CN117230219A - Nucleic acid composition, kit and detection method for detecting isoniazid drug-resistant gene mutation of mycobacterium tuberculosis - Google Patents
Nucleic acid composition, kit and detection method for detecting isoniazid drug-resistant gene mutation of mycobacterium tuberculosis Download PDFInfo
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Classifications
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- 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
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a nucleic acid composition, a kit and a detection method for detecting isoniazid drug-resistant gene mutation of mycobacterium tuberculosis, and relates to the field of biological detection. The nucleic acid composition comprises: a first primer pair with nucleotide sequences shown as SEQ ID No. 1-2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3-4, and a third primer pair with nucleotide sequences shown as SEQ ID No. 5-6. The invention can realize single-tube monomer system detection of nine mutations of KatG, inhA, ahpC and isoniazid drug resistance related genes. The nucleic acid composition, the kit and the detection method provided by the invention have the technical advantages of simplicity and convenience in operation, rapidness and high efficiency, and the detection method has higher detection sensitivity and accuracy.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a nucleic acid composition, a kit and a detection method for detecting isoniazid drug-resistant gene mutation of mycobacterium tuberculosis.
Background
Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), which occurs mostly in the lungs, most commonly in tuberculosis. In China, tuberculosis latent infectors are about 2.5-3 hundred million. The world health organization issues 2021 global tuberculosis report on day 14 of 10 of 2021, and the report shows that the estimated number of new tuberculosis patients in 2020 of China is 84.2 ten thousand, and the estimated tuberculosis incidence is 59/10 ten thousand. If the patient is infected with mycobacterium tuberculosis, the drug resistance of the patient to one or more antitubercular drugs is generated, namely, drug resistant tuberculosis. According to the latest drug resistance monitoring estimation of WHO/IUATLD, in new patients, 10.2% of patients are resistant to at least one antitubercular drug, and 1.1% are multi-drug resistant tuberculosis; of the combination patients, 18.4% were resistant to at least one antitubercular drug with a multiple resistance of 7.0%. Drug-resistant tuberculosis brings great challenges to the prevention and treatment of tuberculosis.
Isoniazid (INH) is a first-line antitubercular drug commonly used in clinic, but due to long treatment course, patients may break treatment, and drug resistance may occur, and the occurrence of drug resistance is also related to genetic mutation of MTB. INH resistance-related mutations mainly occur in the katG and inhA promoter regions, and in addition, there are genes associated with INH resistance such as ahpC, fabG1, kasA, iniA/B/C, ndh, fadE, furA, rv1592c and Rv1772 and their promoter regions. Other two-line drugs commonly used in clinic, such as streptomycin, thiosemicarbazide, cycloserine, ethylsulfanilamide and the like, the drug resistance of MTB to the drugs is relatively stable, and the drug resistance is rarely sensitized after the drug resistance; the rate of sensitization after rifampicin resistance is about 4.2%; whereas, after INH resistance, there may be 88.5% rate of sensitization 36 weeks after drug withdrawal.
The current drug resistance detection technology of mycobacterium tuberculosis comprises a phenotypic drug susceptibility method and a molecular drug susceptibility method. The phenotypic drug-sensitive method is based on culture, and the drug resistance of the tubercle bacillus is detected by observing the growth condition of the tubercle bacillus in a drug-containing culture medium, so that the phenotypic drug-sensitive method is a gold standard for drug resistance detection at present. However, the culture method takes a long time, usually more than several weeks, to obtain the result, and may cause delay for patients who need anti-tuberculosis treatment. The molecular drug sensitivity method is to detect and identify drug resistance gene mutant type of tubercle bacillus by adopting molecular biological technology. The molecular detection has the advantages of being capable of rapidly and sensitively detecting drug-resistant tubercle bacillus from clinical specimens, even specimens coated with perineum and perineum.
Currently, the main tuberculosis drug-resistant molecular diagnostic reagents applied in the market are American Sai Pei Xpert MTB/RIF and a mycobacterium tuberculosis drug-resistant mutation detection kit of Xiamen organisms. The molecular detection method is convenient and rapid, but special detection instruments are required to be arranged for detecting isoniazid drug resistance of the cefpirome, so that the price is high, the cost performance is low, and the popularization and screening and effective prevention and control of tuberculosis are not facilitated; the isoniazid drug-resistant detection reagent for Xiamen is a double-tube double-system, the operation is complex, the detection sample is a separated culture, and the human sputum sample cannot be directly detected.
Therefore, there is an urgent need in the art to develop a method for detecting mycobacterium tuberculosis drug resistance with simple operation, low cost, high detection efficiency, and high sensitivity and accuracy to meet clinical demands.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a nucleic acid composition, a kit and a detection method for detecting mutation of isoniazid drug resistance genes of mycobacterium tuberculosis, so as to realize single-tube monomer system detection of nine mutations of KatG, inhA, ahpC genes related to isoniazid drug resistance.
The invention is realized in the following way:
in a first aspect, the present invention provides a nucleic acid composition for detecting a mutation in a isoniazid resistance gene of mycobacterium tuberculosis, comprising: a first primer pair with nucleotide sequences shown as SEQ ID No. 1-2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3-4, and a third primer pair with nucleotide sequences shown as SEQ ID No. 5-6.
