CN116287328A

CN116287328A - Mycobacterium tuberculosis drug-resistant mutation detection method and kit

Info

Publication number: CN116287328A
Application number: CN202211542219.5A
Authority: CN
Inventors: 徐高连; 晏梦秋; 徐宏; 古宏晨
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2022-12-02
Filing date: 2022-12-02
Publication date: 2023-06-23

Abstract

The application relates to a technology for detecting drug-resistant mutation of mycobacterium tuberculosis, in particular to a method and a kit for detecting drug-resistant mutation of mycobacterium tuberculosis. The method for detecting the tuberculosis drug resistance molecules is simple to establish and operate, high in sensitivity, rapid in detection and low in cost, and the detection cost is reduced by adopting a mode of one-step detection and whole-course tube closing and simultaneously detecting multiple mutations by utilizing a common PCR detection platform. The sensitivity equivalent to the phenotype drug sensitivity method is realized, which is higher than that of the existing molecular drug sensitivity method, and 0.1% drug resistance mutation can be detected, so that the defect of insufficient sensitivity of the existing molecular drug sensitivity method is overcome, and the missed diagnosis rate of tuberculosis drug resistance is reduced.

Description

Mycobacterium tuberculosis drug-resistant mutation detection method and kit

Technical Field

The application relates to a technology for detecting drug-resistant mutation of mycobacterium tuberculosis, in particular to a method and a kit for detecting drug-resistant mutation of mycobacterium tuberculosis.

Background

Tuberculosis is the disease with highest mortality rate of global infectious diseases, and seriously endangers global public health. According to the world health organization estimated, about 20 million people worldwide are infected with mycobacterium tuberculosis, and about 200 ten thousand people die annually from tuberculosis. More seriously, the global drug-resistant tuberculosis epidemic situation is more serious in recent years, and the treatment difficulty of the drug-resistant tuberculosis is greatly increased, thus forming a serious challenge for the prevention and treatment of the tuberculosis. China is one of the world high-burden countries of tuberculosis, the number of tuberculosis patients is inferior to India, and the drug resistance is serious. Therefore, the diagnosis of tuberculosis and the detection of drug resistance are timely and accurately carried out, and the method has great significance for effective treatment and effective control of the transmission of tuberculosis.

The traditional tuberculosis drug resistance detection method is a phenotypic drug sensitivity method, and whether drug resistance is generated to a certain antitubercular drug is judged by observing the growth state of mycobacterium tuberculosis on a drug-containing and drug-free solid or liquid culture medium. The phenotypic drug-sensitive method is a gold standard for detecting the drug resistance of the tuberculosis at present, can detect the drug resistance of the primary first-line and second-line anti-tuberculosis drugs at present, has the sensitivity of 1 percent, is the most sensitive method for detecting the drug resistance of the tuberculosis at present, is also the gold standard for detecting the drug resistance of the tuberculosis, but has high requirements on the biological safety level of a laboratory because the growth speed of the mycobacterium tuberculosis is slow, the detection period of the phenotypic drug-sensitive method usually needs 6 to 8 weeks, which leads to the incapability of reasonably treating the drug-resistant tuberculosis in time and the wide spread of the drug-resistant tuberculosis, and the detection process is to culture living bacteria. In recent years, the world health organization recommends the use of molecular drug sensitivity for the detection of drug resistance in Mycobacterium tuberculosis. The molecular drug sensitivity method is to detect the gene which causes the antituberculosis drug to generate drug resistance by using a molecular amplification technology to judge whether the drug resistance is generated, and the method has the greatest characteristics of greatly shortening the drug resistance detection time and obtaining the detection result in 2 hours at maximum. At present, more tuberculosis drug-resistant mutation detection technologies are applied, including gene sequencing, a linear probe method, a gene chip method, a real-time PCR based on molecular beacons and a melting curve method.

The method for detecting the drug resistance of the mycobacterium tuberculosis based on the gene sequencing technology is characterized in that whether mutation occurs or not is judged by analyzing the sequence of a mutation site related to the drug resistance in a mode of carrying out targeted sequencing or whole genome sequencing on a drug resistance gene, and the method is characterized in that the drug resistance information of various drugs can be obtained.

The world health organization recommended the use of GenoType MTBDRplus and GenoType mtbcrsl kits from Hain corporation, germany, to detect resistance to primary and secondary antitubercular drugs in 2008 and 2016, respectively (Nathavitharana, R.R.Cudahy, P.G.Schumacher, S.G.Steingart, K.R.Pai, M.Denkinger, C.M., accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis.European Respiratory Journal (2017)). The method adopts a linear probe hybridization paper membrane strip technology, wild type probes and mutant probes corresponding to drug-resistant mutation sites are fixed on a test strip, products obtained after PCR amplification are hybridized with the probes on the membrane strip, color development treatment is carried out after multiple elution, and color development conditions of the corresponding probes on the membrane strip are observed to judge drug resistance. Because of the large number of types of tuberculosis-resistant mutation codons that may be generated, it is often necessary to construct multiple mutation probes on the membrane for one mutation site. CFDA-approved tuberculosis drug resistance test kit based on gene chip, which is independently developed by Beijing boao corporation, adopts similar principle, hybridization and imaging of probes and amplicons are carried out on a DNA microarray chip, 13 mutations in the core region of the rifampicin drug resistance gene rpoB, and 4 mutations in the isoniazid drug resistance katG315 site and inhA promoter region can be detected (Zhang, Z.Li, L.Luo, F.Cheng, P.Wu, F.Wu, Z.Hou, T.Zhong, M.Xu, J., rapid and accurate detection of RMP-and INH-resistant Mycobacterium tuberculosis in spinal tuberculosis specimens by CapitalBio) ^TM DNA microarray:A prospective validation study.BMC infectious diseases12,1-7(2012).)。

The world health organization in 2010 recommended the use of Xpert MTB/RIF from Cepheid corporation in the united states for the primary diagnosis of tuberculosis and for the detection of the drug resistance of the first-line antitubercular drug rifampicin (down, s.d. nicol, m.p.,

MTB/RIF assay: development, evaluation and implementation of a new rapid molecular diagnostic for tuberculosis and rifampicin stability. Future microbiology 6,1067-1082 (2011). The method adopts asymmetric real-time PCR to amplify 81 bases of a rifampicin drug resistance determining region, adopts 5 molecular beacons complementarily paired with a wild type to hybridize with an amplicon, and judges whether rifampicin drug resistance occurs or not by delta Ct values of 5 molecular beacon amplification curves if the corresponding molecular beacons have base mutation or disappear relative to fluorescent signals generated by the molecular beacons which have no mutation. The method has the greatest characteristics of integrated detection, namely, nucleic acid extraction, PCR reaction and result processing are integrated into one detection device, the whole process takes about 2 hours, and the operation is simple.

