplification, which comprises the reaction system.
The invention also provides a detection method of the mycobacterium tuberculosis, which comprises the following steps:
selecting a sample;
and (3) extracting DNA: extracting DNA from a sample to be detected;
preparing a reaction system: determining the use amount of each component in the reaction system according to the sample amount N to be detected, wherein the use amount of a primer mixed solution in the reaction system is (N +2) multiplied by 15 mu L, the use amount of Bst DNA polymerase is (N +2) multiplied by 1 mu L, and the use amount of the rest reaction mixed solution is (N +2) multiplied by 4 mu L, vibrating and uniformly mixing the components of the reaction system, and performing instantaneous centrifugation to obtain the reaction system;
sample adding: placing the reaction system in a reaction tube, adding a negative control into a negative control tube, immediately covering a tube cover, adding a positive control into a positive control tube, immediately covering the tube cover, finally respectively adding the extracted nucleic acid into the detection reaction tube, covering the tube cover, and performing instantaneous centrifugation;
nucleic acid isothermal amplification: placing the reaction tube after sample adding in a constant temperature device, keeping the temperature at 65 ℃ and placing for 40-60 minutes;
and (5) judging the result.
According to a preferred embodiment, in the step of formulating the reaction system, the primer Mixture includes a primer composition and dNTP mix.
Based on the technical scheme, the primer composition and the reaction system for detecting the Mycobacterium tuberculosis complex group loop-mediated isothermal amplification have at least the following technical effects:
the invention adopts the loop-mediated isothermal amplification technology to detect the mycobacterium tuberculosis quickly, conveniently, efficiently, specifically and highly sensitively, and the invention breaks away from the dependence on large-scale instruments and even small and medium-sized laboratory instruments and realizes the quick detection on the spot.
The primer composition provided by the invention has good sensitivity and specificity for the loop-mediated isothermal amplification detection of the Mycobacterium tuberculosis complex group, 10 fg/reaction of Mycobacterium tuberculosis genome DNA can be detected at least by adopting the primer composition and the reaction system, and compared with the detection sensitivity of a PCR kit adopted in the prior art which is 100fg-1 pg/reaction, the sensitivity is obviously higher. The primer composition of the present invention can specifically detect Mycobacterium tuberculosis complex including Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium microti and Mycobacterium caprine without cross-reacting with other mycobacteria such as Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium abscessus, Mycobacterium cheloniae, Mycobacterium marmalayi, Mycobacterium gordonae, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium bufonis, Mycobacterium scrofulaceum and Mycobacterium fortuitum.
On the other hand, the reaction system of the embodiment of the invention adopts domestic Bst DNA polymerase, so that the cost of the reagent is greatly reduced, and further, the detection cost is greatly reduced, and the detection cost can be even reduced to 5 yuan/person after large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of primers in a loop-mediated isothermal amplification technique;
FIG. 2 is a graph showing the quantitative ratio of the inner primer, the outer primer and the loop primer in the primer composition for the complex loop-mediated isothermal amplification detection of Mycobacterium tuberculosis of the present invention;
FIG. 3 is a graph showing the sensitivity analysis of the reaction system for the complex loop-mediated isothermal amplification detection of Mycobacterium tuberculosis of the present invention;
FIG. 4 is a schematic explanatory view of the results of the detection method of Mycobacterium tuberculosis of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
Example 1:
this example provides the selection of target genes and the design of primer compositions.
By taking Mycobacterium tuberculosis IS6110 IS-like element (Mycobacterium tuberculosis IS6110 IS-like element) (Genebank number: X17348.1) as a target gene, excavating a gene conserved sequence by using sequence comparison software Clustal X2, designing a primer by using online loop-mediated isothermal amplification primer design software PrimeExplorer V5, and after a series of online and experimental screening optimization, the nucleotide sequence of the selected primer composition IS shown in Table 1. Preferably, the LAMP primers of the present invention can be designed based on IS6110 sequence using existing LAMP Primer design tools/sites including, but not limited to, Primer Explorer, BiosunLAMP, Premierbiosoft, LAMP designer, etc., and can also be designed by the related companies.
