CN117448466A - Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis - Google Patents
Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis Download PDFInfo
- Publication number
- CN117448466A CN117448466A CN202311348261.8A CN202311348261A CN117448466A CN 117448466 A CN117448466 A CN 117448466A CN 202311348261 A CN202311348261 A CN 202311348261A CN 117448466 A CN117448466 A CN 117448466A
- Authority
- CN
- China
- Prior art keywords
- composition
- probe
- katg
- inha
- primer pair
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 99
- 229960003350 isoniazid Drugs 0.000 title claims abstract description 49
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000000523 sample Substances 0.000 claims abstract description 236
- 238000001514 detection method Methods 0.000 claims abstract description 48
- 238000002844 melting Methods 0.000 claims description 71
- 230000008018 melting Effects 0.000 claims description 71
- 108090000790 Enzymes Proteins 0.000 claims description 20
- 102000004190 Enzymes Human genes 0.000 claims description 20
- 238000003753 real-time PCR Methods 0.000 claims description 11
- 238000002474 experimental method Methods 0.000 claims description 10
- 230000003321 amplification Effects 0.000 claims description 8
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 8
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 claims description 4
- 230000002452 interceptive effect Effects 0.000 claims 1
- 206010059866 Drug resistance Diseases 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 101100509674 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) katG3 gene Proteins 0.000 description 35
- 101150013110 katG gene Proteins 0.000 description 35
- 101150005343 INHA gene Proteins 0.000 description 34
- 238000006243 chemical reaction Methods 0.000 description 30
- 238000012360 testing method Methods 0.000 description 23
- 108020004414 DNA Proteins 0.000 description 12
- 201000008827 tuberculosis Diseases 0.000 description 9
- 238000012803 optimization experiment Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 208000015355 drug-resistant tuberculosis Diseases 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 201000009671 multidrug-resistant tuberculosis Diseases 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 238000007400 DNA extraction Methods 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 206010036790 Productive cough Diseases 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000036457 multidrug resistance Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 208000024794 sputum Diseases 0.000 description 2
- 210000003802 sputum Anatomy 0.000 description 2
- LDXJRKWFNNFDSA-UHFFFAOYSA-N 2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)-1-[4-[2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidin-5-yl]piperazin-1-yl]ethanone Chemical compound C1CN(CC2=NNN=C21)CC(=O)N3CCN(CC3)C4=CN=C(N=C4)NCC5=CC(=CC=C5)OC(F)(F)F LDXJRKWFNNFDSA-UHFFFAOYSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000186363 Mycobacterium kansasii Species 0.000 description 1
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 229940072185 drug for treatment of tuberculosis Drugs 0.000 description 1
- 229940124307 fluoroquinolone Drugs 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000000814 tuberculostatic agent Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/32—Mycobacterium
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a composition, a kit and a method for detecting the isoniazid resistance of mycobacterium tuberculosis, wherein the composition for detecting the isoniazid resistance of mycobacterium tuberculosis comprises at least one of a composition 1 and a composition 2; composition 1 is any one set of primer pair 1 and probe 1, primer pair 2 and probe 2, and primer pair 3 and probe 3; composition 2 is any one set of primer pair 4 and probe 4, primer pair 5 and probe 5, and primer pair 6 and probe 6. The invention has the characteristics of simple operation, high sensitivity, good repeatability and high specificity in the isoniazid drug resistance detection of mycobacterium tuberculosis.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a composition, a kit and a method for detecting isoniazid resistance of mycobacterium tuberculosis.
Background
Tuberculosis is a chronic respiratory infectious disease caused by mycobacterium tuberculosis (mycobaTmerium tuberculosis, MTB), a major public health problem worldwide.
Early detection of drug-resistant tuberculosis is very important. The traditional diagnosis method is that after the mycobacterium tuberculosis is smeared and cultured, a drug sensitivity test is carried out, and the drug resistance information of the mycobacterium tuberculosis can be obtained in 1-3 months, and the patient is treated at the moment and only has the drug resistance condition of the patient after a period of treatment, so that the illness state is delayed, and the spread of the drug resistance mycobacterium tuberculosis is possibly caused. WHO pointed out in 2021, "Update on the use of nucleic acid amplification tests to deteTm TB and drug-resistance TB: rapid communication: whether the tuberculosis patient is resistant to rifampicin or not, the drug resistance detection of isoniazid and fluoroquinolone drugs should be carried out in time to guide the treatment decision. Isoniazid is used for various pulmonary tuberculosis in various stages and various tuberculosis outside the lung, and is a first-line medicine for preventing and treating tuberculosis, the continuous utility of the medicine is endangered by the increase of isoniazid drug resistance and multi-drug resistance (MDR) tuberculosis, and the statistical result of the morbidity of isoniazid drug resistance tuberculosis is optimistic. Isoniazid is used as an antituberculosis drug with relatively few side effects and strong bactericidal effect, and its loss will make the treatment effect of tuberculosis much worse. The detection of the isoniazid resistance of the mycobacterium tuberculosis is beneficial to the prediction of drug-resistant tuberculosis, the key to the prevention and treatment of drug-resistant tuberculosis is early discovery, the tuberculosis drug-resistant condition of a patient is confirmed in time, a drug selection scheme is formulated, and then targeted standardized treatment is carried out.