In a preferred embodiment of the invention, the nucleic acid composition further comprises probes comprising a first probe as shown in SEQ ID No.7, a second probe as shown in SEQ ID No.8, a third probe as shown in SEQ ID No.9 and a fourth probe as shown in SEQ ID No. 10;
In an alternative embodiment, the first probe is used to detect the KatG S315T and KatG S315N mutations; the second probe is used for detecting ahpC C-6A and ahpC C-12T mutation; the third probe was used to detect ahpC C-39T, ahpC G-46A and ahpC T-49G mutations; the fourth probe is used for detecting InhAT-8C and InhA C-15T mutation;
in an alternative embodiment, at least one of the first, second, third and fourth probes comprises at least one modified nucleotide group;
in an alternative embodiment, the modified nucleotide group is at least one of (1) to (3):
(1) A phosphate group modified nucleotide group;
(2) Ribosyl group modified nucleotide group.
In an alternative embodiment, the phosphate group modification is modification of the oxygen in the phosphate group, including thio and boronate modifications;
in an alternative embodiment, the ribose group modification is a modification of the 2' -hydroxyl group of the ribose group selected from any of the following modifications: 2 '-fluoro modification, 2' -methoxy modification, 2 '-methoxyethyl modification, 2' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 '-amino modification and 2' -deoxy modification.
In an alternative embodiment, the ribose group modification is a locked nucleic acid modification of the 2' -hydroxyl group of the ribose group;
in an alternative embodiment, base G at position 10 in the nucleotide sequence of the first probe is a locked nucleic acid modifying base; the 19 th base T in the nucleotide sequence of the fourth probe is a locked nucleic acid modified base.
In a preferred embodiment of the application of the invention, the probe is provided with a fluorescent reporter group 5 'and a fluorescent quenching group and/or MGB 3'.
In an alternative embodiment, the fluorescent reporter group is selected from the group consisting of: any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, cy3, cy3.5, NED, TAMRA, ROX, texas Red, cy5, cy5.5 and Quasar 670;
in an alternative embodiment, the fluorescence quenching group is selected from: TAMRA, eclipse, BHQ, dabcyl, lowa BlackTM RQ and Lowa BlackTM FQ;
in an alternative embodiment, the BHQ line is selected from at least one of BHQ0, BHQ1, BHQ2, and BHQ 3.
In a preferred embodiment of the present invention, in any one of the first primer pair, the second primer pair and the third primer pair, the molar ratio of the upstream primer to the downstream primer is independently (1 to 20): (1-20);
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20);
in an alternative embodiment, the molar ratio of the upstream primer to the downstream primer in the first primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer of the second primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer in the third primer pair is independently 1: (1-20).
In an alternative embodiment, the concentration of the first probe, the second probe, the third probe and the fourth probe in the nucleic acid composition is independently 0.01 to 1. Mu.M, preferably 0.1 to 0.5. Mu.M.
In a second aspect, the invention also provides a reagent or kit comprising the nucleic acid composition described above.
In a preferred embodiment of the use of the invention, the reagent or kit further comprises at least one of the following: PCR reaction buffer, DNA polymerase, magnesium ion, dNTP, wild type control, negative control and water;
in an alternative embodiment, the DNA polymerase is selected from at least one of Tth DNA polymerase or a mutant thereof, taq DNA polymerase or a mutant thereof, pfu enzyme or a mutant thereof, and Hawk Z05 polymerase or a mutant thereof;
The concentration of the DNA polymerase is 0.01U/. Mu.l to 5U/. Mu.l;
the working concentration of magnesium ions is 0.5 mM-1.5 mM;
the working concentration of dNTP is 0.1 mM-25 mM;
any one of the first primer pair, the second primer pair and the third primer pair has a concentration of 0.1 mu mol/L to 5 mu mol/L;
in any one of the first primer pair, the second primer pair and the third primer pair, the molar ratio of the upstream primer to the downstream primer is independently (1 to 20): (1-20).
In a third aspect, the invention also provides application of the nucleic acid composition in preparing a detection product of the isoniazid resistance gene mutation of the mycobacterium tuberculosis;
in an alternative embodiment, the detection product is selected from any one of a reagent, a kit and a gene chip.
In a fourth aspect, the invention also provides a method for detecting the isoniazid drug-resistant gene mutation of mycobacterium tuberculosis, which comprises the following steps: PCR detection is carried out on the sample to be detected by adopting the nucleic acid composition or the reagent or the kit;
the detection method is not aimed at diagnosis or treatment of the disease;
in an alternative embodiment, the sample to be tested is selected from at least one of a nucleic acid sample, an environmental sample comprising a nucleic acid sample, a negative control, a positive control, human sputum, sputum solid isolates and cultures thereof, and liquid isolates and cultures thereof.
In a preferred embodiment of the present invention, when PCR detection is performed, the molar ratio of the upstream primer to the downstream primer in any one of the first primer pair, the second primer pair and the third primer pair is independently (1 to 20): (1-20);
in an alternative embodiment, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20);
in an alternative embodiment, the PCR detection is performed with any one of the first, second and third primer pairs, the working concentration of the upstream and downstream primers being independently 0.01 to 5. Mu.M, preferably 0.1 to 1. Mu.M;
in an alternative embodiment, the working concentrations of the first, second, third and fourth probes are independently 0.01 to 1. Mu.M, preferably 0.1 to 0.5. Mu.M, when performing PCR detection;
in an alternative embodiment, the reaction procedure for PCR is as follows: 94-96 ℃ for 15 sec-10 min (pre-denaturation); 94-98 deg.c for 10-30 s, 55-65 deg.c, 20-40 s, 70-74 deg.c for 20-30 s and 30-50 cycles; collecting fluorescent signals at the temperature of 55-56 ℃ for 20-40 s; 94-96 deg.c for 1-3 min and 33-37 deg.c for 1-3 min; and continuously collecting fluorescent signals at 35-90 ℃.
