A primer combination and kit for detecting drug-resistant genes of mycobacterium tuberculosis
By designing specific amplification primer combinations using multiplex PCR targeted capture and nanopore sequencing technology, the problems of long detection time and high cost in Mycobacterium tuberculosis drug resistance analysis have been solved, realizing rapid and low-cost multiplex drug resistance gene detection, which is suitable for ordinary experimental environments.
Patent Information
- Application Number
- CN202211140027.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-20
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2042-09-20
AI Technical Summary
Existing technologies for analyzing drug resistance in Mycobacterium tuberculosis suffer from problems such as long detection times, high costs, and the need for expensive specialized personnel and equipment, which limit the promotion and application of tuberculosis prevention and control, especially in areas with limited resources.
By employing a multiplex PCR targeted capture method combined with nanopore sequencing technology, specific amplification primer combinations were designed. The DNA of the sample to be tested was amplified by multiplex PCR. Taking advantage of the high throughput and long fragment reading characteristics of nanopore sequencing, gene sequence information was rapidly obtained. Combined with bioinformatics analysis, the simultaneous detection of multiple drug resistance genes was achieved.
It enables rapid, low-cost, and convenient detection of drug resistance genes in Mycobacterium tuberculosis, reduces the complexity of experimental procedures, improves detection timeliness and accuracy, is suitable for general laboratory environments, and has good potential for clinical application.
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Figure CN116064864B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of biotechnology, and particularly relates to an amplification primer combination and kit for detecting drug-resistant genes of Mycobacterium tuberculosis. BACKGROUND
[0002] Currently, the global tuberculosis prevention and control is facing great challenges, and its pathogen is called Mycobacterium tuberculosis, which has a very complex drug resistance spectrum (Daley, Thorac Surg Clin 2019, 29: 19-25; Gagneux, Nat Rev Microbiol 2018, 16: 202-213). Two key reasons for the high mortality rate of tuberculosis: multidrug-resistant tuberculosis (MDR-TB), which is infected with Mycobacterium tuberculosis at least resistant to isoniazid and rifampicin; extensively drug-resistant tuberculosis (XDR-TB), which refers to the infection of Mycobacterium tuberculosis, not only resistant to isoniazid and rifampicin, but also resistant to at least one fluoroquinolone and second-line anti-tuberculosis drugs (kanamycin, amikacin, capreomycin, etc.) (Liang et al., Eur J Clin Microbiol Infect Dis 2021, 40: 1851-1861).
[0003] Drug resistance analysis of Mycobacterium tuberculosis is crucial for the full diagnosis and treatment of tuberculosis, and the results of traditional culture-based phenotypic drug susceptibility test (DST) often take 1-3 months. Nucleic acid amplification and other methods have been used in clinical practice, but there are limitations, such as only detecting known gene mutation hotspots; although the second-generation sequencing technology (NGS) has also been widely used, its platform building cost is high, the reagent price is expensive, and professional technical personnel are needed for experimental detection, which greatly limits the promotion and application of sequencing technology in the field of tuberculosis, especially in countries and regions with heavy burden of tuberculosis prevention and control (Dicks et al., Annu Rev Med 2019, 70: 1.1-1.14).
[0004] Nanopore sequencing technology is a newly developed high-throughput sequencing method, which has the advantages of low cost, non-labeling, no base preference, and the ability to cross repetitive genomic regions, and can obtain sequence information in real time (Deamer et al., Nat Biotechnol 2016, 34:518-524; Wang et al., Nat Biotechnol 2021, 39:1348-1365). This technology belongs to single molecule sequencing: when the DNA base passes through the nanopore, the charge changes, thereby temporarily affecting the current intensity flowing through the nanopore (the current change amplitude affected by each base is different), and sensitive electronic equipment detects these changes to identify the passed base; long base sequences can be obtained, the reading speed is fast, the data throughput is super large, and the size of the instrument can be as small as a USB flash disk.