TABLE 1 nucleotide sequences of primer compositions for the Loop-mediated isothermal amplification detection of IS6110
Example 2:
this example investigates the concentration usage of the inner, outer and loop primers in the primer composition of example 1.
In the invention, 6 loop-mediated isothermal amplification primers are designed by taking IS6110 as a target gene, and the initial concentrations and the melting temperatures (Tm) of the outer primer and the inner primer are different, so that the research on the optimal ratio of the inner primer to the outer primer IS very important, and the concentration ratio of the inner primer to the outer primer directly influences the final amplification efficiency and the false positive rate of the kit. The inventors studied the optimum ratio of the inner primer and the outer primer of the screened IS6110 primer using the genomic DNA of the Mycobacterium tuberculosis standard strain H37Rv containing the target gene sequence as a template and using a fluorescence loop-mediated isothermal amplification kit, and the related results are shown in FIG. 2.
As can be seen from FIG. 2, the optimal concentrations of the inner primer, the outer primer and the loop primer in the loop-mediated isothermal amplification primer composition of IS6110 gene selected by the present invention were 1.2. mu.M, 0.2. mu.M and 0.6. mu.M, respectively.
Example 3:
this example 3 was conducted to detect the sensitivity of the primer composition selected in example 2 at the optimum concentration.
According to the optimal concentration ratio of inner primer/outer primer/loop primer obtained in example 2, the inventors diluted the genomic DNA of M.tuberculosis standard strain H37Rv containing the target gene sequence to 1 fg/. mu.L at 100 pg/. mu.L as the highest concentration and 10 Xfold ratio with ultrapure water, and then performed the loop-mediated isothermal amplification experiment at each concentration as the template with the loading amount of 1. mu.L to determine the sensitivity of the loop-mediated isothermal amplification system constructed in the present invention, and the results are shown in FIG. 3.
As can be seen from FIG. 3, the lowest detection limit of the loop-mediated isothermal amplification system constructed based on the target sequence of IS6110 according to the present invention IS 10 fg/reaction using the genomic DNA of the standard strain as a template.
Example 4:
the present embodiment provides a reaction system including the primer composition for the complex loop-mediated isothermal amplification detection of mycobacterium tuberculosis, and the components and concentrations in the reaction system of the present invention are shown in table 2.
TABLE 2 Loop-mediated isothermal amplification reaction System
| Component name | Brand | Volume of | Final concentration |
| Internal primer FIP/BIP | Beijing Qingke Biotechnology Co.,Ltd. | 0.8-2.4μM | |
| Outer primer F3/B3 | Beijing Qingke Biotechnology Co.,Ltd. | 0.1-0.2μM | |
| Loop primer LF/LB | Beijing Qingke Biotechnology Co.,Ltd. | 0.4-1.2μM | |
| Bst DNA Polymerase | Novozan Biotechnology Ltd | 1μL | |
| Tris-HCl(pH 8.8) | Sigma-Aldrich | 20-40mM | |
| KCl | Sigma-Aldrich | 5-15mM | |
| (NH4)2SO4 | Sigma-Aldrich | 10-30mM | |
| Triton X-100 | Sigma-Aldrich | 0.2-1.5％ | |
| Betaine | Sigma-Aldrich | 0.5-1.0M | |
| MgSO4 | Sigma-Aldrich | 5-10mM | |
| dNTP Mixture | Tiangen Biochemical technology Ltd | 1-2mM | |
| Calcein fluorescent dye | Sigma-Aldrich | 0.2-1.0mM | |
| Manganese chloride | Sigma-Aldrich | 5-20mM |
Preferably, the fluorescent dye may be any known/studied fluorescent dye, including but not limited to Calcein/Calcein, SYBR Green, Eva Green, or HNB.