In a fifth aspect, the present invention also provides a system for detecting isoniazid resistance gene mutation in mycobacterium tuberculosis, comprising: the above-described reagent or kit, first means and second means; the first device can respectively perform real-time fluorescent quantitative PCR detection on a DNA sample to be detected, a wild type control and a negative control by adopting a reagent or a kit to obtain a melting curve graph;
the second device includes: the data input module and the data analysis and conclusion output module;
the data input module is configured to input melting curve graph data obtained by the first device; the data analysis and conclusion output module is configured to: judging whether the sample to be detected contains the gene mutation or not and determining the type of the gene mutation according to the change of the Tm difference value of the melting peak of the sample to be detected and the wild type control in each channel.
The invention has the following beneficial effects:
the reagent provided by the invention is based on real-time fluorescence quantitative PCR, combines a multiple asymmetric PCR technology, a Taqman probe technology and a melting curve analysis technology, and detects by taking the KatG gene, the InhA gene and the ahpC gene sequences of isoniazid resistance determining regions of mycobacterium tuberculosis as targets. Realizes one-time specific detection and distinguishes nine mutation sites of KatG S315T, katG S315N, ahpC C-6A, ahpC C-12T, ahpC C-39T, ahpC G-46A, ahpC T-49G, inhA T-8C and InhA C-15T.
The invention also provides a detection reagent and a kit, and a primer for specific amplification is designed aiming at the sequences of various mutation sites, so that multiple asymmetric PCR (polymerase chain reaction) is applied to detect a plurality of sites in a same tube.
The reagent of the invention is used for detecting the Mycobacterium tuberculosis isoniazid drug-resistant national reference plate, the coincidence rate of sensitive reference is 100 percent, the coincidence rate of drug-resistant reference is 100 percent, and the detection limit reference S, SI, SID and precision reference J (1 multiplied by 10) 5 CFU/mL) can be detected.
The nucleic acid composition, the reagent and the kit provided by the invention are suitable for detecting different samples such as human sputum, alveolar lavage fluid or liquid separation culture, can rapidly identify the condition of the isoniazid drug resistance of the mycobacterium tuberculosis in the sample of a suspected or diagnosed tuberculosis patient within 3 hours, and provide more convenient and rapid auxiliary diagnosis and treatment basis for the suspected or diagnosed tuberculosis infection patient.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a typical graph of a FAM channel for isoniazid resistance detection of Mycobacterium tuberculosis;
FIG. 2 is a graph of a typical HEX channel for isoniazid resistance detection of Mycobacterium tuberculosis;
FIG. 3 is a graph of a typical ROX channel for isoniazid resistance detection of Mycobacterium tuberculosis;
FIG. 4 is a graph of a typical CY5 channel for isoniazid resistance detection of Mycobacterium tuberculosis;
FIG. 5 shows the detection result of the common probe of KatG gene of the present invention;
FIG. 6 shows the detection result of the KatG gene locked nucleic acid modified probe of the present invention;
FIG. 7 shows the detection result of the conventional probe for InhA gene of the present invention;
FIG. 8 shows the detection results of the InhA gene-locked nucleic acid-modified probe of the present invention;
FIG. 9 is a graph (FAM channel) showing the detection results of the reagent of the present invention on isoniazid drug-resistant national reference disc drug-resistant reference;
FIG. 10 is a graph (HEX channel) showing the detection results of the reagent of the present invention on isoniazid drug-resistant national reference disc drug-resistant reference;
FIG. 11 is a graph of the results of detection of a reference drug resistance of the agent of the present invention against a reference drug resistance plate of a rifampicin resistant country (ROX channel);
FIG. 12 is a graph showing the results of detection of a reference drug resistance of the agent of the present invention against a reference drug resistance plate of a rifampicin resistant country (CY 5 pathway).
Detailed Description
Reference now will be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. Indeed, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used on another embodiment to yield still a further embodiment.
Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. This technique is well explained in the literature, as is the case for molecular cloning: laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989); oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984); animal cell culture (Animal Cell Culture) (r.i. freshney, 1987); methods of enzymology (Methods in Enzymology) (Academic Press, inc.), experimental immunology handbook (Handbook of Experimental Immunology) (D.M.Weir and C.C.Blackwell, inc.), gene transfer vectors for mammalian cells (Gene Transfer Vectors for Mammalian Cells) (J.M.Miller and M.P.calos, inc., 1987), methods of contemporary molecular biology (Current Protocols in Molecular Biology) (F.M.Ausubel et al, inc., 1987), PCR: polymerase chain reaction (PCR: the Polymerase Chain Reaction, inc., 1994), and methods of contemporary immunology (Current Protocols in Immunology) (J.E.Coligan et al, 1991), each of which is expressly incorporated herein by reference.
In a first aspect, the present invention provides a nucleic acid composition for detecting a mutation in a isoniazid resistance gene of mycobacterium tuberculosis, comprising: a first primer pair with nucleotide sequences shown as SEQ ID No. 1-2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3-4, and a third primer pair with nucleotide sequences shown as SEQ ID No. 5-6.
The primer pair provided by the invention covers nine mutation sites of isoniazid drug resistance genes KatG, ahpC and InhA: katG S315T, katG S315N, ahpC C-6A, ahpC C-12T, ahpC C-39T, ahpC G-46A, ahpC T-49G, inhAT-8C, inhA C-15T. Compared with other existing primer combinations, the primer pair has better detection specificity and sensitivity.
Specifically, the first primer pair covers isoniazid resistance gene KatG, and the detected mutation site comprises: katG S315T, katG S315N.
The second primer pair covers isoniazid resistance gene ahpC, and the detected mutation site comprises: ahpC C-6A, ahpC C-12T, ahpC C-39T, ahpC G-46A, ahpC T-49G.
The third primer pair covers isoniazid resistance gene InhA, and the detected mutation sites comprise InhA T-8C and InhA C-15T.