The Xiamen-induced biology company has independently developed a kit for detecting tuberculosis resistance based on a melting curve analysis technique based on a double-labeled self-quenching probe, which determines whether resistance is generated by analyzing the difference of peak positions of melting curves of the probe after the end of a PCR reaction due to the difference of affinities of hybridization of the probe with wild-type and mutant amplicons (Hu, S.Li, G.Li, H.Liu, X.Niu, J.Quan, S.Wang, F.Wen, H.Xu, Y.Li, Q., rapid detection of isoniazid resistance in Mycobacterium tuberculosis isolates by use of real-time-PCR-based melting curve analysis. Journal of clinical microbiology, 1644-1652 (2014)). The method is characterized by closed tube reaction, does not need to uncap for subsequent analysis of PCR amplification products, reduces cross contamination between samples, and is simpler to operate.

Although the molecular drug sensitivity method can rapidly detect tuberculosis drug resistance, the sensitivity is inferior to that of the phenotype drug sensitivity method, the Xpert MTB/RIF can accurately detect the drug resistance only when the content of a mutant genome in a sample is more than 65%, the sensitivity of the linear molecular probe method is 5-10%, and the sensitivity of the melting curve method is 20-40%. In addition, the molecular drug sensitivity method has the problems of higher detection cost, complicated operation and high requirement on sample quality, and the gene sequencing technology can acquire more comprehensive drug resistance information but needs to rely on special equipment, so that the subsequent data processing workload is large, the requirement on operators is high, the overall detection cost is high, and the method is not clinically popularized and applied at present. The linear molecular probe method and the gene chip method based on probe hybridization need to carry out subsequent hybridization, elution, color development or imaging on PCR amplification products, have complicated operation and longer time consumption, are easy to cause cross contamination between samples due to aerosol, and increase the detection cost by respectively designing hybridization probes for different mutation types. Although Xpert MTB/RIF can realize integrated detection, the operation is simple, the instrument and consumable material cost is expensive, and only the drug resistance of rifampicin, which is a drug, can be detected, and the method is not widely applied in the developing countries with high tuberculosis burden at present. The technology based on melting curve analysis can adopt a closed tube detection mode to avoid cross contamination and simplify the operation process, but the judgment of the peak type of the melting curve is influenced by the quality of a sample, and the peak type is easy to be difficult to judge when the purity of the sample is low.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, an object of the present application is to provide a method and a kit for detecting drug-resistant mutations in mycobacterium tuberculosis, which can achieve low-abundance mutation detection with a sensitivity of 0.1% by using competition between a front primer or a rear primer or a probe and a blocking oligonucleotide.

To achieve the above and other related objects, a first aspect of the present application provides a Mycobacterium tuberculosis drug-resistant mutation detector comprising a pre-primer and a post-primer for specifically amplifying a target sequence, a blocking oligonucleotide adapted to specifically bind to a wild-type target, which is a gene or a gene fragment comprising a Mycobacterium tuberculosis drug-resistant target mutation site, and a probe specifically recognizing the target sequence.

In a second aspect, the application provides the use of the detection object in preparing a mycobacterium tuberculosis drug resistance detection kit.

In a third aspect, the present application provides a kit for detecting a mycobacterium tuberculosis drug resistance mutation site, including the mycobacterium tuberculosis drug resistance detection object for each mycobacterium tuberculosis drug resistance target mutation site, where the mycobacterium tuberculosis drug resistance mutation site includes, but is not limited to, any one or more of rpoB513, rpoB516, rpoB526, rpoB531, katG315, inhA-15, and rpsL 43.

In a fourth aspect, the present application provides a method for detecting drug resistance of mycobacterium tuberculosis, wherein a PCR reaction is performed on a nucleic acid of mycobacterium tuberculosis to be detected by using the detection object or the kit to obtain a solution to be detected; and detecting the liquid to be detected.

Compared with the prior art, the beneficial effects of this application are:

1. compared with the existing molecular drug sensitivity method, the tuberculosis drug resistance detection method has the advantages that the sensitivity is improved to 0.1%, low-abundance mutation can be detected, and the method is equivalent to a gold-standard phenotype drug sensitivity method.

2. By adopting the whole-course closed tube, the tuberculosis drug resistance detection can be rapidly realized within 2 hours in a one-step detection mode, the operation is simple, and the aerosol cross contamination caused by PCR amplified products is avoided.

3. Aiming at the characteristic of complex mutation types of the tuberculosis drug-resistant related sites, a blocking agent capable of effectively distinguishing the wild type from the mutant type is designed, so that one blocking agent can detect multiple mutation types of a single site at the same time, and a corresponding probe is not required to be designed independently according to each mutation type, thereby reducing the detection cost.

Drawings

FIG. 1 is a schematic diagram of the principle of detecting drug resistance mutation of Mycobacterium tuberculosis. Wherein, the base indicated by triangle in A is modified by locked nucleic acid, and underlined is mutation codon to be detected.

FIG. 2 is a schematic representation of the specificity and sensitivity of a seven-site multiplex assay. Wherein, FIG. 2A is a schematic diagram of specificity and sensitivity of rpoB513 site single detection. FIG. 2B is a schematic of the specificity and sensitivity of the rpoB516 site single detection. FIG. 2C is a schematic representation of the specificity and sensitivity of rpoB526 site single detection. FIG. 2D is a schematic of the specificity and sensitivity of the rpoB531 locus singleplex detection. FIG. 2E is a schematic of specificity and sensitivity of katG315 locus singleplex detection. FIG. 2F is a schematic diagram showing the specificity and sensitivity of inhA-15 site single detection. FIG. 2G is a schematic of specificity and sensitivity of rpsL43 site single detection.

FIG. 3 is a schematic diagram of a mixed sample of seven sites for detection of different mutation ratios. Wherein, fig. 3A is a schematic diagram of a mixed sample of rpoB513 site for detecting different mutation ratios. FIG. 3B is a schematic representation of a mixed sample of the rpoB516 site for detection of different mutation ratios. FIG. 3C is a schematic representation of a mixed sample of rpoB526 site detection at different mutation ratios. Fig. 3D is a schematic representation of a mixed sample of rpoB531 locus detection with different mutation ratios. FIG. 3E is a schematic of a mixed sample of katG315 locus for detection of different mutation ratios. FIG. 3F is a schematic diagram of a mixed sample of inhA-15 locus detection with different mutation ratios. FIG. 3G is a schematic representation of a mixed sample of rpsL43 locus detection with different mutation ratios.

FIG. 4 is a schematic representation of a blocker detecting four mutation types at the rpoB526 site. Wherein, FIG. 4A is a schematic diagram of a blocker to detect GTC mutation at rpoB526 site. FIG. 4B is a schematic representation of a blocker detection of the GTA mutation at rpoB526 site. FIG. 4C is a schematic representation of a blocker detection of the GTT mutation at rpoB526 site. FIG. 4D is a schematic representation of a blocker detection of the GCG mutation at rpoB526 site.

