Mycobacterium tuberculosis rifampicin drug resistance gene detection method and kit

Abstract

The invention relates to a method and a kit for detecting a rifampicin drug resistance gene of mycobacterium tuberculosis. The kit comprises polynucleotide primers as shown in SEQ ID NO.1-SEQ ID NO.7. The detection method comprises the following steps: step 1, collecting a sample: collecting a sputum sample of a patient as a test sample; step 2, extracting DNA: extracting DNA of a test sample by using a kit method or a lysis method; 3, preparing a reaction system, and carrying out reaction amplification, namely preparing an amplification reaction system under the guidance of the special primer for the kit by taking the sample DNA in the step 2 as a template, and carrying out specific amplification; step 4, result analysis: in the specific amplification process, due to the difference of amplification target sequences, the basic group contents of amplification products are different; the change of a single basic group can cause the change of the melting temperature of amplified target double chains; the method comprises the following steps: monitoring the change of a fluorescence signal in a temperature rise process in real time, performing data integration and image drawing on a detection result to generate a target product melting curve, and judging the difference of a gene sequence in a target product according to the difference of the melting curve.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection technology, and relates to a method for determining whether a mutation exists in the 81bp "drug resistance determining region" of the rpoB gene of the Mycobacterium tuberculosis complex, and particularly relates to a method and a kit for determining whether a mutation exists in the 81bp "drug resistance determining region" of the rpoB gene of the Mycobacterium tuberculosis complex. Background Art

[0002] Resistance of Mycobacterium tuberculosis complex (MTBC) to anti-TB drugs, particularly resistance to rifampicin (RIF), has become a major challenge to global public health. Drug-resistant TB (including multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB)) complicates treatment options, increases costs, and increases the difficulty of disease transmission and control. In 2022, the total number of TB patients worldwide reached 10.6 million, of which approximately 410,000 were multidrug-resistant or rifampicin-resistant, and only 40% of patients received treatment. In China, there are approximately 30,000 MDR / RR-TB patients, accounting for 7.1% of the global caseload, with a treatment success rate of approximately 63%.

[0003] Rifampicin is a core first-line drug for tuberculosis treatment. Its mechanism of action is to inhibit the β subunit of the DNA-dependent RNA polymerase encoded by the rpoB gene. Approximately 96% of rifampicin-resistant strains harbor mutations within the 81-bp resistance-determining region (RRDR) of the rpoB gene, with common mutations at positions His445, Ser450, and Asp435. Furthermore, mutations at positions outside the RRDR, such as Ile491, may also confer low-level drug resistance, but conventional drug susceptibility testing (AST) is less sensitive for these mutations.

[0004] Research on detecting drug resistance in Mycobacterium tuberculosis (MTB) has made significant progress in recent years, particularly in molecular diagnostic techniques. Traditional drug susceptibility testing (AST) is time-consuming, limiting the early diagnosis of drug-resistant TB. To improve detection efficiency and accuracy, researchers have developed a variety of molecular biological methods. Xpert MTB / RIF is an automated nucleic acid amplification-based detection platform that can detect MTBC and its rifampicin resistance within 2 hours and has been recommended by the World Health Organization as a rapid screening tool. Furthermore, INNO-LiPA Rif.TB accurately detects rpoB gene mutations through line probe hybridization. Gene chip technology relies on simultaneous multi-site detection, enabling parallel analysis of multiple resistance gene mutations, significantly improving detection throughput and efficiency. Furthermore, melting curve analysis (MCA), which monitors changes in fluorescence signal as double-stranded DNA melts during heating to identify DNA sequence differences, has been used to detect MTB drug resistance.

