Mycobacterium drug resistance gene detection primer group, kit and application

Abstract

The invention discloses a mycobacterium drug resistance gene detection primer group, a kit and application, the primer group is specifically designed based on 26 drug resistance genes of first-line and second-line 26 drugs of mycobacterium, drug resistance gene detection can be specifically carried out, the coverage is wide, the sensitivity is high, the specificity is strong, and the primer group and the kit have great significance in guiding clinical medication of mycobacterium infection and can be used for detecting the drug resistance genes of the mycobacterium first-line and second-line 26 drugs of the mycobacterium first-line and second-line 26 drugs of the mycobacterium first-line and second-line 26 drugs of the mycobacterium first-line and second-line 26 drugs. The tuberculosis can be effectively controlled.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pathogen drug resistance detection, in particular to a mycobacterium drug resistance gene detection primer set, a kit and an application. BACKGROUND

[0002] Mycobacterium is a family of 5 genera and more than 200 species of mycobacteria, many of which cause serious diseases in mammals, such as Mycobacterium tuberculosis, which causes a chronic infectious disease. Previously, tuberculosis was the leading cause of death from a single infectious disease in humans, and it is also the leading cause of death in HIV carriers and the main cause of death related to antibiotic resistance.

[0003] Drug-resistant tuberculosis is of many types, of which multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are the two most critical causes of high mortality from tuberculosis. At present, the main mechanisms of drug resistance of Mycobacterium tuberculosis are as follows: (1) changes in cell wall structure and composition; (2) drug efflux pump system; (3) mutation of genes encoding drug targets or enzymes related to drug activity. Clinically, drugs for treating mycobacterial infections generally have first-line drugs and second-line drugs, and two or more drugs are used for combined treatment. Since this is a long-term drug use process, it is extremely easy to develop drug resistance. As can be seen from the above, accurate detection of drug resistance of mycobacteria plays a very important role in guiding clinical correct drug use and effective control of tuberculosis.

[0004] At present, the methods for detecting drug resistance of mycobacteria in clinical practice include smear method, traditional culture method, drug sensitivity test, fluorescent PCR technology, GeneXpert MTB / RIF (Xpert) technology, etc. The drug sensitivity test takes a long time, from sputum sample collection, culture to drug sensitivity test result report, which takes 2.5 months, and cannot meet the timely and accurate diagnosis and treatment of drug-resistant tuberculosis; the fluorescent PCR technology has limited detection range, and can only detect a few mycobacteria and known drug resistance genes and mutation sites, affecting the accuracy of detection; the current molecular technology may not cover all drug resistance genes and mutation types due to low throughput, and may produce false negatives, some mutations such as synonymous mutations (amino acid does not change) and silent mutations (do not affect the expression of encoded proteins) do not cause phenotypic drug resistance, and if detection techniques that do not understand the nature of gene mutations are used, false positives may occur.

[0005] With the rise of next generation sequencing (NGS) technology in recent years, it uses high-throughput sequencing technology, which not only reduces cost, speeds up sequencing, but also maintains high accuracy. Targeted NGS (tNGS) can simultaneously detect multiple target genes by capturing specific genes through targeted capture and then high-throughput sequencing. However, the platform of second-generation sequencing has high cost, expensive reagents, long sequencing time, short read length, and difficulty in covering comprehensive genes, and requires professional technicians 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 treatment. Nanopore sequencing is a new generation of single-molecule real-time electrical signal sequencing technology based on nanopores. It can directly monitor the current change of nucleic acid passing through the protein nanopore in real time, decode these current signals to determine the base sequence, and analyze the sequencing results at the same time, greatly shortening the sequencing time. Moreover, nanopore sequencing has long read length, which can detect drug-resistant genes without splicing, cover the full-length drug-resistant genes, and accurately identify SNP sites. SUMMARY

[0006] The application provides a drug-resistant gene detection primer set, which realizes high sensitivity, high specificity, accurate and rapid detection, and is suitable for drug-resistant gene detection, thereby providing clinical guidance for auxiliary medication.

[0007] Therefore, the application provides the following solutions. In a first aspect, the application provides a mycobacterium drug-resistant gene detection primer set, which is specifically designed for 26 drug-resistant genes of first-line and second-line 26 drugs. The drug-resistant genes are as follows:

[0008] Specifically, the drug-resistant gene detection primer set comprises an amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 1-52, and the correspondence between specific drug-resistant genes and primer pairs is shown in Table 1.