Therefore, providing a detection reagent for detecting isoniazid resistance of mycobacterium tuberculosis with higher sensitivity and higher specificity is important to solve the problems.
Disclosure of Invention
The invention provides a composition, a kit and a method for detecting the isoniazid drug resistance of mycobacterium tuberculosis, which can make detection and judgment of the isoniazid drug resistance of the mycobacterium tuberculosis on a sample to be detected more sensitively and more accurately.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the first aspect of the invention provides a composition for detecting isoniazid resistance of mycobacterium tuberculosis, which comprises at least one of a composition 1 and a composition 2;
the composition 1 is any one set of a primer pair 1 and a probe 1, a primer pair 2 and a probe 2, and a primer pair 3 and a probe 3;
the composition 2 is any one set of a primer pair 4 and a probe 4, a primer pair 5 and a probe 5, and a primer pair 6 and a probe 6;
the primer pair 1 is katG-F1 and katG-R1, the primer pair 2 is katG-F2 and katG-R2, the primer pair 3 is katG-F3 and katG-R3, the probe 1 is katG-P1, the probe 2 is katG-P2, and the probe 3 is katG-P3;
the primer pair 4 is inhA-F1 and inhA-R1, the primer pair 5 is inhA-F2 and inhA-R2, the primer pair 6 is inhA-F3 and inhA-R3, the probe 4 is inhA-P1, the probe 5 is inhA-P2, and the probe 6 is inhA-P3;
the sequence of the inhA-F1 is shown as SEQ ID NO.1, the sequence of the inhA-R1 is shown as SEQ ID NO.2, the sequence of the inhA-P1 is shown as SEQ ID NO.3, the sequence of the inhA-F2 is shown as SEQ ID NO.4, the sequence of the inhA-R2 is shown as SEQ ID NO.5, the sequence of the inhA-P2 is shown as SEQ ID NO.6, the sequence of the inhA-F3 is shown as SEQ ID NO.7, the sequence of the inhA-R3 is shown as SEQ ID NO.8, and the sequence of the inhA-P3 is shown as SEQ ID NO. 9;
the sequence of the katG-F1 is shown as SEQ ID NO.10, the sequence of the katG-R1 is shown as SEQ ID NO.11, the sequence of the katG-P1 is shown as SEQ ID NO.12, the sequence of the katG-F2 is shown as SEQ ID NO.13, the sequence of the katG-R2 is shown as SEQ ID NO.14, the sequence of the katG-P2 is shown as SEQ ID NO.15, the sequence of the katG-F3 is shown as SEQ ID NO.16, the sequence of the katG-R3 is shown as SEQ ID NO.17, and the sequence of the katG-P3 is shown as SEQ ID NO. 18.
In the composition for detecting isoniazid resistance of mycobacterium tuberculosis, preferably, the fluorescent groups of the probe of the composition 1 and the probe of the composition 2 are different from each other and do not interfere with each other.
The composition for detecting isoniazid resistance of mycobacterium tuberculosis preferably, the fluorescent groups of the probe of the composition 1 and the probe of the composition 2 can be selected from any one of FAM, HEX, ROX, VIC, CY 5.5.5, TAMRA, TET, CY3 and JOE without interference.
In a second aspect, the invention provides a kit for detecting isoniazid resistance of mycobacterium tuberculosis, comprising the composition.
The kit for detecting isoniazid resistance of mycobacterium tuberculosis preferably further comprises: mgCl 2 At least one of dNTP and Taq enzyme.
In the kit for detecting isoniazid resistance of mycobacterium tuberculosis, preferably, when the composition only comprises any one of the composition 1 and the composition 2, the primer pair concentration of the composition 1 is 1000nM or the primer pair concentration of the composition 2 is 600nM, the probe concentration of the composition 1 is 60nM or the probe concentration of the composition 2 is 100nM, the Mg2+ concentration is 3mM, the dNTP concentration is 350 mu M, and the Taq enzyme concentration is 2U.
In the kit for detecting isoniazid resistance of mycobacterium tuberculosis, preferably, when the composition comprises the composition 1 and the composition 2, the primer pair concentration of the composition 1 is 1000nM, the primer pair concentration of the composition 2 is 600nM, the probe concentration of the composition 1 is 60nM, the probe concentration of the composition 2 is 100nM, the Mg2+ concentration is 3mM, the dNTP concentration is 350 mu M, and the Taq enzyme concentration is 2U.
In a third aspect, the present invention provides a method for detecting isoniazid resistance of mycobacterium tuberculosis for non-diagnostic purposes, the method comprising the steps of:
extracting DNA of a sample to be detected;
performing fluorescent quantitative PCR detection and/or melting curve experimental detection on the extracted DNA of the sample to be detected by using the composition or the kit;
and analyzing to obtain a detection result.
Preferably, the DNA volume of the sample to be tested used for performing the fluorescent quantitative PCR detection and/or the melting curve experimental detection is 10 mu L, and the MgCl 2 The total volume of dNTPs, and Taq enzyme was 25. Mu.L, and the volume of the composition 1 and/or the composition 2 was 15. Mu.L.
In the method for detecting isoniazid resistance of mycobacterium tuberculosis for non-diagnostic purposes, preferably, the amplification conditions for performing fluorescent quantitative PCR detection are as follows:
due to the adoption of the technical scheme, the invention has the following advantages:
the invention has the characteristics of simple operation, high sensitivity, good repeatability and high specificity in the isoniazid drug resistance detection of mycobacterium tuberculosis.