[0005] High-resolution melting (HRM) technology offers advantages such as ease of operation, low cost, the absence of probes, and a closed system to reduce contamination. It is suitable for large-scale screening and application in resource-limited areas. In MTB drug resistance testing, HRM has been used to detect mutations associated with drugs such as rifampicin (rpoB gene), isoniazid (katG and inhA genes), and fluoroquinolones (gyrA and gyrB genes). Studies have shown that HRM has high sensitivity and specificity for detecting rifampicin and isoniazid resistance in Mycobacterium tuberculosis, reaching 94% and 97%, respectively. In this project, we introduced an ultra-long non-fluorescently labeled probe that can rapidly detect mutations within the 81bp "drug resistance-determining region" (RRDR) of the rpoB gene of the Mycobacterium tuberculosis complex. This probe has broad application prospects and is expected to become a supporting technology for the clinical detection of rifampicin-resistant Mycobacterium tuberculosis. Summary of the Invention

[0006] The present invention provides a method for detecting whether there is a mutation in the 81bp "drug resistance determining region" in the rpoB gene of Mycobacterium tuberculosis complex.

[0007] The method of the present invention includes detecting the rpoB gene sequence of the Mycobacterium tuberculosis complex. Preferably, the present invention selects the IS6110 and IS1081 sequences in the gene as targets for identification and confirmation of the Mycobacterium tuberculosis complex.

[0008] The present invention further provides a reagent used in the detection method and a kit containing the reagent. The kit can be used as a practical product to detect whether there is a mutation in the 81bp "drug resistance determining region" in the rpoB gene of Mycobacterium tuberculosis complex.

[0009] The present invention further provides special primers designed to directly amplify the key drug-resistant rpoB gene of the Mycobacterium tuberculosis complex from a sample using an unlabeled probe combined with HRM technology, determine whether there is a mutation within the 81bp "resistance-determining region" of the rpoB gene, and predict its resistance to the anti-tuberculosis drug rifampicin. The special primers can be used as the core reagent component of the kit described in the present invention.

[0010] The special primers described in the present invention include 3 sets of primers:

[0011] The first primer set consists of a forward primer, a reverse primer, and a 3'-end phosphorylated non-fluorescent probe; the sequences are SEQ ID NO.1-SEQ ID NO.3.

[0012] The second and third sets of primers include a forward primer and a reverse primer respectively:

[0013] Among them, the second set of primers targets the IS6110 target, and the sequence is SEQ ID NO.4-SEQ ID NO.5.

[0014] Among them, the third set of primers targets IS1081 target, and the sequence is SEQ ID NO.6-SEQ ID NO.7.

[0015] The corresponding relationship is shown in Table 1

[0016] Table 1, SEQ ID NO.1-SEQ ID NO.7 are listed as follows:

[0017]

[0018] Note: *3' end phosphorylation

[0019] The detection kit of the present invention comprises any one of the primers SEQ ID NO.1 to SEQ ID NO.7 of the present invention.

[0020] The detection kit provided by the present invention is for detecting whether there is a mutation in the 81bp "drug resistance determining region" of the rpoB gene of the Mycobacterium tuberculosis complex, which contains special primers for detecting the rpoB gene of the Mycobacterium tuberculosis complex and the IS6110 / IS1081 target. The kit also includes a sampling tube, a crude extraction reagent Lysis buffer, a reaction component EvaGreen MasterMix (amplification enzyme, amplification buffer, dNTP and EvaGreen fluorescent dye), a positive control and a negative control. The positive control is a wild-type and mutant positive sample of each detection target, and the negative control is ddH2O. The preparation method of all the above reagents is conventional technology, and it is only necessary to mix the various raw materials evenly at room temperature, without the need for special equipment and conditions. The amount of the detection kit or article included in the present invention is one person's portion, or multiple people's portions, and the multiple people's portions can be 2-1000 people's portions.

[0021] The present invention further provides a kit for detecting whether there is a mutation in the 81 bp "drug resistance determining region" of the rpoB gene of Mycobacterium tuberculosis complex, comprising the steps of using the kit of the present invention.

[0022] like

[0023] Step 1, collect samples:

[0024] Collect sputum samples from patients as test samples.