In a preferred embodiment of the invention, the nucleic acid composition further comprises probes comprising a first probe as shown in SEQ ID No.7, a second probe as shown in SEQ ID No.8, a third probe as shown in SEQ ID No.9 and a fourth probe as shown in SEQ ID No. 10;
In an alternative embodiment, the first probe is used to detect the KatG S315T and KatG S315N mutations; the second probe is used for detecting ahpC C-6A and ahpC C-12T mutation; the third probe was used to detect ahpC C-39T, ahpC G-46A and ahpC T-49G mutations; the fourth probe was used to detect InhAT-8C and InhA C-15T mutations.
In an alternative embodiment, at least one of the first probe, the second probe, the third probe, and the fourth probe comprises at least one modified nucleotide group.
By providing the probe with at least one modified nucleotide group, the stability and activity of the probe is improved, and the modified nucleotide group does not lead to a loss of function of the probe.
In an alternative embodiment, the modified nucleotide group is at least one of (1) to (3):
(1) A phosphate group modified nucleotide group;
(2) Ribosyl group modified nucleotide group.
In an alternative embodiment, the phosphate group modification is modification of the oxygen in the phosphate group, including thio and boronate modifications;
in an alternative embodiment, the ribose group modification is a modification of the 2' -hydroxyl group of the ribose group selected from any of the following modifications: 2 '-fluoro modification, 2' -methoxy modification, 2 '-methoxyethyl modification, 2' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 '-amino modification and 2' -deoxy modification;
In an alternative embodiment, the ribose group modification is a locked nucleic acid modification of the 2' -hydroxyl group of the ribose group. The probe sequence is modified by using locked nucleic acid, so that mismatch can be better distinguished. The probe modified by the locked nucleic acid has stronger distinguishing capability to SNP and low fluorescence background.
In an alternative embodiment, base G at position 10 in the nucleotide sequence of the first probe is a locked nucleic acid modifying base; the 19 th base T in the nucleotide sequence of the fourth probe is a locked nucleic acid modified base.
In a preferred embodiment of the application of the invention, the probe is provided with a fluorescent reporter group 5 'and a fluorescent quenching group and/or MGB 3'.
In an alternative embodiment, the fluorescent reporter group includes, but is not limited to: any of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, cy3, cy3.5, NED, TAMRA, ROX, texas Red, cy5, cy5.5 and Quasar 670.
In an alternative embodiment, the fluorescence quenching groups include, but are not limited to: TAMRA, eclipse, BHQ, dabcyl, lowa BlackTM RQ and Lowa BlackTM FQ;
in an alternative embodiment, the BHQ family includes at least one of BHQ0, BHQ1, BHQ2, and BHQ3 without limitation.
In a preferred embodiment of the present invention, in any one of the first primer pair, the second primer pair and the third primer pair, the molar ratio of the upstream primer to the downstream primer is independently (1 to 10): (1-10).
For example, the molar ratio may be 1: 1. 1: 5. 1: 10. 1: 15. 1:20 or a range between any one or any two thereof;
or the molar ratio may be in the range of between any one or any two of 5:1, 10:1, 15:1, 20:1.
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20).
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer in the first primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer of the second primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer in the third primer pair is independently 1: (1-20).
Further, the person skilled in the art can adjust the molar ratio as needed, and is not limited to the above case.
In an alternative embodiment, the concentration of the first probe, the second probe, the third probe and the fourth probe in the nucleic acid composition is independently in the range of 0.01 to 1. Mu.M, and specifically may be in the range of any one or any two of 0.01. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 1.5. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M; preferably 0.05 to 1. Mu.M. Preferably 0.1 to 0.5. Mu.M.
The nucleic acid composition includes, but is not limited to: the first primer pair, the second primer pair, the third primer pair, the first probe, the second probe, the third probe and the fourth probe are respectively and independently arranged, and the independently arranged primer pairs and probes are combined into a product; or at least two of the first primer pair, the second primer pair and the third primer pair are combined (such as packaging), and at least two of the first probe, the second probe, the third probe and the fourth probe are combined; or all primer pairs and probes may be mixed.
In a second aspect, the invention also provides a reagent or kit comprising the nucleic acid composition described above.
In a preferred embodiment of the use of the invention, the reagent or kit further comprises at least one of the following: PCR reaction buffer, DNA polymerase, magnesium ion, dNTP, wild type control, negative control and water.
The wild controls were: TE solution of KatG, ahpC and InhA three-gene plasmid.
The negative control was: TE solution.
In an alternative embodiment, the DNA polymerase is selected from at least one of Tth DNA polymerase or a mutant thereof, taq DNA polymerase or a mutant thereof, pfu enzyme or a mutant thereof, and Hawk Z05 polymerase or a mutant thereof;
The concentration of the DNA polymerase is 0.01U/. Mu.l to 5U/. Mu.l.
In some embodiments, the DNA polymerase has an action concentration (i.e., working concentration) in a range between any one or any two of 0.01U, 0.015U, 0.02U, 0.025U, 0.03U, 0.035U, 0.04U, 0.045U, 0.05U, 0.055U, 0.06U, 0.065U, 0.07U, 0.075U, 0.08U, 0.085U, 0.09U, 0.095U, 0.1U, 0.2U, 0.3U, 0.4U, 0.5U, 0.6U, 0.7U, 0.8U, 0.9U, and 1U.
The concentration of magnesium ions is 0.5 mM-1.5 mM;
the concentration of dNTPs (in the kit and the reagent) is 0.1mM-25mM;
in some embodiments, the concentration of dNTPs applied in PCR detection is 0.1 to 5mM, and specifically may be in the range of any one or any two of 0.1mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM and 5 mM.