Drawings
FIG. 1-1 is Mg when katG primer 1 set probe was used 2+ Melting curves corresponding to different concentrations;
FIGS. 1-2 are graphs of melting curves corresponding to different concentrations of dNTPs using the 1 st set of primer probes of katG;
FIGS. 1-3 are graphs of melting curves corresponding to different concentrations of primer pair 1 using the 1 st set of primer probes of katG;
FIGS. 1-4 are graphs of melting curves corresponding to probe 1 at different concentrations using the 1 st set of primer probes of katG;
FIG. 2-1 shows Mg when the 2 nd primer set of inhA was used 2+ Melting curves corresponding to different concentrations;
FIG. 2-2 is a graph showing melting curves corresponding to different concentrations of dNTPs when the 2 nd primer set of inhA probe is used;
FIGS. 2-3 are graphs showing the melting curves of primer pair 5 at different concentrations using the 2 nd primer set of inhA;
FIGS. 2 to 4 are graphs showing melting curves corresponding to different concentrations of probe 5 when the 2 nd primer set of inhA probe was used;
FIG. 3-1 is a schematic diagram of the positional relationship of 3 sets of primer probes of katG relative to a target DNA sequence;
FIG. 3-2 is a schematic diagram showing the positional relationship of 3 sets of primer probes of inhA with respect to a target DNA sequence.
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 clearly and completely described below with reference to the accompanying drawings. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
3 sets of primer probes were designed for the katG target gene sequence and the inhA target gene sequence, respectively.
The katG target gene sequence is shown as SEQ ID NO.19, and the inhA target gene sequence is shown as SEQ ID NO. 20.
The object of this example is to provide a composition for detecting isoniazid resistance of Mycobacterium tuberculosis, the composition comprising at least one of composition 1 and composition 2, wherein, composition 1 can be selected from any one of 3 sets of primer probes of katG, and composition 2 can be selected from any one of 3 sets of primer probes of inhA.
The 1 st set of primer probes of katG comprises a primer pair 1 (katG-F1 and katG-R1) and a probe 1 (katG-P1), the 2 nd set of primer probes comprises a primer pair 2 (katG-F2 and katG-R2) and a probe 2 (katG-P2), and the 3 rd set of primer probes comprises a primer pair 3 (katG-F3 and katG-R3) and a probe 3 (katG-P3); the 1 st set of primer probes for inhA includes primer pair 4 (inhA-F1 and inhA-R1) and probe 4 (inhA-P1), the 2 nd set of primer probes includes primer pair 5 (inhA-F2 and inhA-R2) and probe 5 (inhA-P2), and the 3 rd set of primer probes includes primer pair 6 (inhA-F3 and inhA-R3) and probe 6 (inhA-P3). The specific sequences are shown in Table 1.
TABLE 1 primer probe sequence information for katG and inhA
The positional relationship of the 3 sets of primer probes of katG relative to the target DNA sequence is shown in FIG. 3-1; the positional relationship of the 3 sets of primer probes of inhA with respect to the target DNA sequence is shown in FIG. 3-2.
When the composition for detecting isoniazid resistance of mycobacterium tuberculosis of the present application only comprises the composition 1, the fluorescent groups of the probes 1, 2 and 3 in the 3 sets of primer probes of katG are selected from any one of FAM, HEX, ROX, VIC, CY 5.5.5, TAMRA, TET, CY and JOE, and the fluorescent groups of the probes 1, 2 and 3 can be the same or different; when the composition for detecting isoniazid resistance of mycobacterium tuberculosis of the present application only comprises the composition 2, the fluorophores of the probe 4, the probe 5 and the probe 6 in the 3 sets of primer probes of inhA are selected from any one of FAM, HEX, ROX, VIC, CY 5.5.5, TAMRA, TET, CY3 and JOE, and the fluorophores of the probe 4, the probe 5 and the probe 6 can be the same or different; when the composition for detecting isoniazid resistance of mycobacterium tuberculosis of the present application includes the composition 1 and the composition 2, the fluorophores of the selected probes of the composition 1 and the fluorophores of the selected probes of the composition 2 are different from each other and do not interfere with each other.
Preferably, the fluorescent groups of probe 1 (katG-P1), probe 2 (katG-P2) and probe 3 (katG-3) of composition 1 are each FAM, and the fluorescent groups of probe 4 (inhA-P1), probe 5 (inhA-P2) and probe 6 (inhA-P3) of composition 2 are each VIC.
Primer probe feasibility verification experiment:
1) Taking a positive test sample of about 500 mu LkatG and a positive test sample of inhA respectively, if the positive test sample is a sputum sample, adding 500 mu L of 1M NaOH respectively until the test sample is completely liquefied, centrifuging at 10000rpm at room temperature for 5-10min, discarding the supernatant, and if the positive test sample is other samples, directly centrifuging at 10000rpm at room temperature for 5-10min, and discarding the supernatant.
2) The sample to be tested after the treatment in step 1) was extracted according to the nucleic acid extraction kit instructions using a universal magnetic bead DNA extraction kit (DP 307) from Tiangen Biochemical technology (Beijing) Co., ltd, to form a DNA template.