[0025] Step 2, DNA extraction:

[0026] Extract the test sample DNA using a kit method or lysis method.

[0027] Step 3: Configure the reaction system and perform reaction amplification:

[0028] Using the sample DNA in step 2 as a template, prepare an amplification reaction system under the guidance of the special primers in the above kit to perform specific amplification.

[0029] Step 4, result analysis:

[0030] During specific amplification, differences in the target sequence can lead to differences in the base content of the amplified product. A single base change can cause a shift in the melting temperature of the target double strand. By monitoring the changes in the fluorescence signal during the heating process in real time, the test results are integrated and plotted to generate a melting curve for the target product. The differences in the melting curves can be used to determine differences in the gene sequence within the target product.

[0031] Preferably, the detection method of the present invention comprises the following steps:

[0032] Step 1, collect samples:

[0033] Use a dedicated sputum collection tube to collect respiratory specimens from patients.

[0034] Step 2, DNA extraction:

[0035] Extract DNA from the test sample according to the QIAamp Viral DNA Mini kit operating instructions. (The above steps can be completed using other nucleic acid extraction kits or methods to complete DNA extraction from the test sample.)

[0036] In step 3, prepare the reaction system, perform amplification, and perform HRM analysis using the QuantStudio 6Flex Real-Time PCR System. See Tables 2 and 3 for the reaction system and conditions.

[0037] Step 4, data analysis:

[0038] After the reaction, data analysis was performed using QuantStudio 6and 7Flex Real-Time PCR software v1.0. The fluorescence detection software automatically generated melting curves and Tm values ​​for each target amplicon. Comparison with the wild-type control determined the presence of mutations within the 81-bp "resistance-determining region" of the rpoB gene. If the test sample does not have a mutation within the 81-bp resistance-determining region of the rpoB gene, the Tm value of the melting curve will not differ significantly from that of the wild-type control. However, if a mutation is present within the 81-bp region of the rpoB gene critical for drug resistance, the melting curve shape will change accordingly.

[0039] Table 2, reaction system configuration table:

[0040] Element Add Volume EvaGreen 10μL (1μM) Primer SEQ ID NO.1 0.375μL (1μM) Primer SEQ ID NO.2 0.75μL (10μM) Primer SEQ ID NO.3 1μL (10μM) Primer SEQ ID NO.4 0.4μL (1μM) Primer SEQ ID NO.5 0.4μL (1μM) Primer SEQ ID NO.6 0.5μL (1μM) Primer SEQ ID NO.7 0.5μL (1μM) DNA template 2μL Nuclease-free water 4.075μL Total volume 20 μL

[0041] Table 3, reaction conditions flow chart:

[0042]

[0043]

[0044] The special primer set of the present invention is an optimal primer pair that can accurately distinguish whether there is a mutation in the 81bp "drug resistance determining region" of the rpoB gene of the Mycobacterium tuberculosis complex.

[0045] The kit provided by the present invention is used to detect whether there is a mutation in the drug resistance-related region of the rpoB gene of Mycobacterium tuberculosis complex, and the rpoB gene related to rifampicin (first-line recommended drug) resistance is selected as the target gene for detection. First, the rpoB gene is obtained from the GenBank database ( https: / / www.ncbi.nlm.nih.gov / genbank / ) were used to download the fully annotated gene sequences of representative strains of the Mycobacterium tuberculosis complex as reference sequences. A nucleotide sequence BLAST analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) was performed against the NCBI nr database, and the alignment results were downloaded to obtain more target gene sequences. All downloaded sequences were subjected to multiple alignment analysis. Specific amplification primers were designed for rpoB, IS6110, and IS1081 targets using Beacon Designer 8.0 software. Primer specificity was verified using the NCBI online primer tool (https: / / www.ncbi.nlm.nih.gov / tools / primerblast / ). The melting temperatures of the amplified products were predicted using oligocalc (http: / / biotools.nubic.norwestern.edu / oligocalc.hTml) and UMELT online software (https: / / www.DNA.utah.edu / umelt / umelt.hTml). Specific primers can amplify the 81bp "resistance-determining region" of the rpoB gene, forming a main product peak with a higher Tm value. In addition, an ultra-long non-fluorescent labeled probe is designed for the 81bp "resistance-determining region" of the rpoB gene. The probe should perfectly match the 81bp "resistance-determining region" of the rpoB gene, and the 3' end of the probe is phosphorylated to prevent the extension of the probe. Four sets of primer pairs are designed on both sides of the rpoB, IS6110 and IS1081 targets, and the best primer pair that can accurately distinguish between wild type and mutant types is screened. Four non-fluorescent labeled probes of different lengths are designed for testing, and the probe with the largest difference in Tm for different mutant types is selected. Finally, the best primer pair and probe that can accurately distinguish between wild type and multiple mutant types are selected to form the final multiple melting curve analysis system.

