CN117821628A - Kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation and application thereof - Google Patents
Kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation and application thereof Download PDFInfo
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- CN117821628A CN117821628A CN202410127400.2A CN202410127400A CN117821628A CN 117821628 A CN117821628 A CN 117821628A CN 202410127400 A CN202410127400 A CN 202410127400A CN 117821628 A CN117821628 A CN 117821628A
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- 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
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Abstract
The invention provides a kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation and application thereof, belonging to the field of molecular biology detection. The specific sequences of the specific primer group are shown in SEQ ID NO. 1-SEQ ID NO.56, 5 mycobacterium tuberculosis genes are identified, 20 drug resistance genes are detected, the identification of the mycobacterium tuberculosis and the detection of the drug resistance gene mutation are carried out in one primer pool, and the concentrations of different primers in the primer pool are different. The invention utilizes the targeted amplification combined second generation sequencing technology, can simultaneously complete effective detection of 5 tuberculosis mycobacteria genes and 20 genes related to 12 tuberculosis treatment medicines on the same platform, can analyze nucleotide mutation through sequence comparison, reports specific mutation types and mutation proportions, has simple and convenient operation, and has the characteristics of high flux, high sensitivity, strong accuracy, good repeatability and the like.
Description
Technical Field
The invention belongs to the field of molecular biology detection, and particularly relates to a kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation and application thereof.
Background
Tuberculosis is a chronic infectious disease caused by a complex of Mycobacterium tuberculosis (i.e., MTC, including Mycobacterium tuberculosis, mycobacterium bovis, mycobacterium africanum, mycobacterium tenuis), which can involve various organs of the whole body, most frequently seen as pulmonary tuberculosis. Wherein the pathogenic bacteria of tuberculosis are mainly mycobacterium tuberculosis. Mycobacterium tuberculosis is mainly transmitted through air and has long latency. In 2021, the World Health Organization (WHO) reported that 1060 thousands of people all over the world had been infected with tuberculosis, of which 160 ten thousands died from tuberculosis. Especially drug-resistant tuberculosis is the biggest difficulty and serious challenge faced in the modern tuberculosis treatment process.
Drug-resistant tuberculosis is classified into single-drug-resistant tuberculosis, multi-drug-resistant tuberculosis, quasi-broad-drug-resistant tuberculosis and broad-drug-resistant tuberculosis. Among them, multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) are two key causes of high mortality of tuberculosis. The main cause of drug resistance is caused by accumulation of point mutations and insertions/deletions in the drug target gene or drug transferase gene.
Drugs for treating mycobacterium tuberculosis infection are generally classified into first-line drugs and second-line drugs. First-line drugs mainly include Isoniazid (INH), rifampicin (RIF), ethambutol (EMB), pyrazinamide (PZA), and Streptomycin (SM); the second-line medicine mainly comprises fluoroquinolone antibiotics (FQs), kanamycin (Km), amikacin (Am), p-aminosalicylic acid (PAS), ethionamide (ETH) and the like. The most recent groupings of antitubercular drugs used in long-range MDR-TB regimens are: 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.
In the clinical treatment of mycobacterium tuberculosis infection, a treatment regimen combining two or more drugs is generally adopted. Since the treatment of tubercle bacillus infection is a long-term administration process, drug resistance is easily generated. From the above, the detection of drug resistance of mycobacterium tuberculosis is very important for guiding clinical correct medication and effectively controlling tuberculosis.
The diagnosis of drug-resistant tuberculosis is dependent on a phenotypic drug susceptibility test (drug susceptibility test for short) for a long time, and the time period of the test method from the collection and culture of sputum specimens to the report of the drug susceptibility test results is 2.5 months, so that the timely and accurate diagnosis and treatment of drug-resistant tuberculosis cannot be satisfied.
In recent years, drug-resistant tuberculosis molecular diagnosis has been widely used, and the next generation sequencing (next generation sequencing, NGS) technology, namely the second generation sequencing technology, adopts a high-throughput sequencing technology, reduces the sequencing cost, improves the sequencing speed, and maintains high accuracy. With the widespread use of NGS technology, exploring the application of NGS technology in the diagnosis of tuberculosis drug resistance has great value.