Any one of the first primer pair, the second primer pair and the third primer pair has a concentration of 0.1 mu mol/L to 5 mu mol/L;
specifically, the range may be 0.01. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 1.5. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M; preferably 0.05 to 1. Mu.M.
In any one of the first primer pair, the second primer pair and the third primer pair, the molar ratio of the upstream primer to the downstream primer is independently (1 to 20): (1-20).
For example, the molar ratio may be 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: 6. 1: 7. 1: 8. 1:9 and 1:10 or a range between any one or any two of them;
or the molar ratio may be in the range of any one or between any two of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1.
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20).
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer in the first primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer of the second primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer in the third primer pair is independently 1: (1-20).
In a third aspect, the invention also provides application of the nucleic acid composition in preparing a detection product of the isoniazid resistance gene mutation of the mycobacterium tuberculosis;
in an alternative embodiment, the detection product is selected from any one of a reagent, a kit and a gene chip.
In a fourth aspect, the invention also provides a method for detecting the isoniazid drug-resistant gene mutation of mycobacterium tuberculosis, which comprises the following steps: PCR detection is carried out on the sample to be detected by adopting the nucleic acid composition or the reagent or the kit;
the detection method is not aimed at diagnosis or treatment of the disease;
in an alternative embodiment, the sample to be tested is selected from at least one of a nucleic acid sample, an environmental sample comprising a nucleic acid sample, a negative control, a positive control, human sputum, sputum solid isolates and cultures thereof, and liquid isolates and cultures thereof.
In a preferred embodiment of the present invention, when PCR detection is performed, the molar ratio of the upstream primer to the downstream primer in any one of the first primer pair, the second primer pair and the third primer pair is independently (1 to 20): (1-20);
for example, the molar ratio may be 1: 1. 1: 5. 1: 10. a range between any one or any two of 1:15 and 1:20;
or the molar ratio may be in a range between any one or any two of 5:1, 10:1, 15:1 and 20:1.
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20).
In an alternative embodiment, the molar ratio of the upstream primer to the downstream primer in the first primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer of the second primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer in the third primer pair is independently 1: (1-20).
In an alternative embodiment, the PCR detection is performed with any one of the first, second and third primer pairs, the working concentration of the upstream and downstream primers being independently 0.01 to 5. Mu.M, preferably 0.1 to 1. Mu.M;
specifically, the range may be 0.01. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 1.5. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M; preferably 0.05 to 1. Mu.M.
In an alternative embodiment, the concentration of the first probe, the second probe, the third probe and the fourth probe when performing PCR detection is independently in the range of 0.01 to 1. Mu.M, specifically may be any one or any two of 0.01. Mu.M, 0.1. Mu.M, 0.2. Mu.M, 0.5. Mu.M, 1. Mu.M, 1.5. Mu.M, 2. Mu.M, 2.5. Mu.M, 3. Mu.M, 3.5. Mu.M, 4. Mu.M, 4.5. Mu.M, 5. Mu.M; preferably 0.05 to 1. Mu.M. Preferably 0.1 to 0.5. Mu.M.
According to the invention, by analyzing the conserved sequences of the KatG gene, the InhA gene and the ahpC gene of the isoniazid resistance determination region of the mycobacterium tuberculosis, a primer probe combination capable of detecting the isoniazid resistance gene mutation is designed, and a multiplex asymmetric PCR and Taqman probe melting technology is adopted to simultaneously detect nine resistance mutation sites, namely KatG S315T, katG S315N, ahpC C-6A, ahpC C-12T, ahpC C-39T, ahpC G-46A, ahpC T-49G, inhA T-8C, inhA C-15T, in a reaction system, wherein the detection sensitivity can reach 10000CFU/mL, the advantages of high detection specificity, strong sensitivity and the like are achieved, and the rapid detection of the resistance gene mutation can be realized with low cost and wide application range, and the result determination is convenient and intuitive.
In an alternative embodiment, the reaction procedure for PCR is as follows: 94-96 ℃ for 15 sec-10 min; 94-98 deg.c for 10-30 s, 55-65 deg.c, 20-40 s, 70-74 deg.c for 20-30 s and 30-50 cycles; collecting fluorescent signals at the temperature of 55-56 ℃ for 20-40 s; 94-96 deg.c for 1-3 min and 33-37 deg.c for 1-3 min; and continuously collecting fluorescent signals at the temperature of 35-90 ℃ at the heating rate of 0.04 ℃/s.
Judging whether the sample is mutated or not by comparing the difference of melting points (Tm) of melting curves between the detected sample and a wild control; judging the sample as wild type when the difference between the melting point of the detected sample and the melting point of the wild control delta Tm is less than or equal to +/-1 ℃; and judging that the sample is of a drug-resistant mutant type when the difference DeltaTm of the melting point of the detection sample in any one of the channels and the melting point of the wild control are not less than +/-2 ℃, and judging specific drug-resistant mutation site information according to the difference between the corresponding channel and the melting point of the wild type.
In a fifth aspect, the present invention also provides a system for detecting isoniazid resistance gene mutation in mycobacterium tuberculosis, comprising: the above-described reagent or kit, first means and second means; the first device can respectively perform real-time fluorescent quantitative PCR detection on a DNA sample to be detected, a wild type control and a negative control by adopting a reagent or a kit to obtain a melting curve graph;
the second device includes: the data input module and the data analysis and conclusion output module;
the data input module is configured to input melting curve graph data obtained by the first device; the data analysis and conclusion output module is configured to: judging whether the sample to be detected contains the gene mutation or not and determining the type of the gene mutation according to the change of the Tm difference value of the melting peak of the sample to be detected and the wild type control in each channel.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
The NCBI database is used for searching related gene sequences, primer probes are designed according to KatG genes, inhA genes and ahpC genes of isoniazid resistance determination regions of mycobacterium tuberculosis, the primers are designed at the periphery of mutation points, the probe sequences are designed at the mutation points, and the primers and the probes are designed by using software Oligo 7. The probe is modified by adopting a fluorescence reporting group and a fluorescence quenching group. The preferred combined sequences of the amplification primers for each mutation site are shown in Table 1, and the preferred probe sequences are shown in Table 2.
