CN116356056A - Primer group, product and application for detecting drug-resistant genes of mycobacterium tuberculosis - Google Patents
Primer group, product and application for detecting drug-resistant genes of mycobacterium tuberculosis Download PDFInfo
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Abstract
The application relates to the technical field of biology, and particularly provides a primer, a product and application for detecting drug-resistant gene mutation of mycobacterium tuberculosis.
Description
Technical Field
The application relates to the field of biotechnology, in particular to a primer, a product and application related to detection of drug-resistant gene mutation of mycobacterium tuberculosis.
Background
WHO classifies drug-resistant tuberculosis into five categories: isoniazid-resistant tuberculosis, rifampicin-resistant tuberculosis and multi-drug resistant tuberculosis, quasi-broad drug resistant tuberculosis and broad drug resistant tuberculosis.
Once tuberculosis patients have drug resistance, the prognosis is extremely poor, and the problem to be solved in tuberculosis control work is urgent. The WHO issued "MDR-TB and RR-TB treatment guidelines (2018 updated)" in 12 months of 2018, and "drug resistant tuberculosis treatment guidelines (2019 integrated) in 3 months of 2019, and the WHO pointed out that these 2 guidelines were formulated on the basis of evaluating a large amount of up-to-date evidence, and would replace all WHO's treatment guidelines for drug resistant tuberculosis before. For better popularization and use of WHO "MDR-TB and RR-TB therapeutic guidelines (2018 updated edition)" and "drug resistant tuberculosis therapeutic guidelines (2019 integrated edition)", the chinese society of medical science tuberculosis department established "chinese MDR-TB and RR-TB therapeutic expert consensus (2019 edition)". This consensus clearly indicates that phenotypic susceptibility testing (drugsusceptibility testing, DST), including first and second line antitubercular drugs, should be performed prior to treatment in chemotherapy, and that rapid molecular susceptibility testing should be used simultaneously if appropriate. And the antitubercular drugs used in the long-range MDR-TB treatment regimen were reclassified into 3 groups: group A: preferred drugs include levofloxacin (Lfx) or moxifloxacin (Mfx), bedaquiline (Bdq) and linezolid (Lzd). Group B: and the secondary medicaments comprise clofazimine (Cfz) and cycloserine (Cs). Group C: alternative drugs are pyrazinamide (Z), ethambutol (E), delamanid (Dlm), prothioisonicotinamide (Pto), amikacin (Am) or calicheamicin (Cm), p-aminosalicylic acid (p-aminosalicylic acid, PAS), imipenem/cilastatin (imipenem/cilastatin, ipm-Cln) or meropenem (Mpm) in that order. This consensus has clarified the importance of levofloxacin (Lfx) or moxifloxacin (Mfx), bedaquiline (Bdq) and linezolid (Lzd), clofazimine (Cfz), and the like, in the treatment of MDR-TB. The drug resistance of the drugs is reported to appear, and with the application of the drugs, the detection of the drug resistance is urgent, while the products of the drug resistance gene mutation detection of the mycobacterium tuberculosis on the market at present mainly aim at the drug resistance of rifampicin and isoniazid, and drug resistance detection products such as bedaquilin (Bdq), linezolid (Lzd), clofazimine (Cfz) and the like are rarely appearing on the market at present; and the applicable sample types of the drug-resistant tuberculosis gene mutation detection products in the current market are single, about 80% are culture samples, and thus the waiting time of patients is increased undoubtedly. Therefore, comprehensively considering the current market demands and the chemical treatment trend of the future drug-resistant tuberculosis patients, establishing an effective, rapid and applicable sample type-rich drug-resistant tuberculosis detection method is a key for clinical reasonable drug application and epidemic development control.
In view of this, the present application is presented.
Disclosure of Invention
The first object of the application is to provide a specific primer group for detecting the drug-resistant gene mutation of mycobacterium tuberculosis.
A second object of the present application is to provide an application of the primer set for detecting a drug-resistant gene mutation of mycobacterium tuberculosis in preparing a drug-resistant gene detection product of mycobacterium tuberculosis.