According to different detection strategies, high throughput sequencing methods are mainly classified into targeted sequencing (targetedNGS, tNGS), metagenomic sequencing (mNGS), and whole genome sequencing (whole genome sequencingNGS, WGS). the tNGS captures specific genes through targeting and then carries out high-throughput sequencing, and has the characteristic of being capable of simultaneously detecting a plurality of target genes in parallel.
The total number of the detection reagents for the drug-resistant gene mutation of the mycobacterium tuberculosis approved by NMPA at present is 12, wherein 10 detection reagents are used in China and 2 detection reagents are used in abroad. 75% of detection reagents mainly aim at rifampicin and isoniazid drug resistance, and drug resistance gene detection reagents aiming at various antitubercular drugs do not appear. Meanwhile, currently approved reagent for detecting drug-resistant genes of mycobacterium tuberculosis by NMPA is mainly based on a fluorescence melting curve method, a fluorescence quantitative PCR method, a gene chip method, a reverse hybridization method and the like, and drug-resistant gene detection reagent based on NGS technology is not yet marketed. The methods have limitations in detection, not only are the number of mutations detected in the drug-resistant gene limited, but also the specific mutation types cannot be distinguished. Therefore, considering the current market demands comprehensively, it is urgent to develop a detection kit with high efficiency and abundant antitubercular drug types.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation and application thereof. The specific sequences of the specific primer group are shown in SEQ ID NO. 1-SEQ ID NO.56, 5 mycobacterium tuberculosis genes are identified, 20 drug resistance genes are detected, the identification of the mycobacterium tuberculosis and the detection of the drug resistance gene mutation are carried out in one primer pool, and the concentrations of different primers in the primer pool are different. The invention utilizes the targeted amplification combined second generation sequencing technology, can simultaneously complete effective detection of 5 tuberculosis mycobacteria genes and 20 genes related to 12 tuberculosis treatment medicines on the same platform, can analyze nucleotide mutation through sequence comparison, reports specific mutation types and mutation proportions, has simple and convenient operation, and has the characteristics of high flux, high sensitivity, strong accuracy, good repeatability and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a specific primer group for detecting mycobacterium tuberculosis and drug-resistant gene mutation, wherein the mycobacterium tuberculosis genes are IS6110, mpt64, HSP65, rpoB and gyrB, the drug-resistant genes are rpoB, katG, inhA, oxyR-ahpC, embB, embA, pncA, rpsL, rrs, gidB, gyrA, gyrB, eis, rv0678, atpE, rrl, rplC, folC, thyA and alr, and the specific sequences of the specific primer group are shown as SEQ ID NO. 1-SEQ ID NO.56 in the following table 1.
TABLE 1 amplification primer correspondence table
The invention also provides a reagent for detecting mycobacterium tuberculosis and drug-resistant gene mutation, which comprises the specific primer group.
Further, the reagent is a kit.
The invention also provides a construction method of the mycobacterium tuberculosis and drug-resistant gene sequencing library, which is carried out by using the kit.
Furthermore, in the construction method, the identification of the mycobacterium tuberculosis and the detection of the drug-resistant gene mutation are both carried out in a primer pool.
Further, the concentration of the different primers in the specific primer set in the primer pool was different, and the use concentration of each primer was as shown in table 1 above.
Further, the construction method specifically performs multiplex PCR amplification of DNA fragments by using the specific primer set, and then performs end repair, adaptor ligation and library amplification of the amplified products. The specific flow diagram is shown in fig. 1.
Further, the reaction system of the multiplex PCR amplification is as follows: 10. Mu.L of Input DNA, 15. Mu.L of 2X Multiplex PCR Master Mix (BioTechnRabbit), 3. Mu.L of primer mix, 2. Mu.L of nuclease-free water, and 30. Mu.L in total.
The invention also provides a mycobacterium tuberculosis and drug-resistant gene sequencing library constructed by using the construction method.
The invention also provides an application of the specific primer group, the reagent and/or the library construction method in mycobacterium tuberculosis and drug-resistant gene mutation detection.
The method for identifying mycobacterium tuberculosis and detecting drug-resistant gene mutation by using the kit comprises the following steps:
step one, extracting nucleic acid of a clinical sample;
step two, performing multiplex PCR amplification by using the specific primer group;
step three, performing end repair, joint connection and library amplification on the amplified product;
step four, sequencing is performed on an MGISEQ sequencer in a sequencing mode of PE150, and sequencing data is generated.