TABLE 1 primer sequences of the invention
Remarks: f is an upstream primer, and R is a downstream primer.
TABLE 2 Probe sequences of the invention
| Numbering device | Sequence (5 '-3') | SEQ ID No. | Detection site |
| Probe-P1 | TCATCACCAGCGGCATCGAGGT | 7 | KatG S315T、KatG S315N |
| Probe-P2 | ACTGGTGTGATATATCACCTTTG | 8 | ahpC C-39T、ahpC G-46A、ahpC T-49G |
| Probe-P3 | AAGATATATCACACCATATTTA | 9 | hpC C-6A、ahpC C-12T |
| Probe-P4 | ATGCGGCGAGACGATAGGTTGT | 10 | InhA T-8C、InhA C-15T |
Remarks: the underlined base is a modified base of the locked nucleic acid, and the modification of other bases does not achieve the expected detection effect.
The locked nucleic acid is a double-ring nucleic acid derivative, and the 2'-O position and the 4' -C position of ribose form an oxymethylene bridge through the action of shrinkage and are connected into a ring shape, and the ring bridge locks the N configuration in the furanose C3, so that the flexibility of a ribose structure is reduced, and the stability of a local structure of a phosphate skeleton is improved. Locked nucleic acids have sequence specificity, which better distinguishes correctly paired and incorrectly paired sequences than conventional nucleic acids, and LNA-DNA hybrid molecules containing mismatched bases are more unstable than DNA-DNA hybrid molecules containing mismatched bases, and thus the drop in Tm is more pronounced. The invention adopts locked nucleic acid to modify probes with weak distinguishing ability between wild type and mutant type, thereby achieving the recognition ability of single base mismatch.
Example 2
The embodiment provides a reagent for detecting the isoniazid drug-resistant gene mutation of mycobacterium tuberculosis, which specifically comprises the following primers: primer-F1, primer-R1, primer-F2, primer-R2, primer-F3, primer-R3; and (3) probe: probe-P1, probe-P2, probe-P3, probe-P4; PCR reaction buffer; deoxyribonucleoside triphosphates; taq DNA polymerase.
The combinations of primers and probes are shown below.
The PCR reaction solution comprises: PCR reaction buffer solution, 0.1 mmol/L-5 mmol/L deoxyribonucleoside triphosphate, and 0.1 mu mol/L-5 mu mol/L of upstream primer and downstream primer of target gene, wherein the ratio of the primers is 1 (1-20) or 1-20): 1, the probe of the target gene is 0.1 mu mol/L-5 mu mol/L, and 0.01U-1U/. Mu.l Taq DNA polymerase. The specific reagent formulations are shown in the following table:
the screening and system optimization of the primers and the probes are the key points of the invention, and because multiple asymmetric PCR is adopted and multiple probes are also included, the multiple PCR and the mutual inhibition of target fragments are easy to generate, and the results of subsequent melting curves are affected if complex secondary structures are formed between the probes and the primers, so that no melting peak is directly caused. The inventor finally optimizes the reaction system through a large number of fumbling experiments.
The reagent combinations provided in this example allow single-tube monomer line detection of nine mutation sites of KatG gene, ahpC gene and InhA gene: katG S315T, katG S315N, ahpC C-6A, ahpC C-12T, ahpC C-39T, ahpC G-46A, ahpC T-49G, inhA T-8C, inhAC-15T.
Example 3
The embodiment provides a detection method of a mycobacterium tuberculosis isoniazid drug-resistant gene mutation, which comprises the following steps.
1. Sample collection: sputum, mycobacterium tuberculosis, and isolated culture.
2. Sample pretreatment:
2.1 sputum treatment mode: the sample and the liquefaction liquid are mixed in a ratio of 1:1 for liquefaction until the sample reaches the required fluidity. 200. Mu.L of the liquefied solution was used for the subsequent nucleic acid extraction.
2.2 clinical isolation culture treatment protocol: mycobacterium tuberculosis grown on solid medium, bacterial loop 1 was collected with 22SWG standard inoculating loop and resuspended in 400. Mu.L TB DNA extract. Mycobacterium tuberculosis grown in liquid medium is centrifuged at 12000rpm for 15min at 1mL, supernatant is discarded, and precipitate is resuspended in 200 μl TB DNA extract for subsequent nucleic acid extraction.
2.3 alveolar lavage fluid treatment: 1-5 ml of alveolar lavage fluid was centrifuged at 10000rpm for 1 min, the supernatant was discarded, and the pellet was resuspended in 200. Mu.L of TB DNA extract for subsequent nucleic acid extraction.
3. Sample nucleic acid extraction:
commercial nucleic acid DNA extraction kits (e.g., qiagen DNAMini Kit, etc.) can be used and the extraction process should be performed according to the instructions of the commercial kit. The concentration and purity of the template DNA can be determined using a nucleic acid quantitative or ultraviolet spectrophotometer prior to PCR amplification.
4. And (3) sample adding:
4.1 detection reagent configured as in example 2, 10. Mu.L of PCR reagent was added to the PCR tube;
4.2 adding 10 mu L of extracted nucleic acid into a PCR amplification system, uniformly mixing, covering a PCR tube cover, and waiting for machine starting.