[0005] In 2015, when the commercialization of nanopore sequencing technology was just starting, the single molecule accuracy of nanopore sequencing was only about 60%, which limited its application in Mycobacterium tuberculosis sequencing. Although the single molecule accuracy has gradually improved to > 95% in the subsequent period, attempts to sequence Mycobacterium tuberculosis have achieved preliminary results (Dippenaar et al., J Clin Microbiol, 2021, 60(1): e00646-21), but there are still problems in SNP base resolution. Thanks to the upgrading of nanopore sequencing chips and algorithms in the past two years, the overall sequencing yield and quality have been greatly improved, and > 99% accuracy can be achieved. If multiple drug-resistant gene full-length sequences can be multiplexed and synchronized amplified, and the target genes can be rapidly enriched, the experimental operation complexity will be greatly reduced, the gene sequence analysis timeliness will be improved, and combined with the portability of nanopore sequencing equipment, it can be carried out in ordinary experimental environment, and tuberculosis drug resistance analysis will therefore usher in a new breakthrough and application. SUMMARY
[0006] The technical problem to be solved by the present application is to provide an amplification primer combination and kit for detecting multiple drug-resistant genes.
[0007] To solve the above technical problems, the present application provides a special primer combination for Mycobacterium tuberculosis drug-resistant gene detection, which is 20 pairs of primers as shown in Table 1:
[0008] Table 1 Specific amplification primer sequence
[0009]
[0010]
[0011] The present application also provides a Mycobacterium tuberculosis drug-resistant gene detection kit: comprising 20 pairs of primers as described above.
[0012] As the improvement of the detection kit for the Mycobacterium tuberculosis drug resistance gene of the present application: also includes Taq polymerase (multiplex PCR polymerase), buffer, high GC enhancer, dNTPs, positive control and negative control.
[0013] The present application also simultaneously provides a method for detecting the Mycobacterium tuberculosis drug resistance gene using the above-mentioned detection kit:
[0014] Mix 20 pairs of primers to obtain a primer mixture; use multiplex PCR to amplify the DNA of the sample to be tested; then use the high-throughput and long-fragment reading characteristics of the nanopore sequencing technology to obtain the sequence of each gene (read out the ATCG sequence information of each gene one by one), and finally determine the sample to be tested through bioinformatics analysis.
[0015] As the method for detecting the Mycobacterium tuberculosis drug resistance gene of the present application, the following operation steps are included:
[0016] 1) Perform multiplex PCR amplification on the extracted sample DNA;
[0017] In the PCR amplification system: include multiplex amplification primer mixture mixed by 20 pairs of primers, each primer concentration is 100 nM-1 μM; Taq polymerase, concentration is 0.5-3 U / 25 μL; dNTP, concentration is 0.2-1 mM; MgCl2, concentration is 0.5 mM-3 mM; high GC enhancer (to protect the amplification performance of high GC content DNA template), diluted to 1x; the DNA of the sample to be tested as a template;
[0018] The amplification program is 98℃, 60s; 98℃, 20s, 60℃ 30s, 72℃ 120s, cycle 38 times; 72℃, 4min;
[0019] Note: The sequence length of the above PCR amplification product is between 500-3000bp, which can accommodate multiple gene mutation sites or even entire gene fragments;
[0020] 2) Purify the amplification product of step 1) by magnetic bead, and then introduce a tag sequence through a ligation reaction, i.e. each sample corresponds to a unique tag sequence;
[0021] Note: The number can be 6-384 tag sequences, respectively marking 6-384 samples;
[0022] Perform the above steps 1)-2) on each sample DNA respectively;
[0023] 3) Mix the multiplex PCR amplification products corresponding to each sample DNA with the tag sequence obtained in step 2) at the same quality ratio;
[0024] 4) The mixture obtained in step 3) is subjected to a ligation reaction plus motor protein and sequencing adaptor to form a sequencing library;
[0025] 5) The sequencing library obtained in step 4) is added to a nanopore sequencing chip and then loaded into a sequencer for sequencing for 1-24 hours;
[0026] 6) The sequencing data output in step 5) is subjected to base recognition and sequence alignment by using the software provided by the sequencer and open source software to obtain a detection result.