Wherein, the end repair, linker ligation and library amplification are performed using an in-use and on-sale kit, such as those manufactured by Nanjinouzan Biotechnology Co., ltdUniversal DNA Library Prep Kit for MGI(NDM607)。
The raw sequencing data of each sample in the fourth step is not less than 300Mb bases.
After invalid data such as a connector and the like are filtered and quality control analysis is carried out on the off-machine data, the sequence is compared with the target related genes, and the identification condition of the mycobacterium tuberculosis and the mutation condition of the related loci of the drug-resistant genes and the mutation frequency in the detection sample can be known.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention designs primers aiming at 20 genes of 15 antitubercular drugs, and almost comprises all clinical common drug-resistant gene detection targets. Antitubercular drugs are of a variety of species and can help clinicians to provide references for guiding patient medication.
(2) The invention is based on the tNGS technology, and supplements the technical problems of single antitubercular drugs, limited number of drug-resistant gene mutations, low flux and the like which are applicable to the current market.
(3) The invention can finally generate sequencing data with higher uniformity by adjusting the concentration proportion of the primer pool of the multiplex PCR.
(4) The invention uses IS6110, mpt64, HSP65, rpoB and gyrB genes to identify the mycobacterium tuberculosis, has a synergistic effect, can identify the mycobacterium tuberculosis more comprehensively and more accurately, and makes up for false negative results caused by insufficient identification capability of a single gene.
(5) The invention realizes the dual detection of mycobacterium tuberculosis identification and drug-resistant gene mutation, and has short detection period, high sensitivity and good specificity.
(6) The invention can detect as low as 1X 10 2 CFU/mL, the drug resistance ratio is 5% of gene mutation, and the sensitivity is high.
Drawings
FIG. 1 is a schematic flow chart of library construction according to the present invention;
FIG. 2 is a diagram showing primer screening electrophoresis of rpoB, katG, inhA, oxyR-ahpC gene in example 1 of the present invention, wherein M: representing Marker, the bands are 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp and 1500bp from bottom to top; r1: representing the primer rpoB-1 amplification product; r2: representing the primer rpoB-2 amplification product; k1: representing the primer katG-1 amplification product; k2: representing the primer katG-2 amplification product; i1: representing the primer inhA-1 amplification product; i2: representing the primer inhA-2 amplification product; o1: representing the primer oxyR-ahpC-1 amplification product; o2: representing the primer oxyR-ahpC-2 amplification product;
FIG. 3 is a graph showing the result of fluorescence PCR for positive or negative identification of Mycobacterium tuberculosis in example 3 of the present invention;
FIG. 4 is a graph of the results of Sanger sequencing at katG315 site in example 3 of the present invention, the results are represented: the 315th codon of katG gene was mutated from G to C at base 944.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The reagents, kits and instruments used in the following examples are commercially available, and the methods used in the examples are consistent with the methods conventionally used unless otherwise specified.
The technical scheme of the invention is further elaborated in the following in conjunction with examples.
Example 1 screening of primer pairs
(1) 20 tuberculosis drug resistance related genes are determined.
(2) The genome of the mycobacterium tuberculosis H37Rv is taken as a reference genome, and the position information of the related genes on the genome and the full-length information of the genes are obtained in an NCBI database according to the searched gene names.
(3) The 20 drug resistance-related genes include: rpoB, katG, inhA, oxyR-ahpC, embB, embA, pncA, rpsL, rrs, gidB, gyrA, gyrB, eis, rv0678, atpE, rrl, rplC, folC, thyA, alr.
(4) The 15 related tuberculosis therapeutic drugs comprise: rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, bedaquiline, linezolid, clofazimine, cycloserine, amikacin, kanamycin, calicheamicin, para-aminosalicylic acid, fluoroquinolones, clofazimine.
(5) Primer design software was used to design the target sequence primers for the drug resistance related genes rpoB, katG, inhA, oxyR-ahpC, embB, embA, pncA, rpsL, rrs, gidB, gyrA, gyrB, eis, rv0678, atpE, rrl, rplC, folC, thyA, alr.