Example 5:
this example 5 provides a method for detecting mycobacterium tuberculosis, which comprises the following steps:
(1) and selecting a sample. The sample selected includes, but is not limited to, sputum, blood, other bodily fluid samples, tissue samples, or environmental samples. Preferably, for clinical sputum samples, the N-acetyl-L-cysteine-sodium hydroxide method (NALC-NaOH) is used for digestion and sterilization for 15 min; directly centrifuging other body fluid samples to enrich bacteria;
(2) and extracting DNA, namely extracting the DNA from the sample to be detected by adopting a scheme selected by a laboratory.
(3) And (4) preparing a reaction system. The reaction system adopted by the detection is 25 mu L. Determining the sample volume (N) to be detected, determining the use amount of each component in the reaction system according to the sample volume N to be detected, wherein the use amount of a primer mixed solution (containing a primer composition and dNTPs) in the reaction system is (N +2) multiplied by 15 mu L, the use amount of Bst DNA polymerase is (N +2) multiplied by 1 mu L, and the use amount of the rest reaction mixed solution (containing a fluorescent dye and other reagents) is (N +2) multiplied by 4 mu L, vibrating and uniformly mixing the components of the reaction system, and performing instantaneous centrifugation to obtain the reaction system.
(4) And (4) sample adding. Placing 20 μ L of the reaction system in a reaction tube, adding 5 μ L of negative control into the negative control tube, immediately covering the tube cap, adding 5 μ L of positive control (plasmid inserted with target gene) into the positive control tube, immediately covering the tube cap, respectively adding 5 μ L of extracted nucleic acid sample into the detection reaction tube, covering the tube cap, and performing instantaneous centrifugation; the amplification reaction was immediately carried out.
(5) Nucleic acid isothermal amplification: placing the reaction tube with completed sample application in a constant temperature device, keeping the temperature at 65 ℃ and placing for 40-60 minutes, preferably, the reaction time is shortest 40 minutes and is not more than 60 minutes. It should be noted that, before starting amplification, it is ensured that all the reaction tube caps are completely sealed, otherwise the laboratory aerosol contamination is easily caused.
(6) And (4) visualization interpretation of results: after the isothermal amplification is finished, the visual interpretation of the amplification result can be directly finished under visible light or an ultraviolet lamp (385nm) according to the color change. The results are interpreted schematically in FIG. 4. Preferably, the change in color can be detected using a fluorescence detector or a turbidity meter.
As can be seen from fig. 4, the positive control tubes had a clear visible color change relative to the negative control tubes.
In conclusion, the primer composition selected by the loop-mediated isothermal amplification technology based on the target gene of the mycobacterium tuberculosis IS6110 has higher sensitivity and specificity for detecting the mycobacterium tuberculosis, and can detect the genomic DNA of the mycobacterium tuberculosis at the lowest rate of 10 fg/reaction. And has the advantages of rapidness, convenience and lower detection cost. The detection method for mycobacterium tuberculosis can be used for non-diagnostic purposes, including but not limited to environmental sanitation detection, disease overall prevention and control/epidemic trend detection, food and daily necessities detection and the like.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Sequence listing
<110> Sichuan university Hospital in western China
<120> primer composition for Mycobacterium tuberculosis complex group loop-mediated isothermal amplification detection and reaction system
<130> 2020.08.29
<141> 2020-09-04
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Claims (10)
1. The primer composition for detecting the loop-mediated isothermal amplification of the mycobacterium tuberculosis complex IS characterized by comprising at least 3 pairs of primers designed for target genes on the basis of an insertion sequence IS6110 shared by the mycobacterium tuberculosis complex, wherein the at least 3 pairs of primers comprise an external primer F3/B3 with nucleotide sequences shown as SEQ ID No.1 and SEQ ID No.2, an internal primer FIP/BIP with nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4 and a loop primer LF/LB with nucleotide sequences shown as SEQ ID No.5 and SEQ ID No. 6.
2. The primer composition for the detection of Mycobacterium tuberculosis complex LAMP as claimed in claim 1, wherein the concentration of the inner primer is 0.6-2.4 μ M; the concentration of the external primer is 0.1-0.2 mu M; the concentration of the loop primer is 0.4-1.2. mu.M.
