CN116356056B - 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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Classifications
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- 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
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- 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
- C12Q2600/16—Primer sets for multiplex assays
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- 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
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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
- 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
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, which can simultaneously complete accurate detection of SNP loci of a plurality of drug-related 16 genes 53 of tuberculosis treatment in a nucleic acid mass spectrum platform.
Description
Technical Field
The application relates to the technical field of biology, 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 has been proposed.
Disclosure of Invention
The first object of the present application is to provide a specific primer set for detecting drug-resistant gene mutation of Mycobacterium tuberculosis.
The second object of the present application is to provide the application of the primer set for detecting the mutation of the drug-resistant gene of mycobacterium tuberculosis in preparing the drug-resistant gene detection product of mycobacterium tuberculosis.
The 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 application is to provide a method for detecting the drug-resistant gene mutation of mycobacterium tuberculosis, which is used for solving the technical problems of low flux, single type of applicable sample, less applicable antitubercular drugs, high price and the like 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 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 applying 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 a mycobacterium tuberculosis drug-resistant gene mutation detection product.
Compared with the prior art, the application has the following technical advantages:
1) The application carries out strict selection and combination on drug-resistant genes and loci through early-stage experiments, and at least comprises the aspects of primer design thought, screening, grouping and the like in primer design. For example, in order to reduce the pressure of multiple systems 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 screening and optimizing of specific primer sequences, 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 in total 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 drug-resistant gene mutation of the mycobacterium tuberculosis comprises the step of detecting the drug-resistant gene mutation of the mycobacterium tuberculosis in a sample to be detected by using the primer group. The primer group provided by the application can be used for detecting 53SNP loci in total of 16 genes related to various tuberculosis treatment medicines, and has the characteristics of high accuracy, high sensitivity, good repeatability, low cost, short detection period, visual result and the like;
4) The application applies the flight time mass spectrum platform to detection of various SNP loci related to tuberculosis treatment drugs, has broad spectrum applicability, greatly improves detection efficiency, and is particularly suitable for batch detection. The application overcomes the defect of low PCR weight in the prior art, has low cost and is suitable for wide popularization; the application 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, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the 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 a diagram showing the PCR primer screening for the inhA94 site 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 embB497-1 site before and after optimization provided in example 1 of the present application.
FIG. 5 is a mass spectrum of the rpob526-2 locus UEP primer before and after optimization provided in example 1 of the present application.
FIG. 6 is a diagram of the results of the sequencing of the ahpC-10 site in the TB61 sample provided in example 3 of the present application.
FIG. 7 is a diagram showing the results of the sequencing of the TB109 sample EmbB306-1 locus as provided in example 1 of the present application.
FIG. 8 is a diagram showing the results of the analysis of the rpob511-2 locus of the TB28 sample and the sanger sequencing 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 illustrating the present application and should not be construed as limiting the scope of the present application, and that the examples are some examples of the present application but not all examples. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The Massary ARRAY gene analysis technology 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 application is further illustrated by the following specific examples, however, it should be understood that these examples are for the purpose of illustration only in greater detail and are not to be construed as limiting the 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 embodiment of the application is as follows:
EXAMPLE 1 establishment of the reaction System according to the 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:
furthermore, the application establishes PCR and UEP primers suitable for the mass spectrum detection of the nucleic acid of each SNP locus through design and optimization. In order to achieve the aim of combining the sensitivity and the specificity of a multiple 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 template 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 to amplify the Mycobacterium tuberculosis nucleic acid template, respectively, 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 band, 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 application not only screens and optimizes the PCR amplification primer combination of each locus, but also optimizes the UEP extension primer. In the previous tube closing study, the application discovers that the UEP extension primer at the individual site has low extension efficiency or generates non-specific condition, and the application designs different UEP extension primers for comparison test.
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 application are shown in tables 3 and 4 below.
Table 3PCR amplification primer correspondence table:
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table 4UEP extension primer correspondence table:
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3 grouping optimization of primers
The drug-resistant gene disclosed by the application is used for detecting SNP loci, the distribution of the SNP loci of individual genes is relatively close, such as rpob genes, the mutation detection SNP loci related to the genes are relatively concentrated and are uniformly distributed in the range of 507-533, and mutations exist in adjacent 2 bases such as 526CAC > TAC, 526CAC > GAC and the like, if the loci are all positioned in the same tube system, the mutual influence between primers and competition on a template are relatively large, the detection sensitivity is directly influenced, and the grouping optimization is carried out according to the distances between the genes and the loci so as to avoid the 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 acknowledgement 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 type of each site involved in the detection of the present application were diluted in total to three gradients of 50 copies/. Mu.L, 10 copies/. Mu.L, 5 copies/. Mu.L at a concentration of about 103 known concentrations, and each gradient was repeatedly detected 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 of the present application.
(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 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 table provided in the embodiment 1 of the application, then respectively carrying out PCR amplification, SAP digestion, single base extension and mass spectrometry detection analysis according to the operation steps in the embodiment 1, wherein the lowest detection limit of the single cell drug resistant country is 3×10 3 bacteria/mL.
b. Minimum detection limit for non-100% drug resistance
Sensitive bacteria and tolerance with different proportionsMixed bacterial liquid reference of medicinal bacteria (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 amount provided in example 1 of the present application, then performing PCR amplification, SAP digestion, single base extension and mass spectrometry detection analysis respectively according to the operation procedure in example 1, and finally detecting the lowest detection limit of the national reference for drug resistance ratio of the present application to 50% drug resistance ratio and above and the bacterial concentration to be 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:
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because the application 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 the expected value, 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 application 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 or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (4)
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; the drug resistance genes comprise katG, ahpC, inhA, rpoB, rpsL, rrs, embB, pncA, gyrA, gyrB, thyA, rv0678, eis, alr, rplC and rrl;
the primer group comprises 48 amplification primers, and the sequence of the amplification primers is shown as SEQ ID NO. 1-48;
the primer group also comprises 55 extension primers, and the sequence of the extension primers is shown as SEQ ID NO. 49-103;
the primer group also 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;
the primers were divided into 3 groups:
group 1 includes 36 amplification primers, the sequences of which are shown as SEQ ID NO. 1-36; further comprises 25 extension primers, the sequences of which are shown in SEQ ID NO. 49-73;
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; further comprises 17 extension primers, the sequences of which are shown in SEQ ID NO. 73-89;
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; further comprises 15 extension primers, the sequences of which are shown as SEQ ID NO.73 and SEQ ID NO. 90-103.
2. A product for detecting a mutation in a drug-resistant gene of mycobacterium tuberculosis, the product comprising the primer set of claim 1.
3. The product for detecting a drug-resistant gene mutation of mycobacterium tuberculosis according to claim 2, wherein the product further comprises a reagent and/or a device for detecting an index; 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.
4. The use of the primer set of claim 1 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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| 福建省耐多药结核分枝杆菌耐药相关基因突变特征;张媛媛;黄明翔;赵秀芹;潘爱珍;张丽水;万康林;;中国预防医学杂志;第12卷(第05期);第379-382页 * |
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