(6) In order to ensure the detection of the gene loci contained in each gene, the PCR amplification primers of the gene loci contained in each gene are screened and optimized, 2 pairs of primers are designed for each gene, and the length of an amplification product is 200-300bp. The primer was screened using rpoB, katG, inhA, oxyR-ahpC as an example (the primer information is shown in table 2, and a primer pair with high amplification efficiency was screened), and the PCR amplification product was subjected to electrophoresis detection, and the detection result is shown in fig. 2.
TABLE 2rpoB, katG, inhA, oxyR-ahpC primer information
As can be seen from FIG. 2, 2 pairs of PCR primers (R1/R2) designed for rpoB locus region, respectively amplifying nucleic acid of positive sample of Mycobacterium tuberculosis, wherein both R1 and R2 pairs have specific target bands, but the brightness of the R2 band is stronger than that of the R1 primer, which indicates that the amplification efficiency of the R2 primer is high, so that the R2 primer is selected as the final PCR amplification primer for rpoB locus region.
As can be seen from FIG. 2, 2 pairs of PCR primers (K1/K2) designed for katG locus region, respectively amplified nucleic acid of positive sample of Mycobacterium tuberculosis, both pairs of K1 and K2 have specific target bands, but the band brightness of K2 is weaker than that of K1 primer, indicating that the amplification efficiency of K1 primer is high, so K1 primer is selected as final PCR amplification primer for katG locus region.
As can be seen from FIG. 2, 2 pairs of PCR primers (i 1/i 2) designed for the inhA gene locus region, each of which was amplified for a Mycobacterium tuberculosis positive sample nucleic acid, i1 had a specific target band, and i2 had no amplified band, so the i1 primer was selected for the inhA gene locus region as the final PCR amplification primer.
As can be seen from FIG. 2, 2 pairs of PCR primers (o 1/o 2) designed for the oxyR-ahpC gene locus region, respectively, amplify nucleic acid of a positive sample of Mycobacterium tuberculosis, and both o1 and o2 pairs have specific target bands, but the brightness of the bands of o1 is stronger than that of the o2 primer, which indicates that the amplification efficiency of the o1 primer is high, so that the o1 primer is selected for the oxyR-ahpC gene locus region as the final PCR amplification primer.
The method for screening other primers is the same as above, and the primers of the present invention are finally established by using the above method.
Example 2 establishment of multiple System
28 pairs of primers (Table 1) were finally screened using the procedure described in example 1, and these primers were used in accordance with 1:1 (all primers are equal) and form a primer pool, and the positive sample of the mycobacterium tuberculosis is verified according to the following operation steps:
step one, nucleic acid extraction. Extracting nucleic acid by using a radices tenuissimae nucleic acid extraction kit.
Step two, amplifying DNA of positive samples of all the Mycobacterium tuberculosis to be detected by using the mixed primer pool, enriching target gene sequences, and purifying amplified products by using magnetic beads. The amplification system is shown in Table 3.
TABLE 3 multiplex PCR amplification reaction System
And thirdly, sequentially carrying out end repair, linker ligation, library amplification and product purification on the purified product by using a DNA library construction kit (Vazyme). The specific operation method comprises the following steps:
1. end repair
1.1 the End Prep Mix 4 was removed, thawed at room temperature, vortexed for 10s, and centrifuged for 15s instantaneously to prepare an End repair reaction system according to the following Table 4.
TABLE 4 reaction system for repairing terminal
1.2 mixing with a pipette with gentle shaking (mixing without shaking), and briefly centrifuging to collect the reaction solution to the bottom of the tube.
1.3 the PCR tube was placed in a PCR instrument and the reactions shown in Table 5 below were performed.
TABLE 5 end repair reaction procedure
2. Joint connection
2.1 simultaneously taking out Rapid Ligation Buffer, rapidDNAligenase and VAHTSDual UMI Adapters for MGI UDB, thawing at room temperature, and placing on ice for standby.
2.2A reaction mixture was prepared according to the following Table 6.
Table 6 Joint connection reaction System
2.3 mixing with a pipette with gentle blowing (mixing without shaking) and briefly centrifuging to collect the reaction solution to the bottom of the tube.
2.4 the PCR tube was placed in a PCR instrument and the reactions in Table 7 below were performed.