3. The primer composition for the complex loop-mediated isothermal amplification detection of Mycobacterium tuberculosis according to claim 2, wherein the concentrations of the inner primer, the outer primer and the loop primer are 1.2. mu.M, 0.2. mu.M and 0.6. mu.M, respectively.
4. A reaction system comprising the primer composition for the complex loop-mediated isothermal amplification detection of Mycobacterium tuberculosis as described in any one of claims 1 to 3, wherein the primer composition is included in the reaction system in a concentration of: the inner primer FIP/BIP is 0.8-2.4 mu M; the outer primers F3/B were 30.1-0.2. mu.M and the loop primer LF/LB was 0.4-1.2. mu.M.
5. The reaction system as set forth in claim 4, further comprising Bst DNA polymerase, wherein the Bst DNA polymerase is used in an amount of 1 μ L by volume, and the Bst DNA polymerase is selected from polymerases prepared in China.
6. The reaction system of claim 4, further comprising manganese chloride and a fluorescent dye, wherein the fluorescent dye is used in a concentration of 0.2 to 1.0 mM; the concentration dosage of the manganese chloride is 5-20 mM.
7. The reaction system of claim 4, further comprising the following components in concentrations and amounts: Tris-HCl 20-40 mM; KCl 5-15 mM; (NH)4)2SO410-30 mM; triton X-1000.2-1.5%; betaine 0.5-1.0M; MgSO (MgSO)45-10mM and dNTP mix 1-2 mM.
8. A kit for detecting Mycobacterium tuberculosis complex by loop-mediated isothermal amplification, the kit comprising the reaction system of any one of claims 4 to 7.
9. A detection method of Mycobacterium tuberculosis is characterized by comprising the following steps:
selecting a sample;
and (3) extracting DNA: extracting DNA from a sample to be detected;
preparing a reaction system: determining the use amount of each component in the reaction system according to the sample amount N to be detected, wherein the use amount of a primer mixed solution in the reaction system is (N +2) multiplied by 15 mu L, the use amount of Bst DNA polymerase is (N +2) multiplied by 1 mu L, and the use amount of the rest reaction mixed solution is (N +2) multiplied by 4 mu L, vibrating and uniformly mixing the components of the reaction system, and performing instantaneous centrifugation to obtain the reaction system;
sample adding: placing the reaction system in a reaction tube, adding a negative control into a negative control tube, immediately covering a tube cover, adding a positive control into a positive control tube, immediately covering the tube cover, finally respectively adding the extracted nucleic acid sample into the detection reaction tube, covering the tube cover, and performing instantaneous centrifugation;
nucleic acid isothermal amplification: placing the reaction tube after sample adding in a constant temperature device, keeping the temperature at 65 ℃ and placing for 40-60 minutes;
and (5) judging the result.
10. The detection method according to claim 9, wherein the primer Mixture includes a primer composition and dNTP mix in the step of preparing the reaction system.
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| CN115961060A (en) * | 2022-08-22 | 2023-04-14 | 中国海洋大学 | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold |
| CN116987806A (en) * | 2023-09-08 | 2023-11-03 | 南华大学 | LAMP primer group for detecting mycobacterium tuberculosis and kit thereof |
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| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
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| CN111269996A (en) * | 2020-02-10 | 2020-06-12 | 四川大学华西医院 | Mycobacterium tuberculosis complex rapid detection kit and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 刘巍等: "LAMP 技术及其在人类传染病病原体检测的研究进展", 《应用预防医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115961060A (en) * | 2022-08-22 | 2023-04-14 | 中国海洋大学 | Primer probe group, kit, method and application for isothermal amplification detection of mycobacterium tuberculosis based on nanogold |
| CN116987806A (en) * | 2023-09-08 | 2023-11-03 | 南华大学 | LAMP primer group for detecting mycobacterium tuberculosis and kit thereof |
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