[0027] Note: Further drug resistance analysis can be performed according to the sequencing data.
[0028] As a further improvement of the method for detecting the drug resistance genes of Mycobacterium tuberculosis according to the application:
[0029] In the step 5), the sequencing chip and the sequencing instrument can be selected from a variety of options, wherein the sequencing chip and the sequencing instrument of the Oxford Nanopore Company in the United Kingdom can be selected.
[0030] In the step 6), the open source software can be selected from a variety of options, wherein centrifuge, minimap2 and TBProfiler can be selected.
[0031] The application adopts the multiplex PCR targeted capture method combined with the nanopore sequencing technology, which can eliminate the interference of non-target gene sequences such as human genes and simultaneously detect multiple drug resistance genes of Mycobacterium tuberculosis.
[0032] The multiplex PCR targeted capture sequencing adopted in the application contains 20 amplicons. The amplicon sequence length is between 700-2500 bp, which can accommodate multiple gene mutation sites or even entire gene fragments. The nanopore sequencing can completely read these amplicon sequences, and through simple data analysis, the decoding can be quickly performed. The long reading sequencing of the nanopore can analyze the full-length gene, which cannot be achieved by the previous sequencing technology. Due to the increase in reading length, the classification unit differentiation rate is improved compared with the previous technology. The multiplex PCR targeted capture method has the advantages of short library construction cycle, high gene sequence capture rate, good uniformity, high alignment rate, good repeatability and simple operation.
[0033] The establishment of the detection method first designs a specific amplification primer combination, which can be used to amplify 20 genes for identifying Mycobacterium tuberculosis and analyzing various drug resistance conditions (such as rifampicin, isoniazid, streptomycin, ethambutol, fluoroquinolones, kanamycin, amikacin and capreomycin, etc.). After the synthesis of these specific amplification primers, the primers are dissolved and subjected to concentration determination, and are subjected to experimental screening and verification. The specific conditions are shown in Table 1.
[0034] Among them, the whole rrs gene (also known as 16S rRNA gene) is sequenced in full length, including 9 hypervariable fragments (V1-V9) flanked by highly conserved regions, which can be used to confirm Mycobacterium tuberculosis (Callahan et al., Nucleic Acids Res 2019, 47: e103-e103). Sequencing of multiple genes related to anti-tuberculosis drug resistance (katG, inhA, fabG1, ahpC, rpoB, embB, pncA, gyrA, rrs, tylA, eis, etc.) can analyze the drug resistance of Mycobacterium tuberculosis (Dicks et al., Annu Rev Med 2019, 70: 1.1-1.14).
[0035] The PCR reaction reagent system is constructed and contains primers, Taq polymerase, dNTPs, buffer, Mg 2+ , etc. The multiplex amplification primer mixture is mixed by 20 pairs of primers, and the final concentration of each primer is 100 nM-1 muM; the concentration of Taq polymerase is 0.5-3 U / 20 muL; the concentration of dNTP is 0.2-1 mM; the concentration of Mg 2+ is 0.5 mM-3 mM; the high GC enhancer (to ensure the amplification performance of high GC content DNA template) is diluted to 1 times. In order to efficiently amplify the gene fragment, Taq polymerase can be selected from Kapa Taq, Hieff Taq, EpiQuick Taq, HiPer plus Taq, PrimeStar Taq, Phanta Taq, NEB Q5 Taq, etc.
[0036] After the multiplex PCR reaction, the tag sequence is introduced through the ligation reaction, the number can be 6-384, and 6-384 samples are labeled respectively, that is, each sample corresponds to a unique tag sequence.
[0037] The multiplex PCR amplification product of each sample which has been labeled with a tag sequence can be mixed together, and then the motor protein and the sequencing adapter are added by ligation reaction to form a sequencing library, and finally the high-throughput advantage of nanopore sequencing technology is fully utilized for sequencing, and after the machine, the data analysis can be divided into each sample according to the tag.