TABLE 7 linker ligation reaction procedure
3. Post-ligation purification
3.1 the purified beads (VAHTSDNAClean Beads) were removed in advance and equilibrated for at least 30min at room temperature.
3.2 after the PCR was completed, the sample tube was removed and centrifuged briefly.
3.3 60. Mu.L of purified magnetic beads were added to each tube of the sample from step 2.4, vortexed or gently swirled 10 times with a pipette, and thoroughly mixed.
3.4 incubation for 5min at room temperature.
3.5 the PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, after which the solution was clarified (about 5 min) and the supernatant carefully removed.
3.6 keep the PCR tube always placed in the magnetic rack, rinse the beads with 200. Mu.L of freshly prepared 80% ethanol, incubate for 30sec at room temperature, carefully remove the supernatant.
3.7 repeat step 3.6 for a total of two rinses.
3.8 keeping the PCR tube in the magnetic rack all the time, and uncovering the air to dry the magnetic beads for 2-5min until no ethanol remains.
3.9 taking out the PCR tubes from the magnetic rack, adding 22.5 mu L of nuclease-free water into each tube, and standing for 2min at room temperature under vortex oscillation or gentle blowing by using a pipette.
3.10 the PCR tube was briefly centrifuged and placed in a magnetic rack for standing, after the solution was clarified (about 5 min), 20. Mu.L of supernatant was carefully removed to a new EP tube and the beads were not touched.
4. Library amplification
4.1 remove VAHTS HiFi Amplification Mix and VAHTS Unique Dual Barcode Primer for MGI, thaw and mix upside down.
4.2 the reaction mixtures were prepared according to the following Table 7.
Table 8 library amplification reaction System
4.3 mixing with a pipette with gentle blowing (mixing without shaking) and briefly centrifuging to collect the reaction solution to the bottom of the tube.
4.4 the PCR tube was placed in a PCR instrument and the reactions in Table 9 below were performed.
TABLE 9 library amplification reaction procedure
5. Post amplification purification
5.1 the purified beads were removed in advance and equilibrated for at least 30min at room temperature.
5.2 after the PCR is finished, the sample tube is taken out and centrifuged briefly.
5.3 to each tube of sample from step 4.4, 45. Mu.L of purified magnetic beads were added, vortexed or gently swirled 10 times with a pipette and thoroughly mixed.
5.4 incubation for 5min at room temperature.
5.5 the PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, after which the solution was clarified (about 5 min) and the supernatant carefully removed.
5.6 keep the PCR tube always placed in the magnetic rack, rinse the beads with 200. Mu.l freshly prepared 80% ethanol, incubate for 30sec at room temperature, carefully remove the supernatant.
5.7 repeat step 5.6 for a total of two rinses.
5.8 keeping the PCR tube in the magnetic rack all the time, and uncovering the air to dry the magnetic beads for 2-5min until no ethanol remains.
5.9 taking out the PCR tubes from the magnetic rack, adding 62.5 mu L of nuclease-free water into each tube, and standing for 2min at room temperature under vortex oscillation or gentle blowing by using a pipette.
5.10 the PCR tube was briefly centrifuged and placed in a magnetic rack for standing, after the solution was clarified (about 5 min), 60. Mu.L of supernatant was carefully removed to a new EP tube and the beads were not touched.
Step four, using an Equalbit 1X dsDNA HS Assay Kit fluorescent quantitative kit, the library was quantified according to the instructions of the kit. And detecting the fragment distribution of the library using gel electrophoresis. After quality inspection was passed, the library was sequenced on an MGISEQ sequencer in a pattern of PE150 and sequencing data was generated.
And fifthly, filtering invalid data such as a connector and performing quality control analysis on the machine-down data, and comparing the sequence with a target drug resistance related gene to obtain the mutation condition and mutation frequency of the drug resistance gene related site in the detection sample.
The coverage and uniformity results for each gene in the primer pool are shown in Table 10.
TABLE 10 coverage and uniformity information for each gene of primer pools
As can be seen from table 10: the coverage of each gene was 100%. The gene length is distributed between 200 and 300bp. The primer pairs of rrs-2, rrl-3, rrl-2 and Rv0678-2 genes have very few read numbers in a multiple system, the primer pairs of rplC and alr genes have excessive read numbers in the multiple system, and the read numbers of different genes show large differences in the system.