Pcr amplification procedure setup and run:
5.1 PCR amplification was performed on a fluorescent quantitative PCR instrument such as Roche LightCycler, SLAN-96P, etc.
5.2 opening the instrument and placing the instrument into a PCR tube to be detected.
5.3 amplification procedure was set up. The corresponding fluorescence channels FAM channel (Reporter: FAM, quantum: none), HEX channel (Reporter: HEX, quantum: none), ROX channel (Reporter: ROX, quantum: none), CY5 channel (Reporter: CY5, quantum: none) were selected. Sample editing is performed.
5.4 run amplification program.
6. Analysis of results:
6.1 after the reaction is finished, the instrument automatically saves the result and can automatically analyze by utilizing the software of the instrument.
6.2 judging whether the sample is mutated or not by comparing the difference of melting point (Tm) of a melting curve between the detected sample and a wild control (a KatG, ahpC, inhA gene plasmid mixed solution without mutation, which is equivalent to a positive control, namely a quality control); when the difference ΔTm (Tm Sample of -Tm Wild control ) Judging that the strain is wild type at the temperature of less than or equal to +/-1 ℃; judging that the sample in any one of the 4 channels is resistant to mutation when the difference DeltaTm of the melting point of the sample detected in any one of the 4 channels and the melting point of the wild reference is more than or equal to + -2 ℃, and judging that the specific resistant mutation site is believed according to the difference between the corresponding channel and the melting point of the wild reference And (5) extinguishing.
7. Interpretation of the results:
the typical peak diagrams of each channel are shown in fig. 1 to 4, and the melting point difference (Δtm) of the mutant sample in each channel can be referred to in the following table:
note that: "-" means no value;
example 4
This example provides a comparative example of the effect of using locked nucleic acid modified probes and unlocked nucleic acid modified probes.
The common probes and the locked nucleic acid modified probes aiming at the KatG gene and the InhA gene are respectively designed, and the nucleic acid sequences of the locked nucleic acid modified probes are SEQ ID NO.7 and SEQ ID NO.10 respectively. The 10 th base G of SEQ ID NO.7 is a locked nucleic acid modified base; the 19 th base T in SEQ ID NO.10 is a locked nucleic acid modified base. The nucleic acid sequence of the common probe is consistent with the modification of the locked nucleic acid.
Wild type and KatG S315T, katG S315N, inhA T-8C, inhA C-15T mutation sites were detected by the method of example 3.
The detection result shows that after the KatG gene detection probe is modified by the locked nucleic acid, the mutant melting point difference (delta Tm) is similar to that of an unmodified common probe, and the detection result is shown in fig. 5 and 6; after the InhA gene detection probe is modified by the locked nucleic acid, the difference between the melting point of the mutant type and that of the wild type is increased, namely the detection effect of the locked nucleic acid modification probe is better than that of the common probe. The results of the test are shown in the following table, and the dissolution curves are shown in fig. 7 and 8.
And (3) injection: "-" means no value;
example 5
The detection reagent of the embodiment 2 of the invention is adopted to detect the isoniazid drug-resistant gene national reference plate of the mycobacterium tuberculosis.
The reagent of the invention is used for detecting the Mycobacterium tuberculosis isoniazid drug-resistant national reference plate, the coincidence rate of sensitive reference is 100 percent, the coincidence rate of drug-resistant reference is 100 percent, and the detection limit reference S, SI, SID and precision reference J (1 multiplied by 10) 5 CFU/mL) can be detected. The isoniazid drug-resistant national reference plates are shown in the following table, and the detection results are shown in figures 9-12.
Example 6
Clinical application examples of isoniazid drug-resistant gene mutation nucleic acid detection of clinical samples.
A total of 139 sputum samples and isolated cultures were collected from patients suspected of tuberculosis infection at the Ministry of simple yang, chengdu City, and 84 positive sputum samples of Mycobacterium tuberculosis were confirmed. The reagent provided by the invention is used for detecting 84 positive samples of the mycobacterium tuberculosis, wherein 5 positive results of isoniazid drug-resistant mutation exist, and 79 positive results of isoniazid sensitive samples exist. Samples of 5 cases at isoniazid drug-resistant mutation sites were confirmed to be correct by first-generation sequencing.
The results show that the reagent, the kit and the method for detecting the isoniazid resistance of the mycobacterium tuberculosis show good clinical application effect when detecting clinical samples.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A nucleic acid composition for detecting mutations in isoniazid resistance genes of mycobacterium tuberculosis, comprising: a first primer pair with nucleotide sequences shown as SEQ ID No. 1-2, a second primer pair with nucleotide sequences shown as SEQ ID No. 3-4, and a third primer pair with nucleotide sequences shown as SEQ ID No. 5-6.
2. The nucleic acid composition of claim 1, further comprising a probe comprising a first probe as set forth in SEQ ID No.7, a second probe as set forth in SEQ ID No.8, a third probe as set forth in SEQ ID No.9, and a fourth probe as set forth in SEQ ID No. 10;
preferably, the first probe is used to detect KatG S315T and KatG S315N mutations; the second probe is used for detecting ahpC C-39T, ahpC G-46A and ahpC T-49G mutation; the third probe is used for detecting ahpC C-6A and ahpC C-12T mutation; the fourth probe is used for detecting InhA T-8C and InhAC-15T mutation;
Preferably, at least one of the first, second, third and fourth probes contains at least one modified nucleotide group;
preferably, the modified nucleotide group is at least one of (1) to (3):
(1) A phosphate group modified nucleotide group;
(2) A ribosyl group modified nucleotide group;
preferably, the phosphate group modification is modification of the oxygen in the phosphate group, including thio and boration modifications;
preferably, the ribose group modification is to modify 2' -hydroxyl in ribose group, and is selected from any one of the following modifications: 2 '-fluoro modification, 2' -methoxy modification, 2 '-methoxyethyl modification, 2' -2, 4-dinitrophenol modification, locked nucleic acid modification, 2 '-amino modification and 2' -deoxy modification;
preferably, the ribose group modification is a locked nucleic acid modification of the 2' -hydroxyl group in the ribose group;
preferably, the 10 th base G in the nucleotide sequence of the first probe is a locked nucleic acid modified base; and the 19 th base T in the nucleotide sequence of the fourth probe is a locked nucleic acid modified base.