FIG. 5 is a schematic representation of the effect of different length pre-primers on specificity and amplification efficiency. FIG. 5A is a schematic illustration showing the effect of different length pre-primers on WT-10000 copy specificity and amplification efficiency. FIG. 5B is a schematic representation of the effect of different length pre-primers on MUT-100 copy specificity and amplification efficiency. FIG. 5C is a schematic representation of the effect of different length pre-primers on MUT-10 copy specificity and amplification efficiency.

FIG. 6 is a schematic representation of the effect of different concentrations of pre-primers on specificity and amplification efficiency. FIG. 6A is a schematic diagram showing the effect of different concentrations of the pre-primer on WT-10000 copy specificity and amplification efficiency. FIG. 6B is a schematic representation of the effect of different concentrations of the pre-primer on MUT-100 copy specificity and amplification efficiency. FIG. 6C is a schematic representation of the effect of different long concentrations of the pre-primer on MUT-10 copy specificity and amplification efficiency.

FIG. 7 is a schematic representation of the effect of locked nucleic acids of different modified blockers of rpoB513 on specificity and amplification efficiency. FIG. 7A is a schematic representation of the effect of rpoB513 different modified locked nucleic acids on WT-10000 copy specificity and amplification efficiency. FIG. 7B is a schematic representation of the effect of rpoB513 different modified locked nucleic acids on MUT-100 copy specificity and amplification efficiency. FIG. 7C is a schematic representation of the effect of various modified locked nucleic acids of rpoB513 on MUT-10 copy specificity and amplification efficiency.

FIG. 8 is a schematic representation of the effect of locked nucleic acids of different modified blockers of rpoB516 on specificity and amplification efficiency. FIG. 8A is a schematic representation of the effect of various modified locked nucleic acids of rpoB516 on WT-10000 copy specificity and amplification efficiency. FIG. 8B is a schematic representation of the effect of various modified locked nucleic acids of rpoB516 on MUT-100 copy specificity and amplification efficiency. FIG. 8C is a schematic representation of the effect of various modified locked nucleic acids of rpoB516 on MUT-10 copy specificity and amplification efficiency.

FIG. 9 is a schematic representation of the effect of locked nucleic acids of different modified blockers of rpoB526 on specificity and amplification efficiency. FIG. 9A is a schematic representation of the effect of rpoB526 on WT-10000 copy specificity and amplification efficiency of differently modified locked nucleic acids. FIG. 9B is a schematic representation of the effect of rpoB526 on MUT-100 copy specificity and amplification efficiency of differently modified locked nucleic acids. FIG. 9C is a schematic representation of the effect of rpoB526 on MUT-10 copy specificity and amplification efficiency of differently modified locked nucleic acids.

FIG. 10 is a schematic representation of the effect of primers on specificity and amplification efficiency after different lengths. FIG. 10A is a schematic diagram showing the effect of primers on WT-10000 copy specificity and amplification efficiency after different lengths. FIG. 10B is a schematic representation of the effect of primers on MUT-100 copy specificity and amplification efficiency after different lengths. FIG. 10C is a schematic representation of the effect of primers on MUT-10 copy specificity and amplification efficiency after different lengths.

FIG. 11 is a schematic of the effect of different blocker binding temperatures on specificity and amplification efficiency. FIG. 11A is a schematic illustration of the effect of binding temperatures of non-blocker WT10000 to different targets on specificity and amplification efficiency. FIG. 11B is a schematic of the effect of blocker-WT 10000 added binding temperatures to different targets on specificity and amplification efficiency. FIG. 11C is a graph showing the effect of binding temperatures of non-blocker-100 MUT to different targets on specificity and amplification efficiency. FIG. 11D is a graph showing the effect of blocker-100 MUT binding temperatures on specificity and amplification efficiency. FIG. 11E is a schematic of the effect of binding temperatures of non-blocker-10 MUT to different targets on specificity and amplification efficiency. FIG. 11F is a graph showing the effect of blocker-10 MUT binding temperatures on specificity and amplification efficiency.

In order to make the objects, technical solutions and advantageous effects of the present application clearer, the present application is further described below with reference to examples. It should be understood that the examples are presented by way of illustration only and are not intended to limit the scope of the application. The test methods used in the following examples are conventional, unless otherwise indicated, and other advantages and effects of the present application will be readily apparent to those skilled in the art from the disclosure herein.

The inventor of the application finds a method and a kit for detecting drug-resistant mutation of mycobacterium tuberculosis through a great deal of research and study. The tuberculosis drug-resistant mutation detection method established in the application realizes the inhibition of a large amount of wild type backgrounds and the enrichment of low-abundance mutant types based on the competition between the front primer or the rear primer or the probe and the blocking oligonucleotide used in the PCR reaction, thereby improving the detection sensitivity and being capable of detecting seven sites with higher mutation frequencies of common anti-tuberculosis drugs isoniazid, rifampicin and streptomycin.

In one aspect, the application provides a mycobacterium tuberculosis drug resistance mutation detector, which comprises a front primer and a rear primer for specifically amplifying a target sequence, a blocking oligonucleotide, and a probe for specifically recognizing the target sequence, wherein the blocking oligonucleotide is suitable for specifically binding to a wild-type target, and the target is a gene or a gene fragment containing a mycobacterium tuberculosis drug resistance target mutation site.

In the mycobacterium tuberculosis drug-resistant mutation detector provided by the application, the length of the front primer is 13-20 bp, and the length of the rear primer is 13-20 bp. The target binding region corresponding to the front primer or the rear primer or the probe is partially overlapped with the target binding region corresponding to the blocking oligonucleotide, and the base length of the overlapped part is 1-10 bp.

In one embodiment, the mycobacterium tuberculosis may be, for example, H37Rv. The wild-type target in the invention refers to a wild-type sequence or a complementary sequence thereof which does not generate drug resistance mutation and corresponds to a drug resistance target mutation site of mycobacterium tuberculosis. By blocking oligonucleotides suitable for specific binding to wild-type targets is meant that the blocking oligonucleotides specifically recognize and bind to wild-type targets with little or no affinity to the targets where drug resistance mutations occur. For example, the sequence of the blocking oligonucleotide comprises the wild-type sequence of the target mutation site or its complement, and thus is capable of binding specifically only to the wild-type target and not to the mutant target. Preferably, the wild type sequence of the target mutation site or its complement is located in the middle of the blocking oligonucleotide sequence, e.g. upstream and downstream of the wild type sequence of the target mutation site or its complement, further extending 4-9 bases each based on the target sequence. The target may be, for example, a tuberculosis drug resistance-related gene such as ahpC, gyrA, rrs, etc., or a fragment of the above gene containing a target mutation site for drug resistance of interest. The drug resistance of the mycobacterium tuberculosis in the invention can be antituberculosis drugs, such as rifampicin, isoniazid and streptomycin, and can also be other antituberculosis drugs, such as ethambutol, fluoroquinolones, pyrazinamide, betaquinine, linezolid, delamania and the like.