In order to maintain the amplification efficiency of each pair of primers in the primer pool to be uniform, it is necessary to adjust the concentration ratio between the individual primers and to verify the multiple adjusted primer pools with a positive sample of Mycobacterium tuberculosis. The final primer ratios are shown in Table 1. Meanwhile, the results of the coverage and uniformity of the drug resistance gene in the primer pool after the adjustment are shown in Table 11.
Table 11 primer pools adjusted drug resistance Gene detection coverage and uniformity information
Example 3 verification of the System
1 national reference minimum detection limit
The lowest detection limit national reference information is shown in table 12.
Table 12 minimum limit of detection national reference information
The lowest detection concentration of the drug-resistant strain at 1.1100% drug-resistant proportion
S was added according to the bacterial content RD /S ID Serial gradient dilutions were performed to prepare samples of rifampicin, isoniazid resistant strain/mutation at different concentrations. The bacterial content was 1X 10 respectively 4 bacteria/mL, 1×10 3 bacteria/mL, 1×10 2 bacteria/mL,1×10 1 bacteria/mL, 1×10 0 bacteria/mL. Three replicates were made for each concentration gradient; the lowest bacterial liquid concentration detected by all three repetitions is the lowest detection limit of the product. The QC quality control results for each library are shown in table 13 and the drug resistance results are shown in table 14.
Table 13 quality control results of QC of library
TABLE 14 mutation detection information of library
| Concentration (personal bacteria/mL) | Source | Repeat 1 | Repeat 2 | Repeat 3 |
| 1×10 4 | S RD Gradient dilution | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity |
| 1×10 3 | S RD Gradient dilution | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity |
| 1×10 2 | S RD Gradient dilution | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity | Rifampicin drug resistance positivity |
| 1×10 1 | S RD Gradient dilution | Rifampicin drug resistance positivity | Not detected | Rifampicin drug resistance positivity |
| 1×10 0 | S RD Gradient dilution | Not detected | Not detected | Rifampicin drug resistance positivity |
| 1×10 4 | S ID Gradient dilution | Isoniazid drug resistance positive | Isoniazid drug resistance positive | Isoniazid drug resistance positive |
| 1×10 3 | S ID Gradient dilution | Isoniazid drug resistance positive | Isoniazid drug resistance positive | Isoniazid drug resistance positive |
| 1×10 2 | S ID Gradient dilution | Isoniazid drug resistance positive | Isoniazid drug resistance positive | Isoniazid drug resistance positive |
| 1×10 1 | S ID Gradient dilution | Isoniazid drug resistance positive | Isoniazid drug resistance positive | Not detected |
| 1×10 0 | S ID Gradient dilution | Not detected | Not detected | Not detected |
The results show that: finally, the minimum detection limit of the national reference bacterial liquid with single cell drug resistance is 1 multiplied by 10 under the condition of 100 percent drug resistance proportion 2 bacteria/mL.
1.2 minimum detection limits for different Strain concentrations and various drug resistance ratios
Wild strain (S) and drug-resistant strain (S) of Mycobacterium tuberculosis R /S I ) Mixing, and adjusting the ratio of wild strain and drug-resistant strain to obtain bacteria with different strain concentrations and various drug-resistant ratiosAnd (5) a strain mixed solution. Details are shown in Table 15.
TABLE 15 information on bacterial strain mixtures
| Concentration of bacterial liquid | Drug resistance ratio | Drug resistance ratio | Drug resistance ratio | Drug resistance ratio | Drug resistance ratio | Drug resistance ratio |
| 1×10 4 CFU/mL | 0 | 5％ | 20％ | 50％ | 75％ | 100％ |
| 1×10 3 CFU/mL | 0 | 5％ | 20％ | 50％ | 75％ | 100％ |
| 1×10 2 CFU/mL | 0 | 5％ | 20％ | 50％ | 75％ | 100％ |
| 1×10 1 CFU/mL | 0 | 5％ | 20％ | 50％ | 75％ | 100％ |
Three parallel repeats are made for each concentration gradient, and the lowest bacterial liquid concentration detected by the three repeats is the lowest detection limit of the product. The QC quality control results for each library are shown in Table 16, and the mutation detection results for the library are shown in Table 17.