[0038] The application utilizes nanopore sequencing technology to develop a new type of molecular diagnostic kit, which does not need to culture Mycobacterium tuberculosis, directly performs multiplex PCR amplification on sample nucleic acid, then reads sequences by sequencing and analyzes data, so as to quickly analyze the drug resistance of tuberculosis, not only can reduce the comprehensive detection cost of tuberculosis, but also can speed up the diagnosis speed, and has good clinical application potential.
[0039] In conclusion, the application discloses an amplification primer combination and a kit for detecting mycobacterium tuberculosis drug resistance genes. The kit is based on nanopore sequencing technology and contains 20 pairs of primers designed, screened and verified. During detection, the primer mixture is used to amplify the DNA of the sample to be detected by multiplex PCR, each pair of primers amplifies a nucleotide sequence or the whole gene sequence in the reaction process, then the high throughput and long fragment reading characteristics of the nanopore sequencing technology are used to obtain the gene sequences, and finally the sample to be detected is determined through bioinformatics analysis. The kit can complete the amplification and detection of multiple genes under the same reaction condition, does not need to culture mycobacterium tuberculosis, can identify mycobacterium tuberculosis and detect multiple drug resistance genes, and has the characteristics of multiple indexes, rapidness, strong specificity, high sensitivity and the like. BRIEF DESCRIPTION OF DRAWINGS
[0040] The specific embodiments of the application will be further described in detail below with reference to the accompanying drawings.
[0041] Figure 1 The multiplex PCR library construction process of the kit of the application.
[0042] Figure 2 The overall detection process of the kit of the application. DETAILED DESCRIPTION
[0043] The application will be further described below with reference to specific examples, but the protection scope of the application is not limited to the examples:
[0044] The experimental methods used in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents and the like used in the following examples can be obtained from commercial channels unless otherwise specified. The following examples facilitate better understanding of the application, but do not limit the application.
[0045] Example 1, kit composition
[0046] The primer mixture liquid: 20 pairs of primers described in Table 1 are commissioned to be synthesized (Shengong Bioengineering Company), and then a multiplex amplification primer mixture is prepared: the final concentration of each primer in the reaction system is 200nM; multiplex PCR polymerase (NEB Company, USA): NEB Q5 Taq polymerase, the final concentration in the reaction system is 1U / 25μL; 5×buffer (NEB Company, USA): diluted to 1× in the reaction system, which contains 2mM of Mg 2+; 5x High GC Enhancer (NEB, USA): diluted to 1x in the reaction system; dNTPs (Bodin Biotech Co.): various dNTPs with a final concentration of 0.2 mM in the reaction system; positive control: Mycobacterium tuberculosis (H37Rv strain) DNA solution; negative control: TE buffer; PCR product purification reagent: AMPure XP magnetic beads (Beckman, USA), used at a 1:1 volume ratio with the PCR product.
[0047] In addition to the kit, the library construction and nanopore sequencing reagents and chips are also required, which are purchased from Oxford Nanopore Technologies (ONT, UK): tag sequence (molecular barcode adapter) ligation reagent (Native Barcoding Kit 96, SQK-NBD112.96); motor protein and sequencing adapter ligation reagent (Ligation Sequencing Kit, SQK-LSK112); nanopore sequencing chip (Flow Cell R10.4, FLO-MIN112).
[0048] Example 2, application of the kit to cultured known bacterial samples
[0049] Test samples include:
[0050] A total of 9 Mycobacterium tuberculosis (wild type, rifampicin-resistant, isoniazid-resistant, streptomycin-resistant, ethambutol-resistant, fluoroquinolone-resistant, kanamycin-resistant, amikacin-resistant, and capreomycin-resistant), each diluted into 7 different concentrations of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 Each of the 2 non-tuberculosis mycobacteria (Mycobacterium avium, Mycobacterium terrae), 1 each of Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterococcus faecium, and Staphylococcus epidermidis, each with 2 different concentrations of 10 5 , 10 6 The above strains were obtained from existing research basis and cooperative units, and had been confirmed to have drug resistance genes and drug resistance through bacterial culture, drug sensitivity test, Sanger sequencing, etc.