3. The nucleic acid composition of claim 2, wherein the probe is provided with a fluorescent reporter group 5 'and a fluorescent quencher group and/or MGB 3';
Preferably, the fluorescent reporter group is selected from: any one of 5-FAM, 6-FAM, HEX, TET, VIC, JOE, cy3, cy3.5, NED, TAMRA, ROX, texas Red, cy5, cy5.5 and Quasar 670;
preferably, the fluorescence quenching group is selected from: TAMRA, eclipse, BHQ, dabcyl, lowa BlackTM RQ and Lowa BlackTM FQ;
preferably, the BHQ series is selected from at least one of BHQ0, BHQ1, BHQ2, and BHQ 3.
4. The nucleic acid composition of claim 2, wherein in any one of the first, second and third primer pairs, the molar ratio of the upstream primer to the downstream primer is independently (1-20): (1-20);
preferably, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-20);
preferably, the molar ratio of the upstream primer to the downstream primer in the first primer pair is independently 1: (1-20); the molar ratio of the upstream primer to the downstream primer of the second primer pair is independently 1: (1-20); the third primer pair has a molar ratio of upstream primer to downstream primer of independently 1: (1-20);
preferably, the concentration of the first, second, third and fourth probes in the nucleic acid composition is independently 0.01 to 1. Mu.M, preferably 0.1 to 0.5. Mu.M.
5. A reagent or kit comprising the nucleic acid composition of any one of claims 1 to 4.
6. The reagent or kit of claim 5, further comprising at least one of: PCR reaction buffer, DNA polymerase, magnesium ion, dNTP, wild type control, negative control and water;
preferably, the DNA polymerase is selected from at least one of Tth DNA polymerase or a mutant thereof, taq DNA polymerase or a mutant thereof, pfu enzyme or a mutant thereof, and Hawk Z05 polymerase or a mutant thereof;
the working concentration of the DNA polymerase is 0.01U/. Mu.l to 5U/. Mu.l;
the working concentration of the magnesium ions is 0.5 mM-1.5 mM;
the working concentration of the dNTPs is 0.1 mM-25 mM;
any one of the first primer pair, the second primer pair and the third primer pair has the concentration of the upstream primer and the downstream primer of 0.1 mu mol/L to 5 mu mol/L;
in any one of the first, second and third primer pairs, the molar ratio of the upstream primer to the downstream primer is independently (1 to 20): (1-20).
7. Use of the nucleic acid composition of any one of claims 1 to 4 for the preparation of a detection product of a isoniazid resistance gene mutation in mycobacterium tuberculosis;
Preferably, the detection product is selected from any one of a reagent, a kit and a gene chip.
8. A method for detecting a isoniazid drug-resistant gene mutation of mycobacterium tuberculosis, which is characterized by comprising the following steps: performing PCR detection on a sample to be detected using the nucleic acid composition of any one of claims 1 to 4 or the reagent or kit of any one of claims 5 to 6;
the detection method is not aimed at diagnosis or treatment of the disease;
preferably, the sample to be tested is selected from at least one of a nucleic acid sample, an environmental sample containing a nucleic acid sample, a negative control, a positive control, human sputum, sputum solid isolates and cultures thereof, and liquid isolates and cultures thereof.
9. The method according to claim 8, wherein, in performing PCR detection, the molar ratio of the upstream primer to the downstream primer in any one of the first primer pair, the second primer pair and the third primer pair is independently (1 to 20): (1-20);
preferably, the molar ratio of the upstream primer to the downstream primer is independently 1: (1-10);
preferably, in performing PCR detection, the working concentration of any one of the first primer pair, the second primer pair and the third primer pair is independently 0.01 to 5. Mu.M, preferably 0.1 to 1. Mu.M;
Preferably, the working concentrations of the first, second, third and fourth probes are independently 0.01 to 1. Mu.M, preferably 0.1 to 0.5. Mu.M, when performing PCR detection;
preferably, the reaction procedure of the PCR is as follows: 94-96 ℃ for 15 s-10 min; 94-98 deg.c for 10-30 s, 55-65 deg.c, 20-40 s, 70-74 deg.c for 20-30 s and 30-50 cycles; collecting fluorescent signals at the temperature of 55-56 ℃ for 20-40 s; 94-96 deg.c for 1-3 min and 33-37 deg.c for 1-3 min; and continuously collecting fluorescent signals at 35-90 ℃.
10. A system for detecting mutations in isoniazid resistance genes of mycobacterium tuberculosis, comprising: the reagent or kit of any one of claims 5-6, a first device and a second device; the first device can respectively perform real-time fluorescence quantitative PCR detection on a DNA sample to be detected, a wild control and a negative control by adopting the reagent or the kit to obtain a melting curve graph;
the second device includes: the data input module and the data analysis and conclusion output module;
the data input module is configured to input melting curve graph data obtained by the first device; the data analysis and conclusion output module is configured to: judging whether the sample to be detected contains the gene mutation or not and determining the type of the gene mutation according to the change of the Tm difference value of the melting peak of the sample to be detected and the wild type control in each channel.
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