[0046] The method of the present invention can rapidly detect the presence of mutations within the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex. Compared with other existing and similar technologies for detecting mutations within the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex, the technical solution of the present invention has the following advantages:

[0047] Compared to traditional PCR or other molecular detection techniques, the melting curve technology of this invention monitors the changes in the melting curve in real time. The software automatically generates melting curves and Tm values ​​based on different target amplicons. Results are obtained without the need for traditional post-processing procedures (such as electrophoresis). HRM technology is a high-throughput gene screening technology that analyzes PCR products by monitoring the changes in the melting curve in real time. It is not limited by the location and type of mutations in the detection target and does not require the synthesis of expensive sequence-specific probes, greatly reducing detection costs. After the reaction is completed, the experimental results can be quickly and sensitively analyzed directly through the high-resolution melting curve to obtain mutation information in the drug resistance-related regions. Secondly, the present invention uses the saturated dye EvaGreen in the melting curve analysis experiment. Since the saturated dye EvaGreen does not inhibit the PCR reaction, it can be directly added to the PCR reaction system before the reaction starts to participate in the amplification process. After the PCR reaction is completed, there is no need to open the reaction tube to add the dye to directly perform the melting curve analysis, which truly realizes the closed tube operation, avoids the contamination that may be introduced by opening the lid, resulting in false positive results, and improves the accuracy and reliability of the experimental results; finally, the temperature increase and decrease during the analysis process will not cause destructive damage to the DNA structure, and the subsequent cooling can renature the DNA. The renatured DNA can be directly used for subsequent research (such as sequencing verification results), which greatly saves time and manpower and material resources and avoids unnecessary waste.

[0048] Furthermore, this method innovatively combines a non-fluorescent labeled probe with HRM technology. Using only an ultra-long non-fluorescent labeled probe and a pair of amplification primers (the first primer set), it can detect mutations within the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex, covering all important mutational regions associated with rifampicin resistance in the Mycobacterium tuberculosis complex. Because the probe sequence completely matches that of the wild-type rpoB gene, the probe peak exhibits the highest Tm value, enabling rapid identification of the wild-type rpoB gene, the most common type found in rifampicin-resistant Mycobacterium tuberculosis complex. The non-fluorescent labeled probe is 85bp long, and to our knowledge, no probe of this length has yet been optimized in the industry. This method has high sensitivity (100 copies / reaction) and strong specificity, allowing direct application to clinical samples. It is an important technical supplement for detecting mutations within the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex. Furthermore, because the probe does not require fluorescent labeling, the cost is significantly lower than conventional fluorescence quantitative PCR. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the results of detecting whether there is a mutation in the 81bp "resistance-determining region" of the rpoB gene of Mycobacterium tuberculosis complex DETAILED DESCRIPTION

[0050] Examples Based on the present invention, the following detailed descriptions of implementation methods and specific operating procedures are provided. The following specific examples further illustrate the present invention but are not intended to limit the scope of the present invention. The following describes the implementation methods and specific operating procedures for clinical testing of Mycobacterium tuberculosis complex for mutations within the 81bp "resistance-determining region" of the rpoB gene.