In the mycobacterium tuberculosis drug-resistant mutation detector provided by the application, the front primer, the rear primer and the probe do not cover the drug-resistant target mutation site, and the blocking oligonucleotide covers the wild drug-resistant target mutation site. Specifically, none of the pre-primer, the post-primer and the probe-target pairing region comprise a target mutation site, e.g., the 3 'end of the pre-primer terminates upstream of the target mutation site and the 3' end of the post-primer terminates downstream of the target mutation site, with the probe-binding region avoiding the target mutation site. The design enables the detection object to be used for detecting various mutation types possibly occurring in a single codon, accords with the characteristic of complex tuberculosis drug-resistant mutation types, enables a front primer or a rear primer or a probe to detect various mutation types of a site, reduces detection cost, and is suitable for detecting the drug-resistant mutation of mycobacterium tuberculosis with complex mutation types.

In the mycobacterium tuberculosis drug-resistant mutation detection object provided by the application, the probe is selected from one or a combination of more of Taqman probes, molecular beacons and fluorescence resonance energy transfer probes. Taqman probes, molecular beacons, and fluorescence resonance energy transfer probes are all labeled with a fluorescent group and/or a quenching group. A fluorescent group is labeled at the 5 'end of the probe and a quenching group is labeled at the 3' end of the probe. The fluorophore comprises any one or a combination of more of FAM, VIC, JOE, TET, CY, CY5, ROX, texas Red or LC Red 460. The quenching group includes any one or a combination of multiple of MBG, BHQ1, BHQ2, BHQ3, dabcy1, or Tamra.

In the mycobacterium tuberculosis drug-resistant mutation detection provided by the application, the blocking oligonucleotide has blocking modification. Preferably, the blocking modification is located at the 3' end of the blocking oligonucleotide. More preferably, the nucleic acid blocking modification comprises one or more selected from the group consisting of: space, thio, mercapto, amino and uracil bases. Specifically, the 3' -end of the blocking oligonucleotide is NH-substituted ₂ -C6 modification to prevent extension.

In the mycobacterium tuberculosis drug-resistant mutation detection provided by the application, the blocking oligonucleotide also has nucleic acid analogue modification. Specifically, the blocking oligonucleotide is, for example, a blocking agent that recognizes a single base mutation. Preferably, the nucleic acid analogue modification is selected from the group consisting of a combination of one or more of Locked Nucleic Acid (LNA), peptide Nucleic Acid (PNA) and Bridged Nucleic Acid (BNA). In order to effectively distinguish between wild-type and mutant target molecules, the blocking oligonucleotide modifies as few nucleic acid analogues as possible to achieve as high a Tm value as possible, such that the blocking oligonucleotide specifically binds to the wild-type target and the affinity to the mutant target is reduced or diminished.

In the mycobacterium tuberculosis drug resistance mutation detection object provided by the application, the mycobacterium tuberculosis drug resistance target mutation sites comprise, but are not limited to, rifampicin resistance related sites (rpoB 513, rpoB516, rpoB526, rpoB 531), isoniazid resistance related sites (katG 315, inhA-15) and streptomycin resistance related sites (rpsL 43).

In one embodiment, the primer sequence of rpoB513 at the drug-resistant target mutation site of mycobacterium tuberculosis is shown as SEQ ID NO. 1, the primer sequence of rpoB is shown as SEQ ID NO. 2, the blocking oligonucleotide sequence is shown as SEQ ID NO. 19, and the probe sequence is shown as SEQ ID NO. 15.

In one embodiment, the primer sequence of rpoB516 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 3, the primer sequence of rpoB516 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 4, the blocking oligonucleotide sequence of rpoB516 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 20, and the probe sequence of rpoB516 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 15.

In one embodiment, the primer sequence of rpoB526 at the drug-resistant target mutation site of Mycobacterium tuberculosis is shown as SEQ ID NO. 5, the primer sequence of rpoB526 at the drug-resistant target mutation site of Mycobacterium tuberculosis is shown as SEQ ID NO. 6, the blocking oligonucleotide sequence of rpoB526 at the drug-resistant target mutation site of Mycobacterium tuberculosis is shown as SEQ ID NO. 20, and the probe sequence of rpoB526 at the drug-resistant target mutation site of Mycobacterium tuberculosis is shown as SEQ ID NO. 15.

In one embodiment, the primer sequence of rpoB531 of the drug-resistant target mutation site of mycobacterium tuberculosis is shown as SEQ ID NO. 7, the primer sequence of rpoB531 of the drug-resistant target mutation site of mycobacterium tuberculosis is shown as SEQ ID NO. 8, the blocking oligonucleotide sequence of rpoB531 of the drug-resistant target mutation site of mycobacterium tuberculosis is shown as SEQ ID NO. 22, and the probe sequence of rpoB531 of the drug-resistant target mutation site of mycobacterium tuberculosis is shown as SEQ ID NO. 15.

In one embodiment, the pre-primer sequence of the mycobacterium tuberculosis drug-resistant target mutation site katG315 is shown in SEQ ID NO. 9, the post-primer sequence is shown in SEQ ID NO. 10, the blocking oligonucleotide sequence is shown in SEQ ID NO. 23, and the probe sequence is shown in SEQ ID NO. 16.

In one embodiment, the pre-primer sequence of the drug-resistant target mutation site inhA-15 of the mycobacterium tuberculosis is shown as SEQ ID NO. 11, the post-primer sequence is shown as SEQ ID NO. 12, the blocking oligonucleotide sequence is shown as SEQ ID NO. 24, and the probe sequence is shown as SEQ ID NO. 17.

In one embodiment, the primer sequence of rpsL43 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 13, the primer sequence of the primer is shown as SEQ ID NO. 14, the blocking oligonucleotide sequence is shown as SEQ ID NO. 25, and the probe sequence is shown as SEQ ID NO. 18.

In one embodiment, as shown in FIG. 1, when the target is wild-type, the blocking oligonucleotide is perfectly complementarily paired with the wild-type target and has a higher affinity, and thus preferentially binds to the wild-type target such that the 3' end of the pre-primer cannot bind, preventing extension of the DNA polymerase. When the target is mutant, the affinity of the blocking oligonucleotide and the target is greatly reduced due to base mismatch between the two, and is lower than the binding affinity of the pre-primer and the target, so that the pre-primer can be extended by DNA polymerase, and the cleavage of the Taqman probe generates a fluorescent signal.

In another aspect, the application provides the application of the detection object in preparing a mycobacterium tuberculosis drug resistance detection kit. The use is for non-disease diagnosis or therapeutic purposes. In the application provided by the application, the mycobacterium tuberculosis drug resistance detection kit is used for detecting the drug resistance of mycobacterium tuberculosis to antituberculosis drugs. Preferably, the antitubercular drug is selected from one or more of rifampicin, isoniazid, streptomycin, ethambutol, fluoroquinolones, pyrazinamide, beta quinine, linezolid, delamanib.