A third object of the present application is to provide a product for detecting drug-resistant gene mutation of mycobacterium tuberculosis, which is capable of supplementing the technical problems of the product for detecting drug-resistant gene mutation of mycobacterium tuberculosis, which is lack in the prior art, and is capable of high throughput, easy operation and low cost.
The fourth object of the present application is to provide a method for detecting drug-resistant gene mutation of mycobacterium tuberculosis, so as to alleviate the technical problems of low flux, single type of applicable sample, less applicable antitubercular drugs, high price, etc. of the existing detection method.
In order to achieve the above purpose, the present application specifically provides the following technical solutions:
the application firstly provides a primer group for detecting the drug-resistant gene mutation of mycobacterium tuberculosis, wherein the index comprises 53SNP loci in total of 16 genes related to various drugs for treating tuberculosis.
Further, the mutation sites of the drug-resistant genes of the mycobacterium tuberculosis are shown in Table 1.
Further, the primer set comprises 48 PCR amplification primers, the sequences of which are shown as SEQ ID NO.1-SEQ ID NO.48, or have at least 85% identity with SEQ ID NO.1-SEQ ID NO. 48.
Further, the primer set also comprises 55 UEP extension primers, the sequences of which are shown as SEQ ID NO.49-SEQ ID NO.103 or have at least 85% identity with SEQ ID NO.49-SEQ ID NO. 103.
Further, the primer set also comprises 2 internal reference amplification primers, the sequences of which are shown as SEQ ID NO.35-SEQ ID NO.36, or have at least 85% identity with SEQ ID NO.35-SEQ ID NO. 36.
Further, the inner reference primer set also includes an extension primer set forth in SEQ ID NO.73, or has at least 85% identity to SEQ ID NO. 73.
Further, the primer performs grouping to complete PCR amplification of the target gene;
preferably, the PCR amplification systems are divided into 3PCR amplification systems, respectively, groups 1-3: group 1: comprises 36 PCR primers, the sequences of which are shown as SEQ ID NO.1-36 or have at least 85 percent of identity with SEQ ID NO. 1-36; group 2: comprises 22 PCR primers with sequences shown as SEQ ID NO.1-6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.35-46 or at least 85% identity with SEQ ID NO.1-6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO. 35-46; group 3: includes 18 PCR primers with sequences shown as SEQ ID No.1-4, SEQ ID No.7, SEQ ID No.8, SEQ ID No.11, SEQ ID No.12, SEQ ID No.27, SEQ ID No.28, SEQ ID No.31, SEQ ID No.32, SEQ ID No.35, SEQ ID No.36, SEQ ID No.43, SEQ ID No.44, SEQ ID No.47-48, or at least 85% identity with SEQ ID No.1-4, SEQ ID No.7, SEQ ID No.8, SEQ ID No.11, SEQ ID No.12, SEQ ID No.27, SEQ ID No.28, SEQ ID No.31, SEQ ID No.32, SEQ ID No.35, SEQ ID No.36, SEQ ID No.43, SEQ ID No.44, SEQ ID No. 47-48.
Further, the primers are also grouped to complete single base extension;
preferably, it is divided into 3 UEP extension primer systems corresponding to the above groups 1 and 3: group 1: comprises 25 UEP primers with the sequence shown in SEQ ID NO.49-73 or with at least 85% identity with SEQ ID NO. 49-73; group 2: comprises 17 UEP primers, the sequences of which are shown as SEQ ID NO.73-89 or have at least 85 percent of identity with SEQ ID NO. 73-91; group 3: comprising 15 UEP primers with sequences shown as SEQ ID NO.73 and SEQ ID NO.90-103 or at least 85% identity with SEQ ID NO.73 and SEQ ID NO. 90-103.
The application also provides a product for detecting the drug-resistant gene mutation of the mycobacterium tuberculosis, which comprises the primer group for detecting the drug-resistant gene mutation of the mycobacterium tuberculosis.