3) Equal amounts of the katG positive test sample DNA template and the inhA positive test sample DNA template of step 2) were added to the two experimental reaction tubes, respectively, and the reaction systems were prepared according to Table 2, respectively.
4) An equal amount of a known wild-type sample (i.e., a known negative sample) as the experimental reaction tube was added to the control reaction tube, and a reaction system was prepared according to Table 2.
5) And (3) simultaneously carrying out on-machine detection on the two experimental reaction tubes and the control reaction tube, and carrying out PCR amplification and melting curve experiments. The detection instrument can be a molecular diagnosis integrated machine or other real-time fluorescence quantitative PCR instrument manufactured by Kunpeng gene (Beijing) technology Co., ltd, and the detection operation process is carried out according to the instrument instruction.
TABLE 2 feasibility test reaction system
6) And after the detection is finished, judging whether the sample to be detected has mutation or not by comparing the difference of the Tm values of the melting curves between the sample to be detected and the wild sample, namely whether the sample to be detected is a positive sample or not. Specifically, when the melting point of the FAM channel melting curve of the positive test sample of katG is lower than that of the FAM channel melting curve of the wild sample by 2 ℃ or more (delta Tm1 is more than or equal to 2 ℃), judging that the test sample is mutated, wherein the test sample is a positive sample, and the test sample is resistant to isoniazid; when the melting point of the VIC channel melting curve of the positive sample to be tested of inhA is lower than that of the VIC channel melting curve of the wild sample and is higher than or equal to 2 ℃, the mutation of the sample to be tested is judged, the sample to be tested is a positive sample, and the sample to be tested is resistant to isoniazid.
The detection results of the 3 sets of primer probes of katG in the experimental reaction tube are shown in Table 3-1, and the detection results of the 3 sets of primer probes of katG in the control reaction tube are shown in Table 3-2, and the results show that the 3 sets of primer probes of katG can realize the detection of positive samples, and the 3 sets of primer probes of katG have good feasibility; the results of detection of the 3 sets of primer probes for inhA in the experimental reaction tube are shown in Table 4-1, and the results of detection of the 3 sets of primer probes for inhA in the control reaction tube are shown in Table 4-2, and the results show that the 3 sets of primer probes for inhA can realize detection of positive samples, and the 3 sets of primer probes for inhA have good feasibility.
Wherein DeltaTm 1 is the difference between the melting point value of the FAM channel melting curve of the positive sample to be detected and the melting point value of the FAM channel melting curve of the wild sample, and DeltaTm 2 is the difference between the melting point value of the VIC channel melting curve of the positive sample to be detected and the melting point value of the VIC channel melting curve of the wild sample.
TABLE 3-1 detection results of katG 3 sets of primer probes in the experimental reaction tube
| First set of primer probes | △Tm1＝6.23 △Tm2＝0.26 |
| Second set of primer probes | △Tm1＝6.13 △Tm2＝0.39 |
| Second set of primer probes | △Tm1＝5.98 △Tm2＝0.33 |
TABLE 3-2 control of detection results of katG 3 sets of primer probes in reaction tubes
| First set of primer probes | △Tm1＝1.23 △Tm2＝0.41 |
| Second set of primer probes | △Tm1＝0.56 △Tm2＝0.24 |
| Second set of primer probes | △Tm1＝1.03 △Tm2＝0.44 |
TABLE 4-1 detection results of 3 sets of primer probes for inhA in experimental reaction tubes
| First set of primer probes | △Tm1＝0.35 △Tm2＝5.33 |
| Second set of primer probes | △Tm1＝0.43 △Tm2＝5.03 |
| Second set of primer probes | △Tm1＝0.36 △Tm2＝4.98 |
TABLE 4-2 control of detection results of 3 sets of primer probes for inhA in reaction tubes
Example 2
The embodiment aims at providing a kit for detecting isoniazid resistance of mycobacterium tuberculosis.
A kit for detecting isoniazid resistance of Mycobacterium tuberculosis comprises the composition in example 1 and MgCl 2 At least one of dNTP and Taq enzyme.
In addition, the kit also comprises a positive quality control and a negative control 1 wild mycobacterium tuberculosis complex strain and a negative control 2TE.
When the kit is used for detecting isoniazid resistance of mycobacterium tuberculosis on a sample to be detected, the kit has good feasibility as the detection result is recorded in the embodiment 1.
Example 3
The objective of this example is to optimize the concentrations of the components in the kit of example 2, and the optimization experiments were performed at a Taq enzyme concentration of 2U, and the on-machine experiments were performed on the samples to be tested according to the amplification procedure shown in table 5, and the specific optimization experiments are as follows:
TABLE 5 amplification procedure
As shown in Table 3-1 and Table 4-1, 3 sets of primers for katG and 3 sets of primers for inhA in example 1
The detection results of the physical probes on the sample to be detected are not greatly different, so that the kit is formed by a set of primer probes of katG and a set of primer probes of inhA optionally for carrying out an optimization experiment.