[0009] In a second aspect, the application provides a mycobacterium drug-resistant gene detection kit, which comprises the primer set composition of the first aspect.

[0010] Further, a public sequence is added to the 5' end of each primer in the primer set.

[0011] Preferably, the nucleotide sequence of the public sequence is as shown in SEQ ID NO: 53.

[0012] Further, the concentration of each primer in the primer set is 5nM-100nM, preferably 50nM.

[0013] Further, the kit further comprises at least one of multiplex PCR reaction reagent, barcode ligation PCR reaction reagent and nanopore library construction reagent.

[0014] Further, the kit further comprises one or more of nucleic acid extraction reagent, positive quality control and negative quality control.

[0015] Further, the nucleic acid extraction reagent is used for nucleic acid extraction or purification of a sample, wherein the sample is derived from sputum, alveolar lavage fluid or whole blood.

[0016] The third aspect of the present application is to use the primer set of the first aspect or the kit of the second aspect in any of the following: (a) detecting or assisting in detecting mycobacterium drug resistance genes; (b) preparing a product for detecting or assisting in detecting mycobacterium drug resistance genes; (c) detecting or assisting in detecting bacteria containing mycobacterium drug resistance genes; (d) preparing a product for detecting or assisting in detecting bacteria containing mycobacterium drug resistance genes; Wherein, the applications of (a) and (c) are for non-disease diagnosis and treatment purposes.

[0017] The fourth aspect of the present application is to provide a method for preparing a drug resistance gene sequencing fragment, comprising the step of using the primer set of the first aspect to perform PCR amplification on the nucleic acid of a sample.

[0018] The fifth aspect of the present application is to provide a method for detecting mycobacterium drug resistance genes, which is for non-diagnostic purposes, comprising the steps of: using the primer set of the first aspect to amplify the nucleic acid of a sample, then sequencing the amplicon, and comparing the sequencing result with a reference gene to obtain a detection result.

[0019] Further, the detection method comprises using the primer set to perform first-round PCR amplification on the nucleic acid of a sample, and performing second-round PCR amplification after barcode ligation; the first-round PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing process is based on nanopore sequencing method.

[0020] Compared with the prior art, the present application has the following beneficial effects: The drug resistance gene detection primer set provided by the present application is specifically designed based on 26 drug resistance genes of 26 first-line and second-line drugs of mycobacterium, can specifically detect drug resistance genes, has wide coverage, high sensitivity and strong specificity, has important significance for guiding clinical drug use of mycobacterium infection, and is convenient for effective control of tuberculosis.

[0021] The primer set described in the application can be combined with the long read characteristics of nanopore sequencing when detecting mycobacterium drug resistance genes, and the identification is more accurate; relative to ordinary PCR and qPCR, more targets are detected, the throughput is higher, the detection cost is lower, and the detection process is simple to operate, short in time, and convenient to detect. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The drug resistance gene primer amplification banding map described in Example 1 of the application. DETAILED DESCRIPTION

[0023] The technical solutions of the application will be described below in connection with preferred embodiments, obviously, the described embodiments are only a part of the embodiments of the application, not all the embodiments. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

[0024] Example 1

[0025] Design and verification of 26 drug resistance gene specific primers for first-line and second-line 26 drugs.

[0026] 1. Primer pool design: For the 26 drug resistance genes downloaded from the NCBI database, the sequence is extended by about 500 bp on both sides of the full-length sequence, primer 5.0 is used for primer design, the Tm value of the designed primer is uniform (60℃±5℃), the amplicon length is 200-4000 bp, the primer length is 15-25, and the GC content is 40-60%.

[0027] 2. Experimental verification: By establishing a pathogenic reference disc of tuberculosis drug resistance, all primers in the primer pool are amplified and detected, and the primers without amplification bands are removed, the primers are redesigned for supplementation and experimental verification.

[0028] Through a large number of repeated experimental tests, the primer pool in Table 1 is finally obtained, Figure 1 The corresponding drug resistance primer gel map. Experiments prove that there is no cross reaction between each other, the primer sequence interference competition is small, and the overall detection sensitivity and specificity are very good.