Further, the product for detecting the drug-resistant gene mutation of the mycobacterium tuberculosis further comprises a reagent and/or a device for detection;
preferably, the reagent comprises 10 XPCR Buffer, dNTP Mix, mgCl 2 Primer Mix, PCR Enzyme, dUTP, UDG and nuclease free water;
more preferably, the apparatus comprises a mass spectrum of massaraay nucleic acids.
The application also provides a method for detecting the drug-resistant gene mutation of the mycobacterium tuberculosis, which comprises the step of detecting the drug-resistant gene in the sample to be detected by using the primer group.
Further, carrying out PCR amplification and base extension reaction on the genome of the sample to be detected by using the primer group, and then detecting a product obtained by the reaction by using Massarray to determine the genotype of the mutation site of the drug-resistant gene in the sample to be detected;
preferably, the method further comprises the step of dephosphorylating the PCR product prior to the base extension reaction;
preferably, the method further comprises the step of purifying the product of the reaction after the base extension reaction, and then detecting the reaction product using Massarray.
The application also provides application of the primer group in preparation of mycobacterium tuberculosis drug-resistant gene mutation detection products.
Compared with the prior art, the method has the following technical advantages:
1) The application carries out strict selection and combination on the drug-resistant genes and loci through early-stage experiments, and at least comprises multiple aspects of primer design thought, screening, grouping and the like in primer design. For example, in order to reduce the pressure of a multiple system to the greatest extent, the application designs the least amplified primer (24 pairs) to match a large number of extended primers (55 strips); in the aspect of specific primer sequence screening and optimization, the application compares and optimizes each group of primer sequences, and simultaneously skillfully groups the primer sequences; the method not only ensures the optimal detection of a single snp, but also ensures the accurate, specific and sensitive detection of samples under a multiple system, and can realize the rapid and effective detection of the drug-resistant gene mutation of the mycobacterium multijunction in the sample to be detected by using a Massarray system;
2) The system realizes the effective detection of 53SNP loci of 16 genes related to various tuberculosis treatment medicines on the same platform, has high accuracy and high sensitivity, can greatly shorten the detection period, simultaneously reduces the detection cost, and can provide reference for the drug resistance condition of the mycobacterium tuberculosis as the detection result;
3) The method for detecting the mycobacterium tuberculosis drug-resistant gene mutation comprises the step of detecting the mycobacterium tuberculosis drug-resistant gene mutation in a sample to be detected by using the primer group. The method can detect 53SNP loci in total of 16 genes related to various tuberculosis treatment medicines by using the primer set provided by the application, and has the characteristics of strong accuracy, high sensitivity, good repeatability, low cost, short detection period, visual result and the like;
4) The time-of-flight mass spectrum platform is applied to detection of various SNP loci related to tuberculosis treatment medicaments, has broad spectrum applicability, greatly improves detection efficiency, and is particularly suitable for batch detection. The method overcomes the defect of low PCR weight in the prior art, has low cost, and is suitable for wide popularization; the invention is suitable for tuberculosis patients, can detect drug resistance in time, is beneficial to reasonable medication, and can improve the life quality of patients.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram of the PCR primer screening electrophoresis of the alr344 locus provided in example 1 of the present application. Wherein M: representing Marker, the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; 1: the representative template is a mycobacterium tuberculosis amplification product; 2: represents a blank control; alr344-1: representing the amplification product of primer combination 1; alr344-2: representing the primer combination 2 amplification product; alr344-3: representing the amplification product of primer set 3.
FIG. 2 is an electrophoresis chart of the inhA94 site PCR primer screening provided in example 1 of the present application. Wherein M: representing Marker, the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; 1: representing the amplification product of the template mycobacterium tuberculosis; 2: represents a blank control; inhA94 system 1: representing the amplification product of primer combination 1; inhA94 system 2: representing the amplification product of primer combination 2.