[0051] For each sample, the following operations were performed: 400 μL was taken, and the extraction kit was operated according to the instructions (Qiagen, QIAamp UCP Pathogen Mini Kit, Germany), and finally eluted into 50 μL of water. The extracted sample DNA was subjected to multiplex PCR amplification. The PCR amplification system (25 μL) can be prepared according to the final concentration of each component of the kit in Example 1: 5 μL of 5x buffer, 5 μL of 5x high GC enhancer, 2 μL of dNTPs, 1.25 μL of primer mixture, 1.5 μL of water, 0.25 μL of polymerase, and finally 10 μL of sample DNA.
[0052] Reaction conditions: 98°C, 60s; 98°C, 20s, 60°C, 30s, 72°C, 120s, cycle 38 times; 72°C, 4 min. The PCR amplification product was purified by mixing with AMPure XP magnetic beads at a ratio of 1:1, and eluted into 24 μL of water. According to the operating instructions of the ONT company, the molecular barcode adapter ligation reagent (Native Barcoding Kit 96) was used to introduce different tag sequences one by one according to the number of samples.
[0053] Note: This Native Barcoding Kit 96 can only introduce 96 tags at most.
[0054] According to the operating instructions of the ONT company, the sample products labeled with tags were mixed, which facilitated subsequent combined processing; the motor protein and sequencing adapter ligation reagent (Ligation Sequencing Kit) was used to obtain the sequencing library; it was added to the nanopore sequencing chip (Flow Cell R10.4), and the chip was loaded into the sequencer (ONT company, MinION Mk1C), and the MinKNOW software of the instrument was used for process monitoring, and the instrument was run for 3 hours.
[0055] The raw data of nanopore sequencing is in binary fast5 format containing all sequencing signals, and a single sequencing sequence (read) corresponds to a single fast5 file; through the guppy software in the MinKNOW software package, the fast5 format data is converted to fastq format, containing a large number of read base information and its corresponding sequencing quality information. When bioinformatics analysis is performed, each sample needs to be split according to the tag sequence, and the primer sequence of each sample is removed to avoid affecting the accuracy of mutation identification; here, there is no need to eliminate duplicates to avoid reducing the sequencing depth.
[0056] The invention uses centrifuge open source software for classification, the quality threshold is 150, and the low-quality sequence is excluded; the open source software minimap2 is used as the alignment control, which is a very good alignment software designed for three-generation sequences; the TBProfiler open source software is used to predict Mycobacterium tuberculosis drug resistance.
[0057] The software involved in the above is the existing known software, and the actual use is operated according to the requirements of the software, so it is not described in detail.
[0058] Detection results:
[0059] The initial concentration of Mycobacterium tuberculosis sample is 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 bacteria / mL, which is accurately determined as positive and distinguished from wild type, rifampicin resistance, isoniazid resistance, streptomycin resistance, ethambutol resistance, fluoroquinolone resistance, kanamycin resistance, amikacin resistance, capreomycin resistance; and the initial concentration of Mycobacterium tuberculosis sample is 10 1 bacteria / mL, which is lower than the detection limit of 10 2 bacteria / mL, and is determined as negative.
[0060] The initial sample concentration of 10 5 , 10 6 bacteria / mL of two non-mycobacterium (bird mycobacterium, land mycobacterium), as well as escherichia coli, staphylococcus aureus, acinetobacter baumannii, pseudomonas aeruginosa, enterococcus faecium, and staphylococcus epidermidis, are determined as negative.
[0061] Example 3, kit applied to clinical samples
[0062] Test samples include: 4 cases of tuberculosis culture samples (scraping culture into TE buffer), 4 cases of sputum samples, 4 cases of alveolar lavage fluid samples, and 4 cases of blood samples.