[0029] Specifically, the primer pool designed for 26 drug resistance genes is shown in Table 1.

[0030]

[0031] Example 2 Mycobacterium infection drug resistance gene detection primer combination

[0032] The drug resistance gene primer combination used in one round of reaction is composed of a segment sequence and a segment sequence from 5' end to 3' end of each sequence, wherein the segment sequence is a common sequence (SEQ ID NO: 53), and the segment sequence is the forward and reverse sequences of 26 pairs of drug resistance gene specific primers (SEQ ID NO: 1-52). The concentration is 5nM-100nM, preferably 50nM. The primer combination of drug resistance genes rpoB and katG is given as an example, as shown in Table 2.

[0033] Table 2:

[0034] In the primers described in Table 5, the underlined part is the common sequence, and the part without line is the specific primer sequence.

[0035] Example 3 Kit composition

[0036] The kit for detecting mycobacterial drug resistance comprises an amplification reaction solution, a primer pool, and a labeling reagent, and the kit components are shown in Table 3.

[0037] Table 3:

[0038] Example 4 Drug resistance gene detection method and process

[0039] (1) Sample pretreatment and nucleic acid extraction

[0040] Take 200 μl of the sample to be tested, and inactivate the clinical sample before extraction. Place at 60℃ for 30 min. Recommend using nucleic acid extraction or purification reagent (Benay Medical TQ006D-50, TQ007D-64). Refer to the corresponding instruction manual for specific extraction method.

[0041] (2) One round of multiple target-specific reaction

[0042] The extracted nucleic acid and positive and negative controls are subjected to multiple PCR amplification according to the following reaction system: Multiple amplification system A of the kit:

[0043] The reaction conditions of amplification system A are as follows: annealing temperature 60-65℃, preferably 65℃; annealing and extension time 1min-5min, preferably 3min:

[0044] (2) Magnetic bead purification of PCR product

[0045] 2.1 Magnetic bead vortex mixing, room temperature standing for 30 min.

[0046] 2.2 Take a new 200 μΐ eight-syringe tube, purify 20 μΐ PCR product system A from the previous step separately, add 20 μΐ water, then add 32 μΐ (0.8x) magnetic bead purification.

[0047] 2.3 Vortex mix, stand at room temperature for 5 min, centrifuge briefly, place the sample tube on the magnetic stand for 2 min, and carefully remove the supernatant after the solution is completely clarified.

[0048] 2.4 Keep the sample tube in the magnetic stand at all times, add 200 μΐ of freshly prepared 80% ethanol to rinse the magnetic beads, stand at room temperature for 30-60 s, and carefully remove the supernatant.

[0049] 2.5 Repeat step 2.4 once, for a total of two rinses.

[0050] 2.6 Keep the sample in the magnetic stand at all times, and dry the magnetic beads at room temperature for about 2-3 min.

[0051] 2.7 Take the sample out of the magnetic stand, add 10 μΐ of elution buffer, mix thoroughly, stand at room temperature for 2-3 min, centrifuge briefly, place on the magnetic stand for 2 min, and carefully pipette 9 μΐ of the supernatant into a new centrifuge tube after the solution is clarified. If not used immediately, store at -20°C.

[0052] (3) Second round of tag ligation PCR reaction

[0053] Take the first round of reaction products and perform the tag ligation PCR reaction according to the following table, using different tags for different samples.

[0054] Second round of multiplex amplification system B of this kit:

[0055] Reaction conditions of amplification system B: After experimental testing, the annealing temperature is 60-65°C, preferably 65°C; the annealing and extension time is 1 min-5 min, preferably 3 min:

[0056] (4) Magnetic bead purification of PCR products

[0057] 4.1 Vortex mix the magnetic beads, and stand at room temperature for 30 min.

[0058] 4.2 Take a new 200 μΐ eight-syringe tube, purify 15 μΐ of PCR product system B from the previous step, add 25 μΐ water, then add 32 μΐ (0.8x) magnetic bead purification.

[0059] 4.3 Vortex to mix, let stand at room temperature for 5 min, centrifuge briefly, and place the sample tube on the magnetic stand for 2 min. Carefully remove the supernatant after the solution is completely clear.

[0060] 4.4 Keep the sample tube in the magnetic stand at all times. Add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, let stand at room temperature for 30-60 s, and carefully remove the supernatant.