In another aspect, the present application provides a kit for detecting drug-resistant mutation of mycobacterium tuberculosis, which is used for detecting drug-resistant mutation sites of mycobacterium tuberculosis, and comprises the aforementioned drug-resistant detection object of mycobacterium tuberculosis for each drug-resistant target mutation site of mycobacterium tuberculosis. The mycobacterium tuberculosis drug-resistant mutation site includes, but is not limited to, any one or more of rpoB513, rpoB516, rpoB526, rpoB531, katG315, inhA-15, and rpsL 43.

In the kit for detecting the drug-resistant mutation of the mycobacterium tuberculosis, the kit also comprises any one or a combination of a plurality of DNA polymerase, dNTP and PCR buffer solution.

In the kit for detecting the drug-resistant mutation of the mycobacterium tuberculosis, the molar concentration of the blocking oligonucleotide is 10-20 times of that of the front primer or the rear primer in a PCR reaction system using the kit. The Tm value of the blocking oligonucleotide is 75-85℃and the Tm value of the front or rear primer is 50-65 ℃. In the actual use of the kit, the temperature set between the PCR-time-variant temperature and the annealing temperature for blocking preferential binding of the oligonucleotides may be, for example, 70 to 85 ℃.

In another aspect, the present application provides a method for detecting drug resistance of mycobacterium tuberculosis, which comprises performing PCR reaction on nucleic acid of mycobacterium tuberculosis to be detected using the above-mentioned detection substance or the above-mentioned kit to obtain a solution to be detected; and detecting the liquid to be detected. Specifically, the detection may be, for example, fluorescence detection.

The present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

The genetic information used in the examples below is as follows:

gene name	Location	Gene ID
			rpoB	NC_000962.3(759807..763325)	888164
katG	NC_000962.3(2153889..2156111)	885638
			inhA	NC_000962.3(1674202..1675011)	886523
rpsL	NC_000962.3(781560..781934)	888259

Example 1 Rifampicin, isoniazid, and streptomycin resistance seven mutation sites Shan Chongte anisotropy and sensitivity assessment

1. Primer, probe and blocker designs for detection of seven sites

According to the genome sequence of the Mycobacterium tuberculosis H37Rv in Genbank, a primer, a locked nucleic acid blocker and a Taqman probe are designed on a Integrated DNA Technologies (IDT) website aiming at rifampicin resistance related sites (rpoB 513 (CAA), rpoB516 (GAC), rpoB526 (GTG) and rpoB531 (TCG)), isoniazid resistance related sites (katG 315 (AGC) and inhA-15 (C)), and streptomycin resistance related sites (rpsL 43 (AAG)), so that no obvious secondary structure and intermolecular interaction are ensured. The predicted Tm value after modification of the locked nucleic acid by the blocker is preferably higher than 80℃and the mutation site is designed at the middle position of the blocker, and the 3 '-end of the primer and the 5' -end of the blocker are designed to contain overlapping bases so as to form a competitive relationship.

PCR amplification

2.1 extraction of DNA templates: DNA extraction of Mycobacterium tuberculosis isolated and cultured by wild type and mutant type using commercial Mycobacterium tuberculosis nucleic acid extraction kit (QIAGEN, QIAamp DNA microbiome kit (Cat. No. 51704)) to obtain DNA template, quantitative determination by digital PCR, the input of wild type H37Rv genome in a single PCR reaction was 10000 copies, and the input of seven mutant genomes in a single PCR reaction was 100 copies and 10 copies.

2.2 Synthesis of PCR primers.

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction solution, wherein the sequences and the concentrations of the front primer, the rear primer and the probe used for seven sites are shown in table 1, the sequence of the blocking agent is shown in table 2, the concentration of the blocking agent is 1.5 micromole/liter, the concentration of the probe is 0.2 micromole/liter, and the concentration of Taq enzyme is 0.7U,1X PCR buffer,MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

Table 1 sequences, lengths and concentrations of the front primer, rear primer and probe used at seven sites

(underlined indicates the bases of the primer overlapping the blocker)

TABLE 2 sequence, length, number of modified locked nucleic acids and predicted Tm of blocking agents modified with locked nucleic acids at seven sites

(the bolded bases represent locked nucleic acid modifications, the underlined indicates the mutant codons to be detected)

2.4PCR amplification: the reaction procedure for PCR amplification was: pre-denaturation at 95℃for 2 min, 15 cycles of denaturation at 95℃for 1s, 20s at 70℃for 20s at 65℃for annealing extension, and 35 cycles of amplification at 35 cycles of denaturation at 95℃for 1s, 20s at 70℃for 20s at 60℃for annealing extension (acquisition of fluorescent signals). Wherein the sites rpoB513, 516, 526, 531 record fluorescent signals using FAM channel, katG315, inhA-15, and rpsL43 record fluorescent signals using VIC channel.

3. Analysis of results

The detection results of the single weight of seven sites are shown in fig. 2, 10000 copies of wild-type genome of seven sites are amplified to generate strong fluorescent signals under the condition of no blocking agent, 10000 copies of wild-type genome are completely inhibited without amplified signals after the blocking agent is added, and 100 copies and 10 copies of mutant genome can still amplify signals after the blocking agent is added for rpoB513, rpoB526, katG315 and rpsL43 sites, so that mutants as low as 10 copies can be effectively detected. For rpoB516, rpoB531 and inhA-15 sites, 100 copies of the mutant genome could be detected after addition of the blocking agent, and 10 copies could not be stably detected due to inhibition. When 10000 copies of the wild-type genome were mixed with 0/10/100/500/1000/10000 mutant genome at mutation ratios of 0%, 0.1%, 1%, 5%, 10% and 100%, respectively, as shown in FIG. 3, the mixed sample having a mutation ratio of 0.1% was effectively detected at the rpoB513, rpoB526, katG315 and rpsL43 sites, and the mixed sample having a mutation ratio of 1% was effectively detected at the rpoB516, rpoB531 and inhA-15 sites.

Example 2 detection of four mutation types by a blocker at Rifampicin rpoB526 site

Primer, probe and blocker design at rpoB526 site

According to the genome sequence of the mycobacterium tuberculosis H37Rv in Genbank, aiming at a rifampicin drug-resistant related site rpoB526, a primer, a locked nucleic acid blocker and a Taqman probe are designed on a Integrated DNA Technologies (IDT) website, so that no obvious secondary structure and no intermolecular interaction are ensured. The predicted Tm value after modification of the locked nucleic acid by the blocker is preferably higher than 80℃and the mutation site is designed at the middle position of the blocker, and the 3 '-end of the primer and the 5' -end of the blocker are designed to contain overlapping bases so as to form a competitive relationship.

PCR amplification

2.1 construction of plasmid templates: the rpoB gene is cloned into a PUC57 vector in a conventional manner to respectively construct rpoB526 wild type plasmid (with a codon of GTG) and four common mutant plasmids (GTG-GTC/GTA/GTT/GCG), the plasmid templates are quantified by digital PCR, the input amount of the wild type plasmid in a single PCR reaction is 10000 copies, and the input amount of the mutant plasmid in the single PCR reaction is 100 copies and 10 copies.