An optimization experiment was performed using one of the kits of example 2, the kit selected in this experiment was a kit comprising katG 1 st set of primer probes and inhA 2 nd set of primer probes, mg for this kit 2+ Gradient adjusting the concentration to form Mg 2+ The concentrations were 1mM,1.5mM,2mM,2.5mM,3mM,3.5mM,4mM,5mM and respectively detecting the same 1000CFU/mL sample to be detected, wherein the detection results are shown in FIG. 1-1 and FIG. 2-1, and FIG. 1-1 is Mg of FAM channel 2+ FIG. 2-1 shows the corresponding melting curves at different concentrations for the Mg of the VIC channel 2+ The corresponding melting curves at different concentrations can be seen from FIG. 1-1 for Mg 2+ At a concentration of 3.5mM, the peak of the melting curve is highest, and it can be seen from FIG. 2-1 that the melting curve is formed at Mg 2+ The concentration was 2.5mM, the peak of the melting curve was highest, and therefore Mg was finally selected 2+ The optimal reaction concentration was 3mM.
An optimization experiment was performed using one of the kits of example 2, in which the kit used was a kit comprising katG 1 st primer probe and inhA 2 nd primer probe, and the dNTP concentration of the kit was adjusted in a gradient to form a plurality of kits having dNTP concentrations of 200. Mu.M, 250. Mu.M, 300. Mu.M, 350. Mu.M, 400. Mu.M, 500. Mu.M, and the same 1000CFU/mL sample to be tested, respectively, the detection results were shown in FIGS. 1-2 and 2-2, FIGS. 1-2 were melting graphs corresponding to different concentrations of dNTPs of FAM channel, FIGS. 2-2 were melting graphs corresponding to different concentrations of dNTPs of VIC channel, and it was seen from FIGS. 1-2 that the peak of melting curve was highest at the dNTP concentration of 350. Mu.M and the peak of melting curve was highest at the dNTP concentration of 500. Mu.M and the peak of 350. Mu.M was highest at the melting curve was observed from FIGS. 2-2, respectively, so that the final optimum dNTP reaction concentration was selected to be 350. Mu.M.
An optimization experiment was performed using one of the kits of example 2, the kit selected in this experiment was a kit comprising katG 1 st set of primer probes and inhA 2 nd set of primer probes, the concentrations of primer pair 1 and primer pair 5 of the kit were adjusted in a gradient, wherein the concentrations of primer pair 1 and primer pair 5 were identical, a plurality of kits of primer pair 1 and primer pair 5 concentrations of 400nM,600nM,800nM,1000nM,1200nM, respectively, were formed and the same 1000CFU/mL samples to be tested were tested, the results of the tests were shown in FIGS. 1-3 and 2-3, FIGS. 1-3 are melting graphs corresponding to different concentrations of primer pair 1 and primer pair 5 for FAM channel, FIGS. 2-3 are melting graphs corresponding to different concentrations of primer pair 1 and primer pair 5 for VIC channel, and it can be seen from FIGS. 1-3 that the peak of the melting curve is highest at a concentration of 1000nM for primer pair 1, and that the peak of the melting curve is highest at a concentration of 1000nM for primer pair 5, 600nM times, and less different for both, so that the optimal reaction concentrations of primer pair 1 and primer pair 5 are finally selected to be 1000nM and 600nM, respectively.
An optimization experiment was performed using one of the kits of example 2, in which the kit used was a kit comprising katG primer probe 1 and inhA primer probe 2, and the concentrations of probe 1 and probe 5 of the kit were adjusted in a gradient, wherein the concentrations of probe 1 and probe 5 were the same, a plurality of kits were formed with probe 1 and probe 5 concentrations of 40nM,60nM,100nM,200nM,300nM, respectively, and the same sample to be tested was tested, the test results were shown in fig. 1-4 and fig. 2-4, the melting curves corresponding to the probe 1 concentrations of FAM channel were shown in fig. 1-4, the melting curves corresponding to the probe 5 concentrations of VIC channel were shown in fig. 2-4, the peak of the melting curve was the highest at 60nM for probe 1, and the peak of the melting curve was the highest at 100nM for probe 5, that is the optimal reaction concentrations of probe 1 and probe 5 were the peak at 60nM and 100nM for probe 5, respectively, as seen in fig. 2-4.
In summary, the optimal concentration of each component in the kit is determined as follows: the concentration of the primer pair 1 is 1000nM, the concentration of the probe 1 is 60nM, the concentration of the primer pair 5 is 600nM, the concentration of the probe 5 is 100nM, and the concentration of the Mg 2+ The concentration was 3mM, dNTP concentration was 350. Mu. M, taq enzyme concentration was 2U. Wherein MgCl 2 The mixture of dNTP and Taq enzyme is also called a reaction enzyme mixture.
It will be appreciated that when the kit is any set of primer probes comprising katG and any set of primer probes for inhA, the optimum concentrations of the components within the kit are: the concentration of the primer pair of katG was 1000nM, the concentration of the primer pair of inhA was 600nM, the concentration of the probe of katG was 60nM, the concentration of the probe of inhA was 100nM, mg 2+ The concentration was 3mM, dNTP concentration was 350. Mu. M, taq enzyme concentration was 2U. Wherein MgCl 2 The mixture of dNTP and Taq enzyme is also called a reaction enzyme mixture.
Example 4
The purpose of this example is to illustrate a method for detecting isoniazid resistance in Mycobacterium tuberculosis using the composition of example 1 or the kit of example 2.
A method for detecting isoniazid resistance of mycobacterium tuberculosis for non-diagnostic purposes comprises the following steps:
(1) Sample pretreatment: for a viscous sample to be tested, such as sputum, about 500 mu L of the sample is taken, 500 mu L of 1M NaOH is added until the sample to be tested is completely liquefied, the supernatant is discarded after centrifugation at 10000rpm at room temperature for 5-10min, and if the sample to be tested is other samples, the supernatant is discarded after centrifugation at 10000rpm at room temperature for 5-10 min.