[0061] 4.5 Repeat 2.4 once, for a total of two rinses.

[0062] 4.6 Keep the sample in the magnetic stand at all times. Open the lid and dry the magnetic beads at room temperature for about 2-3 min.

[0063] 4.7 Take the sample out of the magnetic stand, add 10 μl of elution buffer, mix well, let stand at room temperature for 2-3 min, centrifuge briefly, place on the magnetic stand for 2 min, and carefully pipette 9 μl of the supernatant into a new centrifuge tube after the solution is clear. If not used immediately, store at -20 °C.

[0064] 4.8 Take 1 μl of the purified product to detect the concentration using the Qubit dsDNA HS Assay Kit.

[0065] (5) Library construction and sequencing

[0066] After the products are pooled in equal amounts, the library is constructed according to the nanopore sequencing process. The present kit can be used in combination with different nanopore platforms for sequencing, including but not limited to the ONT sequencing platform of Oxford Nanopore, Pintbio, Huada, and Jisiteke, etc. all domestic nanopore sequencing platforms. The on-machine operation is performed according to the on-machine operation instruction of the nanopore sequencer.

[0067] (6) Bioinformatics analysis

[0068] The off-machine data is subjected to bioinformatics analysis, comparison with the drug resistance database, and analysis of the detection results.

[0069] Example 5 Sample drug resistance gene detection verification

[0070] The clinical positive samples are subjected to drug resistance detection using the above method. The 26 drug resistance genes are effectively amplified and drug resistance gene reads are obtained according to the detection results of the clinical samples, indicating that all drug resistance genes can be amplified by the corresponding primers and effective sequences are obtained. The drug resistance gene mutation site information is detected by using the drug resistance site bioinformatics analysis process, so as to obtain the drug resistance results. The specific drug resistance detection results of the clinical samples are shown in Table 4. The results of different detection methods are compared, and the consistency of the results of the present application and the clinical drug sensitivity experiment is 100%, indicating that the present method can effectively detect the drug resistance of Mycobacterium tuberculosis.

[0071] Table 4: Results of first-line resistance detection

[0072] While embodiments of the application have been shown and described, it is to be understood that the application is not limited to the details of the embodiments described, since the scope of the application is defined with respect to the appended claims.

Claims

1. A primer set for detecting a drug resistance gene of Mycobacterium, characterized by, The amplification primer pair comprises nucleotide sequences as shown in SEQ ID NO: 1-52.

2. A mycobacterium drug resistance gene detection kit, characterized by, The primer composition of claim 1.

3. The test kit according to claim 2, characterized in that, A common sequence is added to the 5' end of each primer in the primer set.

4. The test kit according to claim 3, characterized in that, The nucleotide sequence of the common sequence is as shown in SEQ ID NO:

53.

5. The test kit according to claim 2, characterized in that, The kit further comprises at least one of multiplex PCR reaction reagent, barcode ligation PCR reaction reagent and nanopore library construction reagent.

6. The test kit according to claim 2, characterized in that, The kit further comprises at least one of nucleic acid extraction reagent, positive quality control and negative quality control.

7. The primer set of claim 1 or the kit of any one of claims 2-6 is used in any one of the following: (a) detecting or assisting in detecting mycobacterium drug resistance genes; (b) preparing a product for detecting or assisting in detecting mycobacterium drug resistance genes; (c) detecting or assisting in detecting bacteria containing mycobacterium drug resistance genes; (d) preparing a product for detecting or assisting in detecting bacteria containing mycobacterium drug resistance genes; wherein, The applications of (a) and (c) are for non-disease diagnosis and treatment purposes.

8. A method for preparing a drug resistance gene sequencing fragment, characterized by, The method comprises the step of performing PCR amplification on sample nucleic acid using the primer set of claim 1.

9. A method for detecting mycobacterial drug resistance genes for non-diagnostic purposes, characterized by the steps of The method comprises: The method comprises the steps of amplifying sample nucleic acid using the primer set of claim 1, then sequencing the amplicon, and comparing the sequencing result with a reference gene to obtain a detection result.

10. The method of claim 9, wherein, The detection method comprises performing first round PCR amplification on sample nucleic acid using the primer set, and performing second round PCR amplification after barcode ligation; the first round PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing process is based on nanopore sequencing method.

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