Table 16 QC quality control results of library
TABLE 17 mutant detection results of library
The results show that: the final national reference for drug resistance detection of Mycobacterium tuberculosis of the present invention has a drug resistance ratio of 5% or more and a bacterial concentration of not less than 1×10 2 CFU/mL。
Verification of Mycobacterium tuberculosis 2 identification
The HSP65, IS6110, rpoB and gyrB genes of the invention were used for identifying Mycobacterium tuberculosis in 6 clinical samples, and the number of reads and total reads of each gene aligned to the genome of Mycobacterium tuberculosis were analyzed. The results are shown in Table 18.
Table 18 results of identification of Mycobacterium tuberculosis by different genes
3 verification of drug resistance gene detection accuracy
The results of mutation detection of drug-resistant genes of Mycobacterium tuberculosis (clinical samples numbered S1-S30 respectively) were carried out on 30 clinical samples using the present invention and are shown in Table 19.
Table 19 results of identification of tuberculosis drug resistance gene mutation in 30 cases of clinical samples
The results show that: 1 sample is positive in tuberculosis but drug-resistant gene related mutation is not detected, 7 samples are not detected in mycobacterium tuberculosis, and the other 22 samples are detected in drug-resistant gene related mutation.
Meanwhile, the identification result of Mycobacterium tuberculosis of the present invention was verified using a fluorescent PCR method, and the result is shown in FIG. 3. The detection result of the drug resistant gene of the present invention was verified by using Sanger sequencing method, and exemplified by the result of katG.Ser315Thr site therein, as shown in FIG. 4 (note: column 3, 944G > C, i.e., the 944 th base of the katG gene was mutated from G to C, and column 4, ser315Thr, i.e., the aforementioned mutation resulted in the amino acid encoded by codon 315 of the katG gene being changed from serine to threonine). The results were consistent with the results of the detection in the present invention.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (10)
1. A specific primer group for detecting mycobacterium tuberculosis and drug-resistant gene mutation IS characterized in that mycobacterium tuberculosis genes are IS6110, mpt64, HSP65, rpoB and gyrB, drug-resistant genes are rpoB, katG, inhA, oxyR-ahpC, embB, embA, pncA, rpsL, rrs, gidB, gyrA, gyrB, eis, rv0678, atpE, rrl, rplC, folC, thyA and alr, and specific sequences of the specific primer group are shown in SEQ ID NO. 1-SEQ ID NO. 56.
2. A reagent for detecting mycobacterium tuberculosis and drug-resistant gene mutation, wherein the reagent comprises the specific primer set according to claim 1.
3. The reagent of claim 2, wherein the reagent is a kit.
4. A method for constructing a sequencing library of mycobacterium tuberculosis and drug-resistance genes, characterized in that the method is carried out by using the kit of claim 3.
5. The method according to claim 4, wherein the identification of Mycobacterium tuberculosis and the detection of the drug-resistant gene mutation are performed in one primer pool.
6. The method according to claim 5, wherein the concentration of the different primers in the specific primer set in the primer pool is different, and the concentrations of each primer are as shown in the following table:
7. the construction method according to claim 6, wherein the construction method is specifically carried out by multiplex PCR amplification of DNA fragments using the specific primer set, followed by end repair, adaptor ligation and library amplification of the amplified products.
8. The method according to claim 7, wherein the multiplex PCR amplification reaction system is: 10. Mu.L of Input DNA, 15. Mu.L of 2X Multiplex PCR Master Mix (BioTechnRabbit), 3. Mu.L of primer mix, 2. Mu.L of nuclease-free water, and 30. Mu.L in total.
9. A mycobacterium tuberculosis and drug-resistant gene sequencing library constructed using the construction method of any one of claims 4 to 8.
10. Use of a specific primer set according to claim 1, a reagent according to any one of claims 2 to 3 and/or a library construction method according to any one of claims 4 to 8 in detection of mutations in a drug-resistant gene of mycobacterium tuberculosis.
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| CN118086546B (en) * | 2024-04-22 | 2024-07-12 | 云南科灿生物科技有限公司 | Kit for detecting mycobacterium tuberculosis bedaquiline drug-resistant gene mutation and detection method |
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