(2) Nucleic acid extraction: and (3) extracting the sample to be detected after the treatment in the step (1) by using a universal magnetic bead DNA extraction kit (DP 307) of Tiangen biochemical technology (Beijing) limited company according to the specification of the nucleic acid extraction kit to form a DNA template.
(3) Sample adding: the relevant reagents were added to the prepared PCR reaction tube according to Table 6, including the 10. Mu.L DNA template of step (2), 15. Mu.L of any one of the compositions (also referred to as primer probe mix) of example 1, and 25. Mu.L of the reaction enzyme mix (MgCl) at the concentrations of the components determined in example 3 2 The mixture of dNTP and Taq enzyme is called a reaction enzyme mixture), the tube cover of the PCR reaction tube is covered tightly, and the PCR reaction tube is subjected to instantaneous low-speed centrifugation.
(4) And (3) PCR amplification detection, namely placing the PCR reaction tube in the step (3) into a fluorescent quantitative PCR instrument for amplification melting curve detection, and generating a melting curve of a sample to be detected. And after the detection is finished, judging whether the sample to be detected has mutation or not by comparing the difference of the Tm values of the melting curves between the sample to be detected and the wild sample, namely whether the sample to be detected is a positive sample or not. Specifically, when the melting points of the FAM channel and the VIC channel of the sample to be detected are lower than the melting point of the wild sample within a preset range (delta Tm is more than or equal to 0 and less than 2 ℃), judging that the sample to be detected is not mutated, wherein the sample to be detected is a negative sample, and the sample to be detected is sensitive to isoniazid resistance: and when the melting point of the sample to be detected in any one of the FAM channel and the VIC channel of the sample to be detected is lower than the melting point of the wild sample by 2 ℃ or more (delta Tm is more than or equal to 2 ℃), judging that the sample to be detected is mutated, wherein the sample to be detected is a positive sample, and the sample to be detected is resistant to isoniazid.
The wild sample is a known negative sample, and when the test of the isoniazid resistance of the mycobacterium tuberculosis of the sample to be tested is performed each time, the test is performed on one fluorescent quantitative PCR instrument according to the above-mentioned experimental method of the embodiment 4, and meanwhile, the same test is performed on the same fluorescent quantitative PCR instrument on the wild sample, so that a more accurate control group is provided when the test result is determined and analyzed in the step (4), and the control group is the melting curve of the wild sample.
TABLE 6 reaction system
| Reagent(s) | Volume of |
| Mixed solution of reactive enzyme | 25μL |
| Primer probe mixed liquid | 15μL |
| DNA template | 10μL |
Example 5
The purpose of this example was to investigate the lowest detected concentration and sensitivity of the composition of example 1 or the kit of example 2.
The operation steps are as follows:
s1: a DNA template of a katG positive test sample was prepared as in the method step (1) in example 4.
S2: and (3) carrying out concentration modulation on the DNA templates of the katG positive sample to be detected in the step (S1) to prepare 4 groups of DNA templates with different concentrations, wherein the concentrations are 1000CFU/mL, 300CFU/mL, 200CFU/mL and 150CFU/mL in sequence.
S3: taking positive samples with different concentrations prepared in the step S2 as samples to be detected, and performing on-machine detection on the samples to be detected by using any one of the kits (the kit used in the experiment is a kit consisting of a 1 st set of primer probes of katG and a 2 nd set of primer probes of inhA) of the example 2 with qualified quality detection, wherein each concentration of the samples to be detected is detected for 10 times. The reaction system of the sample to be tested is prepared according to table 6, the amplification procedure detected by the machine is carried out according to table 5, after amplification is completed, melting curve is generated, statistical analysis results are shown in table 7, wherein DeltaTm 1 is the difference between the melting point value of the FAM channel melting curve of the katG positive sample to be tested and the melting point value of the FAM channel melting curve of the wild sample, and DeltaTm 2 is the difference between the melting point value of the melting curve of the VIC channel of the inhA positive sample to be tested and the melting point value of the VIC channel melting curve of the wild sample.
TABLE 7 statistical information on the DeltaTm values of the results of the lowest detection limit test experiments of katG and inhA
As can be seen from Table 7, the positive detection rate was 100% at a positive sample concentration of 200CFU/mL and 60% at a concentration of 150CFU/mL. The minimum detection limit of the compositions or kits of the present application is thus determined to be 200CFU/mL.
Example 6
The purpose of this example is to verify the specificity of the composition of example 1 or the kit of example 2
The kit of example 2, which was used for quality control, was used to detect the positive samples of katG, inhA positive reference, B1 normal human negative samples, and B2 to B4 clinical positive samples (mycobacterium kansasii, mycobacterium marinus, streptococcus pneumoniae positive samples) of similar species or similar symptoms in this order according to the method of example 4 on the same fluorescent quantitative PCR machine, and the specificity of the kit was examined by analyzing the negative and positive of the detection results. The experimental results are shown in table 8, wherein Δtm1 is the difference between the melting point value of the FAM channel melting curve of each sample and the melting point value of the FAM channel melting curve of the wild-type sample, and Δtm2 is the difference between the melting point value of the VIC channel melting curve of each sample and the melting point value of the VIC channel melting curve of the wild-type sample.