[0063] Because the QIAamp UCP Pathogen Mini kit has an optional menu corresponding to the pretreatment steps of different types of samples, the four samples are pretreated and DNA extracted according to the kit instructions, and finally eluted into 50 μL water; the subsequent experimental procedures are the same as example 2.
[0064] The detection results: the determination results of 16 clinical samples are consistent with the clinical diagnosis results. Among them, 11 cases are positive for Mycobacterium tuberculosis, and 1 case of rifampicin resistance and 1 case of isoniazid resistance are confirmed, as shown in Table 2. The whole nanopore sequencing process takes about 15 hours, which is more than 18 days earlier than the clinical drug sensitivity test to assist the guidance of tuberculosis treatment.
[0065] Table 2: sputum clinical sample results
[0066]
[0067] The above only lists some specific embodiments of the present application. Obviously, the present application is not limited to the above embodiments, and there can be many variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosure of the present application should be considered as falling within the scope of the present application.
Claims
1. A combination of specific primers for detecting drug resistance genes of Mycobacterium tuberculosis in the same reaction, characterized in that 20 pairs of primers as shown below: 。 2. A test kit for detecting drug-resistant genes of Mycobacterium tuberculosis, characterized by: The 20 pairs of primers of claim 1.
3. The kit for detecting the drug resistance gene of Mycobacterium tuberculosis according to claim 2, characterized in that: Also included are Taq polymerase, buffer, high GC enhancer, dNTPs, positive control and negative control.
4. The method for detecting Mycobacterium tuberculosis drug resistance genes using the detection kit of claim 2 or 3 for non-disease diagnosis and treatment purposes, characterized in that: The 20 pairs of primers are mixed to obtain a primer mixture; the DNA of the sample to be tested is amplified by multiplex PCR; then the high-throughput and long-fragment reading characteristics of the nanopore sequencing technology are used to obtain the sequence of each gene; and finally the sample to be tested is determined by bioinformatics analysis.
5. The method for detecting the drug-resistant gene of Mycobacterium tuberculosis according to claim 4, characterized in that The method comprises the following steps: 1) Multiplex PCR amplification of the extracted sample DNA; The PCR amplification system comprises a multiplex amplification primer mixture mixed by the 20 pairs of primers, and the concentration of each primer is 100 nM to 1 μM; Taq polymerase at a concentration of 0.5-3 U / 25 μL; dNTP at a concentration of 0.2-1 mM; Mg 2+ at a concentration of 0.5 mM-3 mM; high GC enhancer diluted to 1x; DNA of the sample to be tested as a template; The amplification program is 98℃, 60s; 98℃, 20s, 60℃ 30s, 72℃ 120s, 38 cycles; 72℃, 4min; 2) The amplification product of step 1) is purified by magnetic beads, and a tag sequence is introduced through a ligation reaction, i.e. each sample corresponds to a unique tag sequence; 3) The multiplex PCR amplification product corresponding to each sample DNA with a tagged sequence obtained in step 2) is mixed in equal quality ratio; 4) The mixture obtained in step 3) is subjected to a ligation reaction to add motor protein and sequencing adapters to form a sequencing library; 5) The sequencing library obtained in step 4) is added to a nanopore sequencing chip and then loaded into a sequencer for sequencing for 1-24 hours; 6) The sequencing data output in step 5) is subjected to base recognition and sequence alignment by the software and open source software of the sequencer to obtain the detection result.
6. The method for detecting Mycobacterium tuberculosis drug resistance genes according to claim 5, characterized in that: In step 1), in order to efficiently amplify the gene fragments, the Taq polymerase is selected from Kapa Taq, Hieff Taq, EpiQuick Taq, HiPer plus Taq, PrimeStar Taq, Phanta Taq or NEB Q5 Taq; In step 6), the open source software is selected from centrifuge, minimap2 or TBProfiler.