2.2 Synthesis of PCR primers: the synthesis method is the existing conventional DNA synthesis method.

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction liquid, wherein the sequences and the concentrations of the pre-primer, the post-primer and the probe used for the rpoB526 site are shown as rpoB526-FP, rpoB526-RP and rpoB-P in table 1, the blocking agent used is rpoB-LNA in table 2, the concentration of the blocking agent is 1.5 micromole/liter, the concentration of the probe is 0.2 micromole/liter, the concentration of Taq enzyme is 0.7U, and the concentration of 1X PCR b uffer，MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

2.4PCR amplification: the reaction procedure for PCR amplification was: pre-denaturation at 95℃for 2 min, 15 cycles of denaturation at 95℃for 1s, 20s at 70℃for 20s at 65℃for annealing extension, and 35 cycles of amplification at 35 cycles of denaturation at 95℃for 1s, 20s at 70℃for 20s at 60℃for annealing extension (acquisition of fluorescent signals). The rpoB526 site uses FAM channel to record fluorescence signals.

3. Analysis of results

The result of detecting four common mutation types by using one blocking agent at rpoB526 site is shown in figure 4, 10000 copies of wild type plasmid are amplified to generate strong fluorescent signals under the condition that no blocking agent is added, 10000 copies of wild type plasmid are completely inhibited after the blocking agent is added, no amplified signals are generated, but 100 copies and 10 copies of mutation types can be effectively detected for the four mutation type plasmid without adding the blocking agent and after adding the blocking agent, and the fact that one blocking agent at a single site can be used for detecting various mutation types possibly generated is disclosed, so that the detection cost is reduced.

Example 3 Effect of different Length Pre-primers on specificity and amplification efficiency

1. Primers, probes and blockers were designed using rpoB526 as an example

According to the genome sequence of the Mycobacterium tuberculosis H37Rv in Genbank, aiming at a rifampicin drug-resistance related site rpoB526, a front primer with different lengths is designed on a Integrated DNA Technologies (IDT) website, as shown in Table 3, a locked nucleic acid blocker and a rear primer are kept unchanged, and no obvious secondary structure and no intermolecular interaction are ensured. The predicted Tm value after modification of the locked nucleic acid by the blocker is preferably higher than 80℃and the mutation site is designed at the middle position of the blocker, and the 3 '-end of the primer and the 5' -end of the blocker are designed to contain overlapping bases so as to form a competitive relationship.

TABLE 3rpoB526 site different length pre-primers

(underlined indicates the bases of the primer overlapping the blocker)

PCR amplification

2.1 construction of plasmid templates: the rpoB gene is cloned into a PUC57 vector in a conventional manner to respectively construct rpoB526 wild type plasmid (the codon is GTG) and mutant plasmid (GTG-GCG), the plasmid template is quantified by digital PCR, the input amount of the wild type plasmid in a single PCR reaction is 10000 copies, and the input amount of the mutant plasmid in the single PCR reaction is 100 copies and 10 copies.

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction liquid, wherein the pre-primers with different lengths used by rpoB526 site are shown in table 3, the concentration of the pre-primer is 0.15 micromolar/liter, the sequences and the concentrations of the post-primer and the probe are shown as rpoB526-RP and rpoB-P in table 1, the blocking agent is rpoB526-LNA in table 2, the concentration of the blocking agent is 1.5 micromolar/liter, the concentration of the probe is 0.2 micromolar/liter, and the concentration of Taq enzyme is 0.7U,1X PCR buffer,MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

3. Analysis of results

The influence of the different length of the front primer on the specificity and the amplification efficiency is shown in figure 5, the used blocking agent and the rear primer are kept unchanged, the length of the front primer is 15/16/17bp, when the length of the front primer is 17bp, 10000 copies of wild type plasmid can not be completely inhibited by the blocking agent, and the specificity of the front primers of 15bp and 16bp can ensure that 10000 copies of wild type plasmid can not generate amplification signals. For mutant plasmids, the front primer with the length of 15bp can detect 100 copies of mutant plasmids after adding the blocker, but cannot detect 10 copies of mutant plasmids, and the front primer with the length of 16bp can detect both 100 copies and 10 copies of mutant plasmids.

Example 4 Effect of different concentrations of the Pre-primer on specificity and amplification efficiency

1. Primers, probes and blockers were designed using rpoB526 as an example

PCR amplification

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction liquid, wherein the front primer of rpoB526 site is rpoB526-FP in table 1 with the concentration of 0.20/0.15/0.10/0.05 micromole/liter respectively, the sequence and concentration of the rear primer and probe are shown as rpoB526-RP and rpoB-P in table 1, the blocking agent is rpoB526-LNA in table 2 with the concentration of 1.5 micromole/liter, the concentration of probe is 0.2 micromole/liter, and the concentration of Taq enzyme is 0.7U,1X PCR buffer,MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

3. Analysis of results

The effect of different concentrations of the pre-primer on the specificity and amplification efficiency is shown in FIG. 6, the concentration of the pre-primer is 0.20/0.15/0.10/0.05. Mu. Mol/liter, respectively, when the length of the pre-primer is 0.20. Mu. Mol/liter, 10000 copies of wild-type plasmid cannot be completely inhibited by the blocker, and the specificity of the remaining three concentrations of pre-primer can ensure that 10000 copies of wild-type plasmid do not generate amplification signals. For mutant plasmids, the 0.05. Mu. Mol/l pre-primer did not detect even 100 copies of mutant plasmids after addition of the blocking agent, the 0.10. Mu. Mol/l pre-primer did detect 100 copies of mutant plasmids after addition of the blocking agent, but 10 copies of mutant plasmids were not detected, and only the 0.15. Mu. Mol/l pre-primer detected both 100 copies and 10 copies of mutant plasmids.

Example 5 Effect of blocking agent modification of locked nucleic acids in different ways on specificity and amplification efficiency

1. Primers, probes and blockers were designed using rpoB513, rpoB516 and rpoB526 sites as examples

According to the genome sequence of the mycobacterium tuberculosis H37Rv in Genbank, aiming at rifampicin drug resistance related sites rpoB513, rpoB516 and rpoB526, primers, a locked nucleic acid blocker and a Taqman probe are designed on a Integrated DNA Technologies (IDT) website, so that no obvious secondary structure and no intermolecular interaction are ensured. The blocking agent modifies the number of different locked nucleic acids to achieve different Tm values, the mutation site is designed at the middle position of the blocking agent as much as possible, as shown in Table 4, and in addition, the 3 '-end of the front primer and the 5' -end of the blocking agent are designed to contain overlapping bases so as to form a competitive relationship.

TABLE 4 blocking agent modification of locked nucleic acids in different ways

(bolded base indicates modified locked nucleic acid)

PCR amplification

2.1 construction of plasmid templates: cloning rpoB genes into a PUC57 vector in a conventional manner to respectively construct rpoB513, rpoB516 and rpoB526 wild-type plasmids (the codon of rpoB513 is CAA, the codon of rpoB516 is GAC, the codon of rpoB526 is GTG) and mutant plasmids (the codon of rpoB513 is CAA-CCA, the codon of rpoB516 is GAC-GGC and the GTG-GCG) of which the input amount of the wild-type plasmids in a single PCR reaction is 10000 copies and the input amount of the mutant plasmids in the single PCR reaction is 100 copies and 10 copies.

2.3 preparing PCR reaction liquid: preparing 20 microliters of PCR reaction solution, wherein the sequence and concentration of the front primer, the rear primer and the probe used at the rpoB513 site are shown as rpoB513-FP, rpoB513-RP and rpoB-P in Table 1, the sequence and concentration of the front primer, the rear primer and the probe used at the rpoB516 site are shown as rpoB516-FP, rpoB516-RP and rpoB-P in Table 1, the sequence and concentration of the front primer, the rear primer and the probe are shown as rpoB526-FP, rpoB526-RP and rpoB-P in Table 1, the blocking agent is shown as Table 4, the blocking agent concentration is 1.5 micromole/liter probe concentration is 0.2 micromole/liter, and the Taq enzyme concentration is 0.7U,1X PCR buffer,MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

3. Analysis of results

The effect of blocking agent modification on specificity and amplification efficiency of locked nucleic acid in different modes is shown in fig. 7-9, and the results of blocking agent modification in different modes of rpoB513, rpoB516 and rpoB526 are shown in fig. 7, 8 and 9, respectively, and blocking agent 1 and blocking agent 2 at rpoB513 site can completely inhibit 10000 copies of wild type plasmid and can detect 100 copies and 10 copies of mutant plasmid. Blocking agent 1 and blocking agent 2 at rpoB516 site both completely inhibited 10000 copies of wild type plasmid, and 100 copies of mutant plasmid could be detected but 10 copies of mutant plasmid could not be detected after blocking agent addition. Blocker 1 of rpoB526 modifies 4 locked nucleic acids, has the lowest Tm value, cannot completely inhibit 10000 copies of wild-type plasmid, blocker 4 modifies 6 locked nucleic acids, has the highest Tm value, 100 copies of mutant plasmid can be detected but 10 copies of mutant plasmid cannot be detected after adding blocker, and blockers 2 and 3 modify 5 and 6 locked nucleic acids respectively, so that 100 copies and 10 copies of mutant plasmid can be detected. The Tm value of the blocker is a measure of the binding energy of the blocker to the target, which indicates that the blocker modifies the locked nucleic acid in such a way as to produce different Tm values, neither because the ability to bind the target is too weak to completely inhibit the wild-type background, nor because the ability to bind the target is too strong to interfere with the detection of mutants.

Example 6 Effect of primers on specificity and amplification efficiency after different Length

1. Primers, probes and blockers were designed using rpoB526 as an example

According to the genome sequence of the mycobacterium tuberculosis in Genbank, aiming at a rifampicin drug resistance related site rpoB526, a primer, a locked nucleic acid blocker and a Taqman probe are designed on a Integrated DNA Technologies (IDT) website, so that no obvious secondary structure and no intermolecular interaction are ensured. The post-primer is designed to have different lengths, and as shown in Table 5, the predicted Tm value after the blocker modifies the locked nucleic acid is preferably higher than 80℃and the mutation site is designed at the middle position of the blocker, and the 3 '-end of the pre-primer and the 5' -end of the blocker are designed to have overlapping bases so as to form a competitive relationship.

TABLE 5 post-primers of different lengths

PCR amplification

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction liquid, wherein the sequence and the concentration of the pre-primer and the probe used for rpoB526 site are shown as rpoB526-FP and rpoB-P in table 1, the post-primer is shown as table 5, the post-primer concentration is 0.1 micromole/liter, the blocker is rpoB526-LNA in table 2, the blocker concentration is 1.5 micromole/liter, the probe concentration is 0.2 micromole/liter, and the Taq enzyme concentration is 0.7U,1X PCR buffer,MgCl ₂ At 1.5 mM, dNTPs at 0.2 mM, template DNA was added at 2.5. Mu.l, and the remaining volume was filled with enzyme-free water.

3. Analysis of results

As shown in FIG. 10, the effect of the primers with different lengths on the specificity and amplification efficiency is that the primer 526RP1 with the length of 16bp cannot completely inhibit 10000 copies of wild type plasmids, the primer 526RP3 with the length of 14bp can detect 100 copies of mutant plasmids after adding a blocking agent, but cannot detect 10 copies of mutant plasmids, and the primer 526RP2 with the length of 15bp can detect 100 copies of mutant plasmids as well as 10 copies of mutant plasmids after adding a blocking agent. Unlike the effect of the front primer of different length, the length of the rear primer affects the overall amplification efficiency without changing the inhibition state of the blocker on the mutant, while changing the length of the front primer changes the competition state with the blocker, thereby changing the inhibition of the blocker on the mutant to different degrees.

Example 7 Effect of different blocking Agents on target binding temperatures on specificity and amplification efficiency

1. Primers, probes and blockers were designed using rpoB526 as an example

According to the genome sequence of the mycobacterium tuberculosis H37Rv in Genbank, aiming at a rifampicin drug-resistant related site rpoB526, a primer, a locked nucleic acid blocker and a Taqman probe are designed on a Integrated DNA Technologies (IDT) website, so that no obvious secondary structure and no intermolecular interaction are ensured. The predicted Tm value after modification of the locked nucleic acid by the blocker is preferably higher than 80℃and the mutation site is designed at the middle position of the blocker, and the 3-terminus of the primer and the 5-terminus of the blocker are designed to contain overlapping bases so as to form a competitive relationship.

PCR amplification

2.3 preparing PCR reaction liquid: preparing 20 microliter PCR reaction liquid, wherein the sequences and the concentrations of the front primer, the rear primer and the probe used for the rpoB526 site are shown as rpoB526-FP, rpoB526-RP and rpoB-P in table 1, the blocking agent used is rpoB-LNA in table 2, the concentration of the blocking agent is 1.5 micromole/liter, the concentration of the probe is 0.2 micromole/liter, and the concentration of Taq enzyme is 0.7U,1X PCR buffer,MgCl ₂ 1.5 mM, 0.2 mM dNTPs, 2.5. Mu.l template DNA was addedThe remaining volume was filled with enzyme-free water.

2.4PCR amplification: the temperature for blocker binding to the target in the reaction procedure of PCR amplification was set to different temperature gradients, 73/76/79/82/85 ℃ respectively, as shown in table 6, with fluorescence signal recorded at rpoB526 site using FAM channel.

TABLE 6 different blocker binding temperatures

3. Analysis of results

The effect of different blocker on the binding temperature of the target on the specificity and amplification efficiency is shown in FIG. 11, when no blocker is added, the amplification efficiency of five temperature gradients is almost unchanged, after the blocker is added, the binding temperature is increased from 73 ℃ to 79 ℃, the inhibition degree of the blocker on the wild type plasmid is gradually increased, but when the blocker is continuously increased to 82 ℃ and 85 ℃, the inhibition on the wild type plasmid is reduced to be equivalent to 76 ℃, the different binding temperatures do not show obvious difference on the amplification of the mutant plasmid, and the effect of changing the binding temperature of the blocker and the target on the whole reaction system is not obvious.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the application. Modifications and variations may be made to the above-described embodiments by those of ordinary skill in the art without departing from the spirit and scope of the present application. Accordingly, it is intended that all equivalent modifications and variations which can be accomplished by persons skilled in the art without departing from the spirit and technical spirit of the present disclosure shall be covered by the claims of this application.

Claims

1. A mycobacterium tuberculosis drug-resistant mutation detector comprising a pre-primer and a post-primer for specifically amplifying a target sequence, a blocking oligonucleotide adapted to specifically bind to a wild-type target, and a probe specifically recognizing the target sequence, the target being a gene or gene fragment comprising a mycobacterium tuberculosis drug-resistant target mutation site.

2. The mycobacterium tuberculosis drug-resistant mutation detector of claim 1, wherein none of the pre-primer, post-primer and probe cover the drug-resistant specific target mutation site, and the blocking oligonucleotide covers a wild-type gene site corresponding to the drug-resistant target mutation site; the probe is selected from one or a combination of more of Taqman probes, molecular beacons and fluorescence resonance energy transfer probes;

and/or, the Tm value of the blocking oligonucleotide is 75-85 ℃, and the Tm value of the front primer or the rear primer is 50-65 ℃;

and/or, the target binding region corresponding to the pre-primer or post-primer or probe partially overlaps with the target binding region corresponding to the blocking oligonucleotide; the base length of the overlapping part is 1-10 bp;

and/or the length of the front primer is 13-20 bp, and the length of the rear primer is 13-20 bp.

3. The mycobacterium tuberculosis drug-resistant mutation detector of claim 1, wherein the blocking oligonucleotide has a blocking modification; preferably, the blocking modification is located at the 3' end of the blocking oligonucleotide; more preferably, the blocking modification is selected from the group consisting of one or more of a Spacer, a thio group, a thiol group, an amino group, and a uracil base;

optionally, the blocking oligonucleotide further comprises a nucleic acid analogue modification; preferably, the nucleic acid analogue is selected from the group consisting of one or more of a locked nucleic acid, a peptide nucleic acid and a bridged nucleic acid.

4. A mycobacterium tuberculosis drug-resistant mutation detector as described in any of claims 1-3, wherein the mycobacterium tuberculosis drug-resistant target mutation site includes, but is not limited to

Any of rpoB513, rpoB516, rpoB526, rpoB531, katG315, inhA-15, and rpsL 43.

5. The Mycobacterium tuberculosis drug-resistant mutation detection material as described in claim 4, wherein,

the primer sequence of rpoB513 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 1, the primer sequence of rpoB513 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 2, the blocking oligonucleotide sequence of rpoB513 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 19 or SEQ ID NO. 28, and the probe sequence of rpoB513 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 15;

The front primer sequence of the drug-resistant target mutation site rpoB516 of the mycobacterium tuberculosis is shown as SEQ ID NO. 3, the rear primer sequence is shown as SEQ ID NO. 4, the blocking oligonucleotide sequence is shown as SEQ ID NO. 20 or SEQ ID NO. 29, and the probe sequence is shown as SEQ ID NO. 15;

the primer sequence of rpoB526 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 5, the primer sequence of rpoB526 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 6, the blocking oligonucleotide sequence of rpoB526 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 21 or SEQ ID NO. 31, and the probe sequence of rpoB526 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 15;

the front primer sequence of rpoB531 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 7, the rear primer sequence is shown as SEQ ID NO. 8, the blocking oligonucleotide sequence is shown as SEQ ID NO. 22, and the probe sequence is shown as

SEQ ID NO. 15;

the front primer sequence of the drug-resistant target mutation site katG315 of the mycobacterium tuberculosis is shown as SEQ ID NO. 9, the rear primer sequence is shown as SEQ ID NO. 10, the blocking oligonucleotide sequence is shown as SEQ ID NO. 23, and the probe sequence is shown as

SEQ ID NO. 16;

the front primer sequence of the drug-resistant target mutation site inhA-15 of the mycobacterium tuberculosis is shown as SEQ ID NO. 11, the rear primer sequence is shown as SEQ ID NO. 12, the blocking oligonucleotide sequence is shown as SEQ ID NO. 24, and the probe sequence is shown as

SEQ ID NO. 17;

the primer sequence of rpsL43 of the drug-resistant target mutation site of the mycobacterium tuberculosis is shown as SEQ ID NO. 13, the primer sequence of the primer is shown as SEQ ID NO. 14, the blocking oligonucleotide sequence is shown as SEQ ID NO. 25, and the probe sequence is shown as

SEQ ID NO. 18.

6. Use of the test substance according to any one of claims 1 to 5 for the preparation of a kit for detecting drug resistance of mycobacterium tuberculosis.

7. The use according to claim 6, wherein the mycobacterium tuberculosis resistance detection kit is for detecting the resistance of mycobacterium tuberculosis to antitubercular drugs; preferably, the antitubercular drug includes, but is not limited to, one or more of rifampin, isoniazid, streptomycin, ethambutol, fluoroquinolones, pyrazinamide, beta quinine, linezolid, delamanib.

8. A kit for detecting the drug resistance of Mycobacterium tuberculosis, which is used for detecting the drug resistance mutation sites of Mycobacterium tuberculosis, and comprises the drug resistance detection object of Mycobacterium tuberculosis according to any one of claims 1 to 5 aiming at each drug resistance target mutation site of Mycobacterium tuberculosis, wherein the drug resistance mutation sites of Mycobacterium tuberculosis comprise but are not limited to

Any one or more of rpoB513, rpoB516, rpoB526, rpoB531, katG315, inhA-15, and rpsL 43.

9. The mycobacterium tuberculosis resistance detection kit of claim 8, wherein the kit further comprises a combination of any one or more of DNA polymerase, dntps, PCR buffer;

and/or, in a PCR reaction system using the kit, the molar concentration of the blocking oligonucleotide is 10 to 20 times that of the pre-primer or the post-primer.

10. A method for detecting drug resistance of mycobacterium tuberculosis, which comprises the steps of carrying out PCR reaction on nucleic acid of mycobacterium tuberculosis to be detected by adopting the detection object according to any one of claims 1 to 5 or the kit according to any one of claims 8 to 9 to obtain liquid to be detected; and detecting the liquid to be detected.