TABLE 8 statistical information on the detection results DeltaTm values of the test experiments for each sample
/>
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A composition for detecting isoniazid resistance of mycobacterium tuberculosis, which is characterized by comprising at least one of a composition 1 and a composition 2;
the composition 1 is any one set of a primer pair 1 and a probe 1, a primer pair 2 and a probe 2, and a primer pair 3 and a probe 3;
the composition 2 is any one set of a primer pair 4 and a probe 4, a primer pair 5 and a probe 5, and a primer pair 6 and a probe 6;
the primer pair 1 is katG-F1 and katG-R1, the primer pair 2 is katG-F2 and katG-R2, the primer pair 3 is katG-F3 and katG-R3, the probe 1 is katG-P1, the probe 2 is katG-P2, and the probe 3 is katG-P3;
the primer pair 4 is inhA-F1 and inhA-R1, the primer pair 5 is inhA-F2 and inhA-R2, the primer pair 6 is inhA-F3 and inhA-R3, the probe 4 is inhA-P1, the probe 5 is inhA-P2, and the probe 6 is inhA-P3;
the sequence of the inhA-F1 is shown as SEQ ID NO.1, the sequence of the inhA-R1 is shown as SEQ ID NO.2, the sequence of the inhA-P1 is shown as SEQ ID NO.3, the sequence of the inhA-F2 is shown as SEQ ID NO.4, the sequence of the inhA-R2 is shown as SEQ ID NO.5, the sequence of the inhA-P2 is shown as SEQ ID NO.6, the sequence of the inhA-F3 is shown as SEQ ID NO.7, the sequence of the inhA-R3 is shown as SEQ ID NO.8, and the sequence of the inhA-P3 is shown as SEQ ID NO. 9;
the sequence of the katG-F1 is shown as SEQ ID NO.10, the sequence of the katG-R1 is shown as SEQ ID NO.11, the sequence of the katG-P1 is shown as SEQ ID NO.12, the sequence of the katG-F2 is shown as SEQ ID NO.13, the sequence of the katG-R2 is shown as SEQ ID NO.14, the sequence of the katG-P2 is shown as SEQ ID NO.15, the sequence of the katG-F3 is shown as SEQ ID NO.16, the sequence of the katG-R3 is shown as SEQ ID NO.17, and the sequence of the katG-P3 is shown as SEQ ID NO. 18.
2. The composition for detecting isoniazid resistance in mycobacterium tuberculosis according to claim 1, wherein the fluorescent groups of the probe of the composition 1 and the probe of the composition 2 are different from each other and do not interfere with each other.
3. The composition for detecting isoniazid resistance in mycobacterium tuberculosis according to claim 2 wherein the fluorescent groups of the probe of composition 1 and the probe of composition 2 can be selected from any one of FAM, HEX, ROX, VIC, CY 5.5.5, TAMRA, TET, CY3 and JOE without interfering with each other.
4. A kit for detecting isoniazid resistance in mycobacterium tuberculosis comprising the composition of any one of claims 1-3.
5. The kit for detecting isoniazid resistance in mycobacterium tuberculosis according to claim 4, further comprising: mgCl 2 、dNTP、TaqAt least one of the enzymes.
6. The kit for detecting isoniazid resistance in mycobacterium tuberculosis according to claim 5, wherein when the composition includes only any one of the composition 1 and the composition 2, the primer pair concentration of the composition 1 is 1000nM or the primer pair concentration of the composition 2 is 600nM, the probe concentration of the composition 1 is 60nM or the probe concentration of the composition 2 is 100nM, the Mg 2+ The concentration was 3mM, the dNTP concentration was 350. Mu.M, and the Taq enzyme concentration was 2U.
7. The kit for detecting isoniazid resistance in mycobacterium tuberculosis according to claim 5, wherein when the composition comprises the composition 1 and the composition 2, the primer pair concentration of the composition 1 is 1000nM, the primer pair concentration of the composition 2 is 600nM, the probe concentration of the composition 1 is 60nM, the probe concentration of the composition 2 is 100nM, and the Mg 2+ The concentration was 3mM, the dNTP concentration was 350. Mu.M, and the Taq enzyme concentration was 2U.
8. A method for detecting isoniazid resistance of mycobacterium tuberculosis for non-diagnostic purposes, the method comprising the steps of:
extracting DNA of a sample to be detected;
performing fluorescent quantitative PCR detection and/or melting curve experimental detection on the DNA of the extracted sample to be tested using the composition of any one of claims 1-3 or the kit of any one of claims 4-7;
and analyzing to obtain a detection result.
9. The method for detecting isoniazid resistance of Mycobacterium tuberculosis according to claim 8, wherein the DNA volume of the sample to be detected used in performing the fluorescent quantitative PCR detection and/or the melting curve experiment detection is 10. Mu.L, and the MgCl 2 The total volume of dNTP, and Taq enzyme was 25. Mu.L, the composition 1 and/orThe volume of the composition 2 was 15. Mu.L.