[0051] Example 1: Method of using the kit

[0052] 1) Use the sample collection tube provided in the kit to collect the patient's sputum sample.

[0053] 2) Extract genomic DNA from the sample according to the QIAamp Viral DNA Mini kit instructions. (The above steps can be performed using other nucleic acid extraction kits or methods.)

[0054] 3) Using the sample genomic DNA obtained in the above steps as a template, a dedicated primer set was used to detect mutations within the 81-bp resistance-determining region of the rpoB gene of the Mycobacterium tuberculosis complex. A 20 μL reaction system consisted of: 10 μL of EvaGreen Master Mix, the optimal amplification concentrations of each primer in the reaction system (Table 2), 2 μL of sample genomic DNA, and ddH2O to a total of 20 μL. Both positive and negative control reaction tubes were added to each test reaction.

[0055] 4) Amplification and HRM analysis were performed using the QuantStudio 6Flex Real-Time PCR System. Amplification reaction conditions were as follows: incubation at 95°C for 10 minutes, followed by annealing at 95°C for 15 seconds and extension at 60°C for 1 minute, for a total of 30 cycles. After completion of the amplification reaction, the sample was incubated at 40°C for 1 minute, followed by a slow ramp at 0.025°C / s to 95°C, while fluorescence signals were continuously collected.

[0056] 5) After the reaction, data analysis was performed using QuantStudio 6and 7Flex Real-Time PCR software v1.0. The fluorescence detection software automatically generated melting curves and Tm values ​​for each target amplicon. By comparing the results with those of the wild-type control, the presence of mutations within the 81-bp "resistance-determining region" of the rpoB gene was determined. If the test sample does not have a mutation within the 81-bp "resistance-determining region" of the rpoB gene, the melting curve will remain unchanged compared to the wild-type control. If a mutation is present within the 81-bp region of the rpoB gene critical for drug resistance, the melting curve will change accordingly.

[0057] Example 2: The superiority of the method and kit of the present invention is further demonstrated by the following experimental data:

[0058] 1. Accuracy of the method

[0059] In this study, we collected 40 pure culture samples from respiratory clinics for double-blind testing. All samples were sequenced to determine whether mutations were present within the 81-bp resistance-determining region of the rpoB gene. This method was then compared with ours. The results showed a 95% concordance between ours and the sequencing method, further demonstrating the specificity of our approach.

[0060] 2. Sensitivity of the method

[0061] To accurately assess the sensitivity of the method, we conducted a series of experiments using wild-type and mutant plasmids to prepare DNA from various concentrations of Mycobacterium tuberculosis complex strains, creating a gradient of simulated infection samples. Through systematic, reproducible testing (n > 10) of these samples at varying concentrations, we confirmed that the method can achieve a sensitivity of 100 genome copies per reaction. This level of sensitivity fully meets the requirements of clinical application and ensures effective detection even in low bacterial loads.

[0062] 3. Anti-cross-reactivity ability of the method

[0063] To evaluate the cross-infection resistance of the present method in clinical applications, 17 mycobacterial species were used to assess cross-reactivity. A total of 17 samples were collected: Mycobacterium kansasii (n=1), Mycobacterium intracellulare (n=1), Mycobacterium chelonae (n=1), Mycobacterium fortuitum (n=1), Mycobacterium gordonii (n=1), Mycobacterium marinum (n=1), Mycobacterium smegmatis (n=1), Mycobacterium avium (n=1), Mycobacterium scrofulae (n=1), Mycobacterium toadense (n=1), Mycobacterium abscessus (n=1), Mycobacterium malmoe (n=1), Mycobacterium flavescens (n=1), Mycobacterium thermotolerans (n=1), Mycobacterium ulcerans (n=1), Mycobacterium chelonae (n=1), and Mycobacterium occulta (n=1). The results demonstrated that the present method exhibited no cross-reactivity with other mycobacteria, demonstrated high specificity, and was suitable for the detection of Mycobacterium tuberculosis complex infections.

[0064] Example 3: Advantages of Ultra-Long Unlabeled Probes

[0065] In order to evaluate the performance of the probe of the present invention, we designed a series of experiments to test a series of probes of different lengths covering the 81bp "resistance-determining region" of the rpoB gene for performance testing, with lengths of 81bp, 83bp, 85bp, 90bp, 100bp, 110bp, and 115bp, respectively. Through the tests of cross-infection and accuracy, the 85bp ultra-long probe effectively detects whether there is a mutation in the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex while minimizing the incidence of false positives. Through systematic evaluation of accuracy and cross-reaction, the experiment verified that even in the presence of complex common clinical pathogens, the probe of this method can still effectively detect whether there is a mutation in the 81bp "resistance-determining region" of the rpoB gene of the Mycobacterium tuberculosis complex, without being significantly interfered with by other pathogens. This result not only proves the high accuracy of the method of the present invention, but also demonstrates its reliability and robustness in clinical settings.

Claims

1. A kit for detecting whether there is a mutation in the 81bp resistance-determining region of the rpoB gene of Mycobacterium tuberculosis complex, characterized in that: The kit contains a detection reagent that can accurately distinguish whether there is a mutation in the 81bp drug resistance determining region of the rpoB gene.

2. The kit according to claim 1, wherein The detection reagent includes primer sequences of SEQ ID NO.1 to SEQ ID NO.

7.

3. The kit according to claim 2, wherein The primers detect whether there is a mutation in the 81bp drug resistance determining region of the rpoB gene.

4. The kit according to claim 3, wherein The primers include three sets of primers. The first set of primers consists of a forward primer, a reverse primer, and a 3'-end phosphorylated non-fluorescent labeled probe. The second and third sets of primers respectively include a forward primer and a reverse primer. The second and third sets of primers are respectively targeted at IS6110 and IS1081 targets.

5. The method for using the kit according to claim 1, wherein: The following steps are involved: Step 1, collect samples: Collect sputum samples from patients as test samples; Step 2, DNA extraction: Extract the test sample DNA using a kit method or lysis method; Step 3: Configure the reaction system and perform reaction amplification: Using the sample DNA from step 2 as a template, prepare an amplification reaction system under the guidance of the dedicated primers in the above kit to perform specific amplification; Step 4, result analysis: During the specific amplification process, due to the differences in the amplified target sequences, there are differences in the base content of the amplified products. The change of a single base can cause a change in the melting temperature of the amplified target double strand. By real-time monitoring of the changes in the fluorescent signal during the heating process, the detection results are integrated and imaged to generate the target product melting curve. The differences in the gene sequences in the target products are judged based on the differences in the melting curves.

6. The method of use according to claim 5, characterized in that: The following steps are involved: Step 1, collect samples: Use a dedicated sputum collection tube to collect respiratory specimens from patients. Step 2, DNA extraction: Use the kit method or lysis method to extract the test sample DNA, Step 3: Configure the reaction system, perform amplification, and perform amplification and melting curve analysis on the QuantStudio 6Flex Real-Time PCR System. The reaction system and reaction conditions are as follows: Reaction conditions flow: Step 4, data analysis: After the reaction, data analysis was performed using QuantStudio 6and 7Flex Real-Time PCR software v1.

0. The fluorescence detection software automatically generated melting curves and Tm values ​​for each target amplicon. These values ​​were compared with those of the wild-type control to determine whether a mutation existed within the 81-bp resistance-determining region of the rpoB gene. If the test sample did not harbor a mutation within the 81-bp resistance-determining region of the rpoB gene, the Tm value of the melting curve would be similar to that of the wild-type control. However, if a mutation existed within the 81-bp region of the rpoB gene critical for drug resistance, the shape of the melting curve would change accordingly.

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