FIG. 3 is a PCR primer screening electrophoresis chart of the inhA94 promoter region provided in example 1 of the present application. M: representing Marker, the bands are 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom; 1: representing the amplification product of the template mycobacterium tuberculosis; 2: represents a blank control; inhA-1: representing the amplification product of primer combination 1; inhA-2: representing the primer combination 2 amplification product; inhA-3: representing the amplification product of primer set 3.
FIG. 4 is a mass spectrum of the primer of the UEP at the position of the embB497-1 provided in example 1 of the present application before and after optimization.
FIG. 5 is a mass spectrum of the rpob526-2 locus UEP primer provided in example 1 of the present application before and after optimization.
FIG. 6 is a diagram of the results of the sequence analysis of the ahpC-10 site in TB61 sample, as provided in example 3 of the present application.
FIG. 7 is a diagram of the results of the TB109 sample embB306-1 site mass spectrum and sanger sequencing provided in example 1 of the present application.
FIG. 8 is a diagram of the results of the analysis of the rpob511-2 locus of the TB28 sample provided in example 1 of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application, and that the examples are some, but not all, examples of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.
The Massary ARRAY gene analysis technology in the application is based on MALDT-TOF time-of-flight mass spectrometry, the target gene is enriched through PCR amplification, the extension primer extends 1 base on the target fragment, and finally the genotype of the detected locus can be obtained through comparing the difference of the single base extension product quality.
The present application is further illustrated below by specific examples, but it should be understood that these examples are for the purpose of illustration only in greater detail and should not be construed as limiting the present application in any way.
The main reagent information used in the examples of the present application is as follows:
the main instrument information used in the embodiments of the present application is as follows:
example 1 reaction System set-up in the present application
Through early test exploration and simultaneously combining with clinical requirements, the application finally determines the detection range of the drug-resistant gene mutation of the mycobacterium tuberculosis, covers the detection of 53SNP loci of 16 genes related to various drugs for treating tuberculosis, and the specific locus information is shown in Table 1.
Table 1 details of the mutation sites of the drug-resistant genes:
further, the present application establishes PCR and UEP primers suitable for mass spectrometric detection of nucleic acids at each SNP site by design and optimization. In order to achieve the sensitivity and specificity of the multiplex system, the application considers from various aspects: for example, the system pressure is considered, the adjacent sites are amplified by adopting the same pair of primers, and fewer amplification primer pairs are designed as much as possible; to ensure the detection specificity and sensitivity of each site, the primers of each site are optimized in a comparison way; to avoid interference between primers, the grouping is performed according to the requirement that the interaction between sites is low (not simple partitioning).
Regarding the optimization design, exemplary experiments are as follows:
1. the PCR primer screening of the alr344 site, the inhA94 site and the inhA promoter region is exemplified (the screening of the primer with good specificity and excellent amplification efficiency)
In order to ensure detection of each site, the application screens and optimizes PCR amplification primers of each site, taking the screening of the PCR amplification primers of alr344 site, inhA94 site and inhA promoter region as an example, respectively designing 2-3 groups of PCR amplification primers (the primer information is shown in the table 2) at each site in the early stage, then carrying out PCR amplification reaction according to the PCR amplification steps of the steps, and carrying out electrophoresis detection on PCR amplification products after the reaction is finished, wherein the detection results are shown in the figures 1-3.
Table 2alr344 site, inhA94 site, inhA promoter region PCR primer information:
as can be seen from FIG. 1, 3 sets of PCR primers designed for the alr344 locus region amplify the Mycobacterium tuberculosis nucleic acid templates respectively, the 1 st set of primers have no purpose amplification band, and the 2 nd set and the 3 rd set of primers have purpose amplification bands, but the 2 nd set of primers have obviously weaker brightness than the 3 rd set of primers, which means that the 2 nd set of primers have low amplification efficiency, so the 3 rd set of primers are selected as the final PCR amplification primers for the alr344 locus region.
As can be seen from FIG. 2, 2 sets of PCR primers designed for the inhA94 locus region are used for amplifying the Mycobacterium tuberculosis nucleic acid template respectively, and the 1 st set of primers and the 2 nd set of primers have target amplified bands, and the band brightness is almost unchanged, but the 1 st set of primers have nonspecific bands between 1000 and 2000bp, so the 2 nd set of primers are selected as the final PCR amplified primers for the inhA94 locus region.
As can be seen from FIG. 3, 3 sets of PCR primers designed for the inhA promoter region amplify the Mycobacterium tuberculosis nucleic acid templates, respectively, the 1 st set of primers and the 2 nd set of primers have no target amplified bands, and only the 3 rd set of primers have target amplified bands, so the 3 rd set of primers are selected as the final PCR amplified primers for the inhA promoter region.
2. Optimizing UEP extension primers at the EmbB497-1, rpob526-2 sites (influence of different UEP primer pair specificities and extension efficiencies)
In order to improve the detection sensitivity and specificity, the PCR amplification primer combination of each site is screened and optimized, and the UEP extension primer is also optimized. In earlier tube-closing studies, the present application found that the UEP extension primers at individual sites were either inefficient or nonspecifically generated, and compared to different UEP extension primers designed for this application.
The UEP extension primer (5'-TCCTGACCGTGGTGTTCGCCGAC-3') of the embB497-1 locus is non-specific for C base when detecting the embB497 CAG > AAG mutant plasmid; after the UEP is redesigned (5'-CCCTGACCGTGTGGTTCGCTGAC-3'), when the embB497 CAG > AAG mutant plasmid is detected again, the non-specific situation of C base does not appear at the embB497-1 locus, and the result is shown in FIG. 4; as can be seen from FIG. 4, when the mutant plasmid of EmbB497 CAG > AAG is detected after the adjustment of the UEP primer of EmbB497-1 site, the A base can be accurately detected at the EmbB497-1 site without the nonspecific extension of C base, so that the final UEP primer of EmbB497-1 site (5'-CCCTGACCGTGTGGTTCGCTGAC-3') after the adjustment is selected as the final UEP primer of the site.
When the UEP extension primer (5'-TGCAGCGCCGACAGTCGGCGCTTG-3') of the rpob526-2 locus detects a clinical sample TB-123, no extension product is found at the rpob526-2 locus, and the extension efficiency of the UEP is extremely poor; after redesigning the UEP primer (5'-GGAACCCGCTCGTGGGGTTGACCC-3') at the rpob526-2 site, the same clinical sample TB123 is detected, the detection result is shown in fig. 5, and it can be seen from fig. 5 that after UEP replacement, the extension product of a base exists at the 526-2 site, the detection result shows that the rpob526-2 site is a base, which is consistent with the sanger sequencing result, and the extension efficiency is close to 1.0, so that the adjusted rpob526-2 site UEP primer (5'-GGAACCCGCTCGTGGGGTTGACCC-3') is selected as the final UEP primer at the site.
From the above experiments, it was finally established that the primers of the present invention are shown in tables 3 and 4 below.
Table 3PCR amplification primer correspondence table:
table 4UEP extension primer correspondence table:
3 grouping optimization of primers
The drug-resistant gene related to the application detects SNP loci, the distribution of individual gene SNP loci is relatively close, such as rpob genes, mutation related to the genes detects SNP loci, the mutation related to the genes is relatively concentrated and is distributed in 507-533 bp, mutation exists in adjacent 2 bases such as 526CAC > TAC, 526CAC > GAC and the like, if the loci are all located in the same tube system, the mutual influence between primers and competition of the templates are relatively large, the detection sensitivity can be directly influenced, and the application carries out grouping optimization according to the distance between the genes and the loci in order to avoid interference between the primers as much as possible. The specific components are as follows 3 groups: group 1 includes SEQ ID NO.1-36 amplification primers; group 2 includes SEQ ID NO.1-6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.35-46 amplification primers; the amplification primers of the 3SEQ ID No.1-4, SEQ ID No.7-8, SEQ ID No.11-12, SEQ ID No.27-28, SEQ ID No.31-32, SEQ ID No.35-36, SEQ ID No.43-44 and SEQ ID No. 47-48. Further, set 1 also includes SEQ ID NO.49-73 extension primers; set 2 further comprises SEQ ID NO.73-89 primer extensions; group 3 also includes SEQ ID NO.73, SEQ ID NO.90-103 extension primers.
Example 2 minimum detection limit validation of the present application
After confirming the optimal reaction system in this example, each detection site was subjected to the minimum limit confirmation test using a national reference for a Mycobacterium tuberculosis rifampicin resistance gene detection reagent and a national reference for a Mycobacterium tuberculosis isoniazid resistance gene detection reagent, which were synthesized by the division of bioengineering (Shanghai).
The verification scheme is as follows:
(1) The wild type and mutant forms of each site involved in the detection of the present application were diluted in total at about 103 plasmid gradients of known concentration to three gradients of 50 copies/. Mu.L, 10 copies/. Mu.L, 5 copies/. Mu.L, each of which was repeated 2 times; the lowest detection limit of the product is determined by taking the lowest concentration which can be detected by 100%, and then repeating the detection for 5 times under the concentration, and detecting all the 5 times. The specific verification process comprises the following steps: the amplification primer MIX and the extension primer MIX were first prepared according to the system addition scale provided in example 1 of the present application, and then PCR amplification, SAP digestion, single base extension and mass spectrometry detection analysis were performed, respectively, according to the procedure in example 1, with a final plasmid minimum detection limit of 10 copies/. Mu.l.
(2) National reference minimum detection limit
The lowest detection limit national reference information is shown in table 5 below.
Table 5 minimum detection limit national reference information:
a. single cell drug resistance minimum detection limit
The single cell drug-resistant bacteria liquid reference (94002 ', 94023') is diluted to 1X 10 according to different bacteria content before detection 4 bacteria/mL, 3×10 3 bacteria/mL, 10 3 Three gradients of bacteria/mL, each gradient was repeatedly detected 2 times; the lowest concentration that can be detected at 100% is taken as the lowest detection limit of the product, and thenAt this concentration, the detection is repeated 5 times, and the concentration is determined to be the lowest detection limit of the product when all the 5 times are detected. The specific verification process comprises the following steps: the amplification primer MIX and the extension primer MIX were first prepared according to the system addition scale provided in example 1 of the present application, and then PCR amplification, SAP digestion, single base extension and mass spectrometry detection analysis were performed, respectively, according to the procedure in example 1, with the lowest detection limit of the single cell resistant country of the present application being 3×10 3 bacteria/mL.
b. Minimum detection limit for non-100% drug resistance
Mixed bacterial liquid reference of sensitive bacteria and drug-resistant bacteria with different proportions (S and S) I/R ) Before detection, the bacteria are prepared according to different bacterial contents and various drug resistance ratios, and the details are shown in the following table 6.
Table 6 preparation of mixed bacterial solutions of sensitive bacteria and drug-resistant bacteria in different proportions:
each gradient was repeatedly tested 2 times; the lowest detection limit of the product is determined by taking the lowest concentration which can be detected by 100%, and then repeating the detection for 5 times under the concentration, and detecting all the 5 times. The specific verification process comprises the following steps: firstly preparing an amplification primer MIX and an extension primer MIX according to the system addition scale provided in example 1 of the present application, then respectively performing PCR amplification, SAP digestion, single base extension and mass spectrometry detection analysis according to the operation procedure in example 1, wherein the lowest detection limit of the national reference for detecting the drug resistance ratio of the present application is 50% drug resistance ratio or more and the bacterial concentration is not lower than 1×10 3 CFU/mL。
Example 3 clinical sample testing
In this example, 97 clinical samples were validated after confirmation of the optimal reaction system. Including 73 isolate culture samples, 17 sputum samples, and 7 alveolar lavage samples. Comparing the detection result with the sanger sequencing result, and verifying that the total coincidence rate is more than 95%.
The specific verification scheme is as follows:
the amplification primer MIX and the extension primer MIX were first prepared according to the system addition scale provided in example 1 of the present application, and then PCR amplification, SAP digestion, single base extension, and mass spectrometry detection analysis were performed according to the procedure in example 1, respectively, with the detection results shown in the following table.
Table 7 clinical sample test results:
because the invention has more detection sites, the debugging process is only illustrated by taking part of detection sites as an example in the design and optimization analysis of a PCR system. Figures 6-8 show the results of detection mass spectrometry of a portion of the sites of a portion of the clinical sample and the results of sanger sequencing, respectively.
In summary, comparison of the massaray results and Sanger results of 97 samples shows (table 7), the total compliance rate of the system verification experiment of the application is 100%, the effect exceeds expectations, and the clinical value is very remarkable.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.
Claims (10)
1. A primer group for detecting drug-resistant gene mutation of mycobacterium tuberculosis, which is characterized in that the primer is aimed at a drug-resistant gene SNP locus.
2. The primer set of claim 1, wherein the drug-resistant gene comprises katG, ahpC, inhA, rpoB, rpsL, rrs, embB, pncA, gyrA, gyrB, thyA, rv0678, eis, alr, rplC and rrl.
3. The primer set of any one of claims 1-2, wherein the primer set comprises 48 amplification primers having the sequences shown in SEQ ID nos. 1-48.
4. The primer set of claim 3, wherein the primer set further comprises 55 extension primers having the sequences shown in SEQ ID NOS.49-103.
5. The primer set according to claim 4, wherein the primer set comprises an internal reference amplification primer, and the sequence of the internal reference amplification primer is shown as SEQ ID NO. 35-36; the primer group also comprises an internal reference extension primer, and the sequence of the internal reference extension primer is shown as SEQ ID NO. 73.
6. The primer set of claim 5, wherein the primers are divided into 3 groups:
group 1 includes 36 amplification primers, the sequences of which are shown as SEQ ID NO. 1-36;
group 2 includes 22 amplification primers with sequences shown as SEQ ID NO.1-6, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO. 35-46;
group 3 includes 18 amplification primers, the sequences of which are shown as SEQ ID NO.1-4, SEQ ID NO.7-8, SEQ ID NO.11-12, SEQ ID NO.27-28, SEQ ID NO.31-32, SEQ ID NO.35-36, SEQ ID NO.43-44, and SEQ ID NO. 47-48.
7. The primer set of claim 6, wherein:
the group 1 further comprises 25 extension primers, and the sequences of the extension primers are shown as SEQ ID NO. 49-73;
the group 2 further comprises 17 extension primers, and the sequences of the extension primers are shown as SEQ ID NO. 73-89;
the group 3 further comprises 15 extension primers, and the sequences of the extension primers are shown as SEQ ID NO.73 and SEQ ID NO. 90-103.
8. A product for detecting a mutation in a drug-resistant gene of mycobacterium tuberculosis, comprising the primer set of any one of claims 1-6.
9. The product for detecting a drug-resistant gene mutation of Mycobacterium tuberculosis according to claim 8,
the product further comprises reagents and/or equipment for detecting an indicator; preferably, the reagent comprises: 10 XPCR Buffer, dNTP Mix, mgCl 2 Primer Mix, PCR Enzyme, dUTP, UDG and nuclease free water; the device is a massaray nucleic acid mass spectrum.
10. Use of the primer set according to any one of claims 1 to 6 for preparing a kit for detecting drug-resistant gene mutation of mycobacterium tuberculosis.
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| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
| CN113817850A (en) * | 2021-09-07 | 2021-12-21 | 中国农业科学院农业基因组研究所 | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof |
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| KR102123621B1 (en) * | 2019-01-30 | 2020-06-16 | 주식회사 창 헬스케어 | Detection of Non-tuberculosis Mycobacterium, Mycobacterium tuberculosis, and Multidrug-resistant Mycobacterium tuberculosis using real-time polymerase chain reaction and melting temperature measurement |
| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
| CN113817850A (en) * | 2021-09-07 | 2021-12-21 | 中国农业科学院农业基因组研究所 | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof |
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