10. The method for detecting isoniazid resistance of mycobacterium tuberculosis according to claim 9 wherein the amplification conditions for performing fluorescent quantitative PCR detection are:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311348261.8A CN117448466A (en) | 2023-10-18 | 2023-10-18 | Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311348261.8A CN117448466A (en) | 2023-10-18 | 2023-10-18 | Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117448466A true CN117448466A (en) | 2024-01-26 |
Family
ID=89592085
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311348261.8A Pending CN117448466A (en) | 2023-10-18 | 2023-10-18 | Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117448466A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011140237A2 (en) * | 2010-05-04 | 2011-11-10 | The Government Of The Usa Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Process for detection of multidrug resistant tuberculosis using real-time pcr and high resolution melt analysis |
| CN107034277A (en) * | 2017-04-27 | 2017-08-11 | 厦门大学 | A kind of method for detecting low abundance gene mutation |
| CN114150076A (en) * | 2021-06-30 | 2022-03-08 | 江苏硕世生物科技股份有限公司 | Primer probe and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid |
-
2023
- 2023-10-18 CN CN202311348261.8A patent/CN117448466A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011140237A2 (en) * | 2010-05-04 | 2011-11-10 | The Government Of The Usa Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Process for detection of multidrug resistant tuberculosis using real-time pcr and high resolution melt analysis |
| CN107034277A (en) * | 2017-04-27 | 2017-08-11 | 厦门大学 | A kind of method for detecting low abundance gene mutation |
| CN114150076A (en) * | 2021-06-30 | 2022-03-08 | 江苏硕世生物科技股份有限公司 | Primer probe and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid |
Non-Patent Citations (4)
| Title |
|---|
| MELISSA V RAMIREZ等: "Rapid Detection of Multidrug-Resistant Mycobacterium tuberculosis by Use of Real-Time PCR and High-Resolution Melt Analysis", J CLIN MICROBIOL, vol. 48, no. 11, 30 November 2010 (2010-11-30), pages 4003 - 4009, XP008134977, DOI: 10.1128/JCM.00812-10 * |
| 杨彩虹等: "高分辨率熔解曲线技术用于结核分枝杆菌临床分离株异烟肼耐药性的快速检测", 中国人兽共患病学报, vol. 33, no. 05, 15 May 2017 (2017-05-15), pages 403 - 412 * |
| 柳清云等: "双通道实时荧光PCR熔解曲线法检测结核分枝杆菌药物耐药相关基因突变", 中华检验医学杂志, vol. 36, no. 1, 31 January 2013 (2013-01-31), pages 1 - 2 * |
| 胡思玉: "实时PCR在结核分枝杆菌异烟肼耐药突变分子诊断中的应用研究", 中国博士学位论文全文数据库医药卫生科技辑, no. 8, 15 August 2014 (2014-08-15), pages 061 - 3 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP4116440A1 (en) | Rt-pcr detection method and kit for novel coronavirus | |
| CN111004862B (en) | Primer and probe for rapidly detecting and identifying cryptococcus and application thereof | |
| CN112410472B (en) | Primer probe combination for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus and detection kit | |
| CN111733291A (en) | Method and kit for detecting novel coronavirus nucleic acid by digital PCR (polymerase chain reaction) | |
| CN113201594A (en) | Method for rapidly detecting food-borne Burkholderia gladioli | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| CN113862393A (en) | Method for rapidly detecting cryptococcus gatherensis | |
| CN114381509B (en) | Plasma miRNA marker related to non-tuberculous pneumonia and application thereof | |
| KR20230057778A (en) | Method and Kit for Diagnosing Bovine tuberculosis using PNA | |
| CN109321651A (en) | A kind of composition, kit, sample treatment and application detecting people CYP2D6 gene pleiomorphism | |
| CN116219071B (en) | Multiplex qPCR kit for simultaneously detecting and identifying type I, type IIa and type IIb monkey poxviruses | |
| CN106916903A (en) | The real-time fluorescence RT PCR detection methods and kit of mycobacterium tuberculosis 85B mRNA | |
| WO2020134950A1 (en) | Gene mutation/fusion combination and kit for identification of benign and malignant pulmonary nodules | |
| CN110607381A (en) | Mycobacterium tuberculosis detection kit and method | |
| CN117448466A (en) | Composition, kit and method for detecting isoniazid resistance of mycobacterium tuberculosis | |
| CN112501349B (en) | Primer probe composition and kit for synchronously detecting 8 children digestive tract infection fungi and parasite-related pathogens | |
| CN112501321B (en) | Method for molecular typing of mycobacterium tuberculosis | |
| CN117230222A (en) | Composition, kit and method for detecting quinolone resistance of mycobacterium tuberculosis | |
| CN110241231A (en) | Detect composition, kit, method and the application of CYP2C19 gene pleiomorphism | |
| CN116024360B (en) | Primer combination for mycobacterium tuberculosis complex identification and drug-resistant gene mutation detection and application thereof | |
| CN117070645A (en) | Composition, kit and method for detecting aminoglycoside drug resistance of mycobacterium tuberculosis | |
| CN112458194B (en) | Primer probe combination and detection kit for mycoplasma pneumoniae and drug-resistant mutation thereof | |
| CN113564240B (en) | HLA-B27 allele detection method and detection kit | |
| CN116769945B (en) | Primer and fluorescent probe for detecting mycobacterium tuberculosis complex and application of primer and fluorescent probe | |
| CN117106865B (en) | High-sensitivity detection method based on double amplification and double LNA probe specific recognition DNA mutation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |