Composition and kit for detecting mycobacterium tuberculosis and drug resistance thereof

Abstract

The invention provides a composition and a kit for detecting mycobacterium tuberculosis and drug resistance thereof. Specifically, the invention provides a composition of primers and probes for detecting mycobacterium tuberculosis and drug resistance thereof, and genes targeted by the primers and the probes comprise an IS6110 insertion sequence, an IS1081 insertion sequence, a 16s rRNA sequence, an rpoB gene, a katG gene, an inhA gene, an ahpC gene, a gyrA gene and a gyrB gene.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, the present invention relates to a composition and a kit for detecting Mycobacterium tuberculosis and its drug resistance. Background Art

[0002] Mycobacterium tuberculosis (MTB) is the main pathogen that causes tuberculosis in humans and belongs to the Mycobacterium tuberculosis complex (MTBC). Tuberculosis is a chronic infectious disease in humans or animals that is transmitted through the respiratory tract and mainly invades the lungs, with pulmonary tuberculosis being the most common. Most patients present with systemic symptoms such as low fever, fatigue, and night sweats, and respiratory manifestations such as cough, sputum, and hemoptysis. Tuberculosis is a public health problem that seriously endangers public health. At the same time, nontuberculous mycobacteria (NTM) diseases are also increasing rapidly. The clinical manifestations of NTM disease are similar to those of tuberculosis, and in the absence of species identification results, it is often misdiagnosed as tuberculosis. A large number of studies and clinical data have shown that NTM is resistant to most general anti-tuberculosis drugs and has a very high natural resistance rate. If misdiagnosed as tuberculosis, it often leads to treatment failure. Therefore, the detection of MTB and NTM differentiation is extremely important for the early diagnosis, differential diagnosis, individualized effective treatment and control of transmission of tuberculosis. In addition, due to the irregular anti-tuberculosis treatment and the abuse of immunosuppressants, drug-resistant tuberculosis occurs frequently.

[0003] The drug resistance of Mycobacterium tuberculosis includes many categories, such as rifampicin (RFP) resistance, isohexadecanidine (INH) resistance, fluoroquinolone (FLQ) resistance, etc. In clinical practice, different treatment strategies are often used for different types of Mycobacterium tuberculosis resistance. If misdiagnosed as the wrong type of Mycobacterium tuberculosis resistance, it may lead to the use of wrong treatment methods, which will not only delay the effective treatment of patients, but also create a greater risk of transmission. However, the traditional detection method of tuberculosis resistance test is mainly drug sensitivity test, which is complicated to operate and has a culture cycle of at least 4 weeks, which is not conducive to the early diagnosis of Mycobacterium tuberculosis resistance.

[0004] Therefore, there is an urgent need in the art for a technology and product that can accurately distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria and accurately determine the drug resistance category of Mycobacterium tuberculosis. Summary of the invention

[0005] In view of the lack of detection methods, reagents and kits for accurately distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria and accurately judging the drug resistance category of Mycobacterium tuberculosis in the art, the inventors have conducted in-depth research and provided a combination of primers and probes suitable for distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria and accurately judging the drug resistance category of Mycobacterium tuberculosis. The combination of the primers and probes is used for detection, which has good accuracy, can distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria, and can also accurately identify the drug resistance category of Mycobacterium tuberculosis. At the same time, the detection only requires the collection of sputum samples, and the detection process is simple, which is particularly suitable for screening early Mycobacterium tuberculosis and its drug resistance in a large population.

[0006] The object of the present invention is to provide a composition and a kit for detecting Mycobacterium tuberculosis and its drug resistance.

[0007] In a first aspect of the present invention, a composition of primers and probes for detecting Mycobacterium tuberculosis and its drug resistance is provided, wherein the genes targeted by the primers and probes include: IS6110 insertion sequence, IS1081 insertion sequence, 16s rRNA sequence, rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene.

[0008] In one or more embodiments, the primers and probes are used to detect mutations in genes associated with drug resistance for treating tuberculosis, comprising or consisting of mutations selected from the group consisting of:

[0009] mutations in the rpoB gene 516GAC>GTC, 531TCG>TTG, 533CTG>CCG, 511CTG>CCG, 513CAA>AAA, 522TCG>CAG, 526CAC>GAC, 526CAC>CTC, 526CAC>CGC, 526CAC>TAC, and 531TCG>TGG;

[0010] mutations of 315AGC>ACC and 315AGC>ACA in the katG gene;

[0011] mutations of -15C>T, -8T>C, and -8T>A in the inhA gene;

[0012] mutations of -32G>A and -10C>A in the ahpC gene;

[0013] mutations of 88GGC>TGC, 90GCG>GTG, 94GAC>GGC, 94GAC>AAC, and 94GAC>GCC in the gyrA gene;

[0014] Mutations 551GGG>AGG and 540GAA>GAC in the gyrB gene.

[0015] In one or more embodiments, mutations in the rpoB gene are associated with rifampicin resistance, mutations in the katG gene, the inhA gene, and the ahpC gene are associated with isoniazid resistance; and mutations in the gyrA gene and the gyrB gene are associated with fluoroquinolone resistance.

[0016] In one or more embodiments, the primer sequence for the rpoB gene is shown in SEQ ID NOs: 1-2, the primer sequence for the katG gene is shown in SEQ ID NOs: 14-15, the primer sequence for the inhA gene is shown in SEQ ID NOs: 18-19, the primer sequence for the ahpC gene is shown in SEQ ID NOs: 23-24, the primer sequence for the gyrA gene is shown in SEQ ID NOs: 27-28, the primer sequence for the gyrB gene is shown in SEQ ID NOs: 34-35, the primer sequence for the IS6110 insertion sequence is shown in SEQ ID NOs: 38-39, the primer sequence for the IS1081 insertion sequence is shown in SEQ ID NOs: 41-42, and / or the primer sequence for 16s rRNA is shown in SEQ ID NOs: 44-45.

[0017] In one or more embodiments, the sequences of the upstream primer and the downstream primer for the rpoB gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the sequences of the upstream primer and the downstream primer for the katG gene are shown in SEQ ID NO:14 and SEQ ID NO:15, respectively; the sequences of the upstream primer and the downstream primer for the inhA gene are shown in SEQ ID NO:18 and SEQ ID NO:19, respectively; the sequences of the upstream primer and the downstream primer for the ahpC gene are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the sequences of the upstream primer and the downstream primer for the gyrA gene are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively; the sequences of the upstream primer and the downstream primer for the gyrB gene are shown in SEQ ID NO:34 and SEQ ID NO:35, respectively; the sequences of the upstream primer and the downstream primer for the IS6110 insertion sequence are shown in SEQ ID NO:38 and SEQ ID NO:39, respectively; the sequences of the upstream primer and the downstream primer for the IS1081 insertion sequence are shown in SEQ ID NO:39 and SEQ ID NO:31, respectively. NO:41 and SEQ ID NO:42; the sequences of the upstream primer and downstream primer for 16s rRNA are shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.

[0018] In one or more embodiments, the probe sequence for the rpoB gene is shown in SEQ ID NOs:3-13, the probe sequence for the katG gene is shown in SEQ ID NOs:16-17, the probe sequence for the inhA gene is shown in SEQ ID NOs:20-22, the probe sequence for the ahpC gene is shown in SEQ ID NOs:25-26, the probe sequence for the gyrA gene is shown in SEQ ID NOs:29-33, the probe sequence for the gyrB gene is shown in SEQ ID NOs:36-37, the probe sequence for the IS6110 insertion sequence is shown in SEQ ID NO:40, the probe sequence for the IS1081 insertion sequence is shown in SEQ ID NO:43, and the probe sequence for 16s rRNA is shown in SEQ ID NO:46.

[0019] In one or more embodiments, the 5' end and 3' end of the probe are modified with a fluorescent group and a quencher group, respectively.

[0020] In one or more embodiments, the fluorescent group includes: FAM, HEX, ROX, CY5 and / or CY5.5.

[0021] In one or more embodiments, the quencher group includes: BHQ1, BHQ2 and / or MGB.

[0022] In one or more embodiments, the probe for rpoB gene is labeled with FAM fluorescent marker, the probes for katG gene, inhA gene and ahpC gene are labeled with HEX fluorescent marker, the probes for gyrA gene and gyrB gene are labeled with ROX fluorescent marker, the probes for IS6110 insertion sequence and IS1081 insertion sequence are labeled with ROX fluorescent marker, and the probe for 16srRNA is labeled with CY5 fluorescent marker.

[0023] In one or more embodiments, the composition further comprises primers and probes for internal reference genes, external reference genes and / or discriminant genes.

[0024] In one or more embodiments, the internal reference gene includes: a ribonuclease P (RNase P) gene.

[0025] In one or more embodiments, the external reference gene includes: Bacillus subtilis 16s rRNA gene.

[0026] In one or more embodiments, the discriminant gene is a conserved region outside the drug resistance determining region RRDR of the rpoB gene.

[0027] In one or more embodiments, the primer sequence for the internal reference gene is as shown in SEQ ID NO:47-48, more preferably including the upstream primer shown in SEQ ID NO:47 and the downstream primer shown in SEQ ID NO:48; the probe sequence for the internal reference gene is as shown in SEQ ID NO:49.

[0028] In one or more embodiments, the primer sequence for the external reference gene is as shown in SEQ ID NO:50-51, more preferably including the upstream primer shown in SEQ ID NO:50 and the downstream primer shown in SEQ ID NO:51; the probe sequence for the external reference gene is as shown in SEQ ID NO:52.

[0029] In one or more embodiments, the primer sequence for the discriminant gene is as shown in SEQ ID NO:53-54, more preferably including the upstream primer shown in SEQ ID NO:53 and the downstream primer shown in SEQ ID NO:54; the probe sequence for the external reference gene is as shown in SEQ ID NO:55.

[0030] In one or more embodiments, the primer and probe combination comprises:

[0031] Composition 1, comprising or consisting of the following probes:

[0032] Probes for the rpoB gene as shown in SEQ ID NOs:6-13, probes for the katG gene as shown in SEQ ID NOs:16-17, probes for the inhA gene as shown in SEQ ID NOs:20-22, probes for the ahpC gene as shown in SEQ ID NOs:25-26, probes for the IS6110 insertion sequence as shown in SEQ ID NO:40, probes for the IS1081 insertion sequence as shown in SEQ ID NO:43, and probes for 16s rRNA as shown in SEQ ID NO:46;

[0033] Composition 1 also includes or consists of the following primers:

[0034] Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 14-15 for the katG gene, primers as shown in SEQ ID NOs: 18-19 for the inhA gene, primers as shown in SEQ ID NOs: 23-24 for the ahpC gene, primers as shown in SEQ ID NOs: 38-39 for the IS6110 insertion sequence, primers as shown in SEQ ID NOs: 41-42 for the IS1081 insertion sequence, and primers as shown in SEQ ID NOs: 44-45 for 16s rRNA;

[0035] Composition 2, comprising or consisting of the following probes:

[0036] Probes for the rpoB gene as shown in SEQ ID NOs: 3-5, probes for the gyrA gene as shown in SEQ ID NOs: 29-33, and probes for the gyrB gene as shown in SEQ ID NOs: 36-37;

[0037] Composition 2 also includes or consists of the following primers:

[0038] Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 27-28 for the gyrA gene, and primers as shown in SEQ ID NOs: 34-35 for the gyrB gene;

[0039] Wherein, composition 1 and composition 2 are not mixed in the same reaction system.

[0040] In one or more embodiments, the internal reference gene is set in composition 2, and / or the discriminant gene is set in composition 2, and / or the external reference gene is set in composition 1 and composition 2 simultaneously, separately or sequentially.

[0041] The second aspect of the present invention provides use of the composition according to any embodiment of the present invention in preparing a detection reagent, a kit or a detection device for detecting Mycobacterium tuberculosis and its drug resistance.

[0042] In one or more embodiments, the concentration of the primer and probe combination is 100nM to 300nM, preferably 100-150nM, 100-200nM, 100-250nM, 150-200nM, 150-250nM, 200-250nM, 150-300nM or 200-300nM.

[0043] In one or more embodiments, the detection reagents further include one, more or all of the following reagents:

[0044] The liquefied liquid more preferably comprises sodium hydroxide, more preferably 30% to 50% sodium hydroxide;

[0045] A neutralization inactivation solution, more preferably comprising isopropanol, Triton X-100 and glacial acetic acid, more preferably 10% to 40% isopropanol, 2% to 20% Triton X-100 and 50% to 80% glacial acetic acid;

[0046] Lysis and binding solution, more preferably comprising sodium perchlorate and magnetic bead solution, more preferably 100% sodium perchlorate and 10 μL magnetic bead solution;

[0047] The washing solution more preferably comprises ethanol, more preferably 80% ethanol;

[0048] The eluent more preferably comprises Tris-HCl, more preferably 0.0001 to 1 M Tris-HCl;

[0049] Reagents used in PCR reactions.

[0050] In one or more embodiments, the kit includes the detection reagent described in any one of the embodiments.

[0051] In one or more embodiments, the primer and probe composition is present in the kit in a lyophilized form, and more preferably, composition 1 and composition 2 are two lyophilized reagents placed in different chambers or containers.

[0052] In one or more embodiments, the detection device further comprises: a nucleic acid analysis instrument, preferably a PCR instrument.

[0053] The third aspect of the present invention provides a detection reagent, a kit or a detection device for detecting Mycobacterium tuberculosis and its drug resistance, wherein the detection reagent, the kit or the detection device comprises the composition described in any embodiment of the present invention.

[0054] In one or more embodiments, the detection reagent, kit or detection device is as described in any embodiment of the present invention.

[0055] A fourth aspect of the present invention provides a method for detecting Mycobacterium tuberculosis and its drug resistance, the method comprising:

[0056] 1) contacting the sample to be tested with a detection reagent, a kit or a detection device comprising the primer and probe combination described in any embodiment of the present invention;

[0057] 2) obtaining the test results of the sample to be tested in 1);

[0058] 3) According to the test results in 2), analyzing whether Mycobacterium tuberculosis is positive and analyzing the type of drug resistance of Mycobacterium tuberculosis.

[0059] In one or more embodiments, the sample to be tested includes: plasma, serum, sputum, urine, feces, body fluid; preferably, the sample to be tested is a sputum sample.

[0060] In one or more embodiments, the detection reagent, kit or detection device is as described in any embodiment of the present invention.

[0061] In one or more embodiments, step 2) of the method further includes: analyzing the test results of internal references and / or external references: if the test results of the internal references and / or external references meet the requirements, step 3) is performed; if the test results of the internal references and / or external references do not meet the requirements, step 3) is not performed.

[0062] In one or more embodiments, the requirements for the detection results of the internal reference and / or external reference are: the internal reference and / or external reference present a typical S-shaped amplification curve and the Ct value of the internal reference and / or external reference is ≤35; preferably, the typical S-shaped amplification curve includes an S curve that has not reached the plateau phase.

[0063] In one or more embodiments, whether Mycobacterium tuberculosis is positive is analyzed based on the detection results of 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence, and the type of drug resistance of Mycobacterium tuberculosis is analyzed based on the detection results of rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene, gyrB gene and discriminant gene.

[0064] In one or more embodiments, the detection results of 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence are analyzed separately to see whether they meet the requirements. If only 16s rRNA meets the requirements, the sample to be tested is considered to be a sample of non-tuberculous mycobacteria; if 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence all meet the requirements, the sample to be tested is considered to be a sample positive for Mycobacterium tuberculosis; if 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence all do not meet the requirements, the sample to be tested is considered to be a sample negative for Mycobacterium.

[0065] In one or more embodiments, the detection result of analyzing 16s rRNA meets the requirements that: 16s rRNA presents a typical S-shaped amplification curve (including an S curve that has not reached a plateau phase) and Ct≤35.

[0066] In one or more embodiments, the detection results for 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence all meet the requirements that: Mycobacterium 16s rRNA presents a typical S-shaped amplification curve with Ct≤35, IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve with Ct≤30; preferably, the typical S-shaped amplification curve includes an S curve that has not reached a plateau phase.

[0067] In one or more embodiments, the presence of mutations is analyzed based on the detection results of the rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene, gyrB gene and the discriminant gene, and the type of drug resistance of Mycobacterium tuberculosis is determined based on the type of mutated gene.

[0068] In one or more embodiments, if there is an rpoB gene mutation, the drug resistance type is rifampicin (RFP) resistance; if there is an inhA, katG and / or ahpC gene mutation, the drug resistance type is isohexamidine (INH) resistance; if there is a gyrA and / or gyrB gene mutation, the drug resistance type is fluoroquinolone (FLQ) drug resistance.

[0069] In one or more implementation schemes, the detection results of the drug resistance genes rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene and the discriminant gene are analyzed respectively to see whether they meet the requirements. If they meet the requirements, it is considered that the drug resistance gene has a mutation; if they do not meet the requirements, it is considered that the drug resistance gene does not have a mutation.

[0070] In one or more embodiments, the requirements for the detection results of the drug-resistant gene are: the mycobacterium 16s rRNA presents a typical S-type amplification curve and Ct≤35, the IS6110 insertion sequence and the IS1081 insertion sequence present a typical S-type amplification curve and Ct≤30, the discriminant gene presents a typical S-type amplification curve and Ct≤35, the drug-resistant gene presents a typical S-type amplification curve and Ct value≤35, and the Ct difference between the corresponding drug-resistant gene mutant detection target and the discriminant gene is less than 4; preferably, the typical S-type amplification curve includes an S curve that has not reached the plateau phase.

[0071] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. DETAILED DESCRIPTION

[0072] The inventors designed a large number of primers and probes for the IS6110 and IS1081 insertion sequences specific to the Mycobacterium tuberculosis complex, the mycobacterium-specific fragment 16s rRNA sequence, the rifampicin resistance gene rpoB gene, the isoniazid resistance gene katG gene, the inhA gene and the ahpC gene, and the fluoroquinolone resistance gene gyrA gene and the gyrB gene. After thousands of attempts at primer and probe combinations, they finally screened a primer and probe combination that has extremely excellent accuracy in distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria and accurately determining the drug resistance category of Mycobacterium tuberculosis, which is conducive to the early diagnosis of Mycobacterium tuberculosis and its drug resistance.

[0073] Herein, the "IS6110" refers to the insertion sequence 6110 that exists only in the Mycobacterium tuberculosis complex, and its Genomic Sequence can be referenced to NC_000962.3 (3486509..3487711).

[0074] Herein, the "IS1081" refers to the insertion sequence 1081 that exists only in the Mycobacterium tuberculosis complex, and its Genomic Sequence can be referenced to NC_000962.3 (2828556..2829803, complement).

[0075] Herein, the "16s rRNA" refers to the mycobacterium-specific fragment 16s rRNA sequence, and its Genomic Sequence can be referenced to NC_000962.3 (1471846..1473382).

[0076] Herein, the "drug resistance gene" or "drug resistance gene" generally refers to a mutation in the gene that is related to drug resistance to tuberculosis drugs. The "drug resistance" can be single drug resistance to a certain tuberculosis drug, or multiple drug resistance to multiple tuberculosis drugs. Drugs for treating tuberculosis that may produce drug resistance include, but are not limited to: rifampicin (RFP), isoniazid (INH), fluoroquinolone (FLQ) drugs (such as ofloxacin, ciprofloxacin, moxifloxacin, gatifloxacin, etc.), pyrazinamide (PZA), streptomycin (SM), ethambutol (EMB) and aminoglycosides (such as amikacin, kanamycin, amikacin, etc.).

[0077] Herein, the "rpoB" refers to the rifampicin resistance gene rpoB gene, and its Genomic Sequence can refer to NC_000962.3 (759807..763325). Mutations associated with rifampicin resistance in the rpoB gene include but are not limited to: 516GAC>GTC, 531TCG>TTG, 533CTG>CCG, 511CTG>CCG, 513CAA>AAA, 522TCG>CAG, 526CAC>GAC, 526CAC>CTC, 526CAC>CGC, 526CAC>TAC, and 531TCG>TGG.

[0078] Herein, the "katG" refers to the isoniazid resistance gene katG gene, and its Genomic Sequence can refer to NC_000962.3 (2153889..2156111, complement). Mutations in the katG gene associated with isoniazid resistance include but are not limited to: 315AGC>ACC and 315AGC>ACA.

[0079] Herein, the "inhA" refers to the isoniazid resistance gene inhA gene, and its Genomic Sequence can be referenced to NC_000962.3 (1674202..1675011). Mutations associated with isoniazid resistance in the inhA gene include but are not limited to: -15C>T, -8T>C, and -8T>A.

[0080] Herein, the "ahpC" refers to the isoniazid resistance gene ahpC gene, and its Genomic Sequence can be referenced to NC_000962.3 (2726193..2726780). Mutations associated with isoniazid resistance in the ahpC gene include but are not limited to: -32G>A and -10C>A.

[0081] Herein, the "gyrA" refers to the fluoroquinolone resistance gene gyrA gene, and its Genomic Sequence can be referenced to NC_000962.3 (7302..9818). Mutations associated with isoniazid resistance in the gyrA gene include but are not limited to: 88GGC>TGC, 90GCG>GTG, 94GAC>GGC, 94GAC>AAC and 94GAC>GCC.

[0082] Herein, the "gyrB" refers to the fluoroquinolone resistance gene gyrB gene, and its Genomic Sequence can be referenced to NC_000962.3 (5240..7267). Mutations in the gyrB gene associated with isoniazid resistance include but are not limited to: 551GGG>AGG and 540GAA>GAC.

[0083] Combinations of primers and probes

[0084] Herein, the "primer" refers to a nucleic acid molecule with a specific nucleotide sequence that guides synthesis at the start of nucleotide polymerization. Primers are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region, and its function is to serve as the starting point of nucleotide polymerization. In vitro artificially designed primers can be used for polymerase chain reaction (PCR), qPCR, sequencing and probe synthesis, etc. Usually, primers are designed to be 50-150bp, 60-140bp, 70-130bp or 80-120bp in length for amplification. Herein, the primers are usually primers for specific genes or for a certain region or fragment of the gene.

[0085] Herein, the "probe" refers to a single-stranded nucleic acid with a known nucleotide sequence, which has a nucleotide sequence structure that is substantially complementary to the target nucleic acid and can form a double strand with the "target nucleic acid". The "probe" can carry a detectable marker. The detectable marker includes (but is not limited to): various radioactive parts, enzymes, prosthetic groups, fluorescent markers, bioluminescent markers, metal particles, protein-protein binding pairs, protein-antibody binding pairs, etc. Examples of fluorescent markers include, but are not limited to: FAM, HEX, ROX, CY5, CY5.5, etc. Examples of bioluminescent markers include, but are not limited to, luciferase (such as bacteria, fireflies, click beetles, etc.), luciferin, photoproteins, etc. Different detectable markers can be set for different genes, so that different genes can be distinguished. For example, fluorescent markers of different colors can be set for different genes for distinction.

[0086] The invention provides a composition of primers and probes for detecting Mycobacterium tuberculosis and its drug resistance, wherein the genes targeted by the primers and probes include: IS6110 insertion sequence, IS1081 insertion sequence, 16s rRNA sequence, rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene.

[0087] In one or more embodiments, the primers and probes are used to detect mutations in genes associated with drug resistance for treating tuberculosis, including:

[0088] mutations in the rpoB gene 516GAC>GTC, 531TCG>TTG, 533CTG>CCG, 511CTG>CCG, 513CAA>AAA, 522TCG>CAG, 526CAC>GAC, 526CAC>CTC, 526CAC>CGC, 526CAC>TAC, and 531TCG>TGG;

[0089] mutations of 315AGC>ACC and 315AGC>ACA in the katG gene;

[0090] mutations of -15C>T, -8T>C, and -8T>A in the inhA gene;

[0091] mutations of -32G>A and -10C>A in the ahpC gene;

[0092] mutations of 88GGC>TGC, 90GCG>GTG, 94GAC>GGC, 94GAC>AAC, and 94GAC>GCC in the gyrA gene;

[0093] Mutations 551GGG>AGG and 540GAA>GAC in the gyrB gene.

[0094] In one or more embodiments, mutations in the rpoB gene are associated with rifampicin resistance, mutations in the katG gene, the inhA gene, and the ahpC gene are associated with isoniazid resistance; and mutations in the gyrA gene and the gyrB gene are associated with fluoroquinolone resistance.

[0095] In one or more embodiments, the primer sequence for the rpoB gene is shown in SEQ ID NOs: 1-2 (preferably including the upstream primer shown in SEQ ID NO: 1 and the downstream primer shown in SEQ ID NO: 2), the primer sequence for the katG gene is shown in SEQ ID NOs: 14-15 (preferably including the upstream primer shown in SEQ ID NO: 14 and the downstream primer shown in SEQ ID NO: 15), the primer sequence for the inhA gene is shown in SEQ ID NOs: 18-19 (preferably including the upstream primer shown in SEQ ID NO: 18 and the downstream primer shown in SEQ ID NO: 19), the primer sequence for the ahpC gene is shown in SEQ ID NOs: 23-24 (preferably including the upstream primer shown in SEQ ID NO: 23 and the downstream primer shown in SEQ ID NO: 24), the primer sequence for the gyrA gene is shown in SEQ ID NOs: 27-28 (preferably including the upstream primer shown in SEQ ID NO: 27 and the downstream primer shown in SEQ ID NO: 28), and the primer sequence for the gyrB gene is shown in SEQ ID NOs: The primer sequences for the IS6110 insertion sequence are shown in SEQ ID NOs:38-39 (preferably including the upstream primer shown in SEQ ID NO:38 and the downstream primer shown in SEQ ID NO:39), the primer sequences for the IS1081 insertion sequence are shown in SEQ ID NOs:41-42 (preferably including the upstream primer shown in SEQ ID NO:41 and the downstream primer shown in SEQ ID NO:42), and the primer sequences for 16s rRNA are shown in SEQ ID NOs:44-45 (preferably including the upstream primer shown in SEQ ID NO:44 and the downstream primer shown in SEQ ID NO:45).

[0096] In one or more embodiments, the probe sequence for the rpoB gene is shown in SEQ ID NOs: 3-13 (preferably, the probe is labeled with FAM fluorescence), the probe sequence for the katG gene is shown in SEQ ID NOs: 16-17 (preferably, the probe is labeled with HEX fluorescence), the probe sequence for the inhA gene is shown in SEQ ID NOs: 20-22 (preferably, the probe is labeled with HEX fluorescence), the probe sequence for the ahpC gene is shown in SEQ ID NOs: 25-26 (preferably, the probe is labeled with HEX fluorescence), the probe sequence for the gyrA gene is shown in SEQ ID NOs: 29-33 (preferably, the probe is labeled with ROX fluorescence), the probe sequence for the gyrB gene is shown in SEQ ID NOs: 36-37 (preferably, the probe is labeled with ROX fluorescence), the probe sequence for the IS6110 insertion sequence is shown in SEQ ID NO: 40 (preferably, the probe is labeled with ROX fluorescence), and the probe sequence for the IS1081 insertion sequence is shown in SEQ ID NO: 41. NO:43 (preferably the probe is fluorescently labeled with ROX), and the probe sequence for 16s rRNA is shown in SEQ ID NO:46 (preferably the probe is fluorescently labeled with CY5). The probe can be modified with a fluorescent group and a quenching group, respectively. For example, the 5' end and the 3' end of the probe are modified with a fluorescent group and a quenching group, respectively. The fluorescent group includes, but is not limited to, FAM, HEX, ROX, CY5, and CY5.5. The quenching group includes, but is not limited to, BHQ1, BHQ2, and MGB.

[0097] In the present invention, when multiple primers and probes for multiple genes are used, the inventors innovatively split all primers and probes into two or more compositions, thereby reducing the mutual interference between different primers and probes and improving the accuracy of the detection results. In one or more embodiments, the composition of primers and probes includes:

[0098] Composition 1, comprising or consisting of the following probes:

[0099] Probes for the rpoB gene as shown in SEQ ID NOs:6-13, probes for the katG gene as shown in SEQ ID NOs:16-17, probes for the inhA gene as shown in SEQ ID NOs:20-22, probes for the ahpC gene as shown in SEQ ID NOs:25-26, probes for the IS6110 insertion sequence as shown in SEQ ID NO:40, probes for the IS1081 insertion sequence as shown in SEQ ID NO:43, and probes for 16s rRNA as shown in SEQ ID NO:46;

[0100] Composition 1 also includes or consists of the following primers:

[0101] Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 14-15 for the katG gene, primers as shown in SEQ ID NOs: 18-19 for the inhA gene, primers as shown in SEQ ID NOs: 23-24 for the ahpC gene, primers as shown in SEQ ID NOs: 38-39 for the IS6110 insertion sequence, primers as shown in SEQ ID NOs: 41-42 for the IS1081 insertion sequence, and primers as shown in SEQ ID NOs: 44-45 for 16s rRNA;

[0102] Composition 2, comprising or consisting of the following probes:

[0103] Probes for the rpoB gene as shown in SEQ ID NOs: 3-5, probes for the gyrA gene as shown in SEQ ID NOs: 29-33, and probes for the gyrB gene as shown in SEQ ID NOs: 36-37;

[0104] Composition 2 also includes or consists of the following primers:

[0105] Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 27-28 for the gyrA gene, and primers as shown in SEQ ID NOs: 34-35 for the gyrB gene;

[0106] Wherein, composition 1 and composition 2 are not mixed in the same reaction system.

[0107] In order to determine the drug resistance of the rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and / or gyrB gene, a discriminant gene can be set. The discriminant gene is usually a conserved region outside the resistance-determining region of the rpoB resistance gene. In some specific embodiments, the discriminant gene is located in a conserved region outside the resistance-determining region RRDR of the rpoB gene of the Mycobacterium tuberculosis complex. Specifically, the composition of primers and probes also includes: primers and probes for discriminant genes, for example, primers as shown in SEQ ID NO:53-54 for discriminant genes (preferably including upstream primers as shown in SEQ ID NO:53 and downstream primers as shown in SEQ ID NO:54) and probes as shown in SEQ ID NO:55. Preferably, the discriminant gene is set in composition 2.

[0108] It should be noted that when detecting the IS6110 insertion sequence, the IS1081 insertion sequence, the 16s rRNA sequence, the rpoB gene, the katG gene, the inhA gene, the ahpC gene, the gyrA gene and the gyrB gene, internal references and / or external references may be used.

[0109] The internal reference is a gene present in the sample to be tested, whose expression is not interfered by the target gene. As an internal reference, it can be used to detect the quality of the sample to be tested, calibrate the expression level of the target gene, and / or provide a reference for real-time fluorescence quantitative curve analysis. In one or more embodiments, the internal reference can be, for example, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribonuclease P (RNase P), etc. Specifically, the composition of primers and probes also includes: primers and probes for internal reference genes, for example, primers as shown in SEQ ID NO:47-48 for the RNase P internal reference gene (preferably including the upstream primer shown in SEQ ID NO:47 and the downstream primer shown in SEQ ID NO:48) and the probe as shown in SEQ ID NO:49. Preferably, the internal reference gene is set in composition 2.

[0110] The external reference is a gene that does not exist in the sample to be tested, which is artificially added to the sample to be tested (such as a sputum sample) and can be used as a quality control product to detect whether the entire experimental process is normal and whether the result is credible. In one or more embodiments, the external reference can be, for example, Bacillus subtilis subsp.Natto, Bacillus subtilis subsp.Subtilis, Bacillus subtilis subsp.spizizenii. Specifically, the composition of the primers and probes also includes: primers and probes for external reference genes, for example, primers as shown in SEQ ID NO:50-51 for the external reference gene of Bacillus subtilis subsp.spizizenii (preferably including the upstream primer shown in SEQ ID NO:50 and the downstream primer shown in SEQ ID NO:51) and the probe as shown in SEQ ID NO:52. Preferably, the external reference genes are set in composition 1 and composition 2 simultaneously, separately or sequentially.

[0111] Detection reagents, kits

[0112] The present invention also provides a detection reagent for detecting Mycobacterium tuberculosis and its drug resistance, the reagent comprising the combination of primers and probes of the present invention. The concentration of the primers and probes can be 100nM to 300nM, such as 100-150nM, 100-200nM, 100-250nM, 150-200nM, 150-250nM, 200-250nM, 150-300nM or 200-300nM.

[0113] The detection reagent may also include (but not limited to): a liquefaction solution, preferably including sodium hydroxide, such as 30% to 50% sodium hydroxide; a neutralization inactivation solution, preferably including isopropanol, Triton X-100 and glacial acetic acid, such as 10% to 40% isopropanol, 2% to 20% Triton X-100 and 50% to 80% glacial acetic acid; a lysis binding solution, preferably including sodium perchlorate and a magnetic bead solution, such as 100% sodium perchlorate and 10 μL magnetic bead solution; a washing solution, preferably including ethanol, such as 80% ethanol (the washing solution can be set according to the number of washing times required, for example, if three washings are required, three sets of washing solutions can be set); an eluting solution, preferably including Tris-HCl, such as 0.0001 to 1 M Tris-HCl; reagents for PCR reaction, preferably including an amplification buffer, dNTPs (including modified dNTPs), a polymerase or a nicking enzyme (such as Taq DNA polymerase) and a Mg-containing 2+ of solution.

[0114] The present invention also provides a kit for detecting Mycobacterium tuberculosis and its drug resistance, wherein the kit comprises the combination of the primer and the probe described in the present invention, or the detection reagent described in the present invention.

[0115] The kit may also include a system for storing, transporting, or delivering reagents (e.g., probes, primers, enzymes, etc. in appropriate containers) and / or coordinating materials (e.g., buffers, written instructions for performing the assessment, etc.) from one location to another. For example, the kit may include one or more housings (e.g., boxes) containing the relevant reagents and / or coordinating materials. These contents may be used simultaneously or separately. The kit includes a composition of primers and probes of the present invention (preferably, composition 1 and composition 2 are used separately).

[0116] The components of the kit can, for example, be present in one or more containers, for example, all components can be present in different chambers of a container as needed (e.g., composition 1 and composition 2 are located in different chambers of the same container), or, for example, all components are present in different containers (e.g., composition 1 and composition 2 are located in different containers). The components can be, for example, dried (e.g., powdered) or in a stable buffer (e.g., chemically stable, thermally stable). The dried components can be prepared, for example, by lyophilization, vacuum and centrifugal assisted drying and / or ambient drying. In a specific embodiment, composition 1 and composition 2 are present in lyophilized form and are lyophilized, freeze-dried, or in a buffer in different containers or different chambers of the same container. More specifically, when the composition of primers and probes described in the present invention is applied to a PCR reaction system, the concentration of the primers and probes can be 100nM to 300nM, for example, 100-150nM, 100-200nM, 100-250nM, 150-200nM, 150-250nM, 200-250nM, 150-300nM or 200-300nM.

[0117] For ease of operation, the kit may be provided with multiple reagent chambers and a driving device that can connect the reagent chambers at different angles. The driving device can be used to extract nucleic acids from the sample to be tested, enrich nucleic acids (for example, enrich the nucleic acids to be tested by reverse transcription or magnetic bead adsorption), and PCR amplification to achieve integration from nucleic acid extraction to test result output. Preferably, the kit can also cooperate with a nucleic acid detection analyzer to achieve fully enclosed automatic output of samples to gene test results, and realize point of care testing (POCT) of Mycobacterium tuberculosis and its drug resistance.

[0118] In addition, the kit may also include instructions for use and / or analysis software for describing the use of the kit of the present invention in detecting Mycobacterium tuberculosis and its drug resistance.

[0119] Applications and methods

[0120] The present invention also provides the use of the combination of primers and probes described in the present invention in preparing a detection reagent, a kit or a detection device for detecting Mycobacterium tuberculosis and its drug resistance. The present invention also provides the use of the detection reagent and kit described in the present invention in preparing a detection device for detecting Mycobacterium tuberculosis and its drug resistance.

[0121] The present invention also provides a method for detecting Mycobacterium tuberculosis and its drug resistance, comprising:

[0122] 1) contacting the sample to be tested with a detection reagent comprising the primer and probe combination;

[0123] 2) obtaining the test results of the sample to be tested in 1);

[0124] 3) According to the test results in 2), analyzing whether Mycobacterium tuberculosis is positive and analyzing the type of drug resistance of Mycobacterium tuberculosis.

[0125] For the detection with internal reference and / or external reference, step 2) of the method further includes: analyzing the detection results of the internal reference and / or external reference: if the detection results of the internal reference and / or external reference meet the requirements, step 3) is performed; if the detection results of the internal reference and / or external reference do not meet the requirements, step 3) is not performed. The requirements that the detection results of the internal reference and / or external reference meet can be: the internal reference and / or external reference present a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase) and the Ct value of the internal reference and / or external reference is ≤35.

[0126] When analyzing whether Mycobacterium tuberculosis is positive, a comprehensive judgment can be made based on the test results (such as Ct value) of 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence. For example, the test results of 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence can be analyzed separately to see whether they meet the requirements. If only 16s rRNA meets the requirements, the sample to be tested is considered to be a sample of non-tuberculous mycobacteria; if 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence all meet the requirements, the sample to be tested is considered to be a sample positive for Mycobacterium tuberculosis; if 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence all do not meet the requirements, the sample to be tested is considered to be a sample negative for Mycobacterium. The requirements for the test results of 16s rRNA analysis to meet can be: 16s rRNA presents a typical S-type amplification curve (including an S curve that has not reached the plateau phase) and Ct≤35. The requirements that the detection results of analyzing 16s rRNA, IS6110 insertion sequence, IS1081 insertion sequence and discriminant gene all meet can be: 16s rRNA presents a typical S-shaped amplification curve (including an S curve that has not reached a plateau phase) and Ct≤35, and both the IS6110 insertion sequence and the IS1081 insertion sequence present a typical S-shaped amplification curve (including an S curve that has not reached a plateau phase) and the Ct value is ≤30.

[0127] When analyzing the types of drug resistance of Mycobacterium tuberculosis, a comprehensive judgment can be made based on the detection results of the drug resistance genes (rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene) and the discriminant genes (such as Ct value). For example, the detection results of the rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene can be analyzed separately to see whether they meet the requirements. If they meet the requirements, it is considered that the drug resistance gene has a mutation. If they do not meet the requirements, it is considered that the drug resistance gene does not have a mutation. The requirements for the detection results of the analysis of drug-resistant genes may be: Mycobacterium 16srRNA presents a typical S-shaped amplification curve (including an S-shaped curve that has not reached the plateau phase) and Ct≤35, IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve (including an S-shaped curve that has not reached the plateau phase) and Ct≤30, the discriminant gene presents a typical S-shaped amplification curve (including an S-shaped curve that has not reached the plateau phase) and Ct≤35, the drug-resistant gene presents a typical S-shaped amplification curve (including an S-shaped curve that has not reached the plateau phase) and Ct value≤35, and the Ct difference between the corresponding drug-resistant gene mutant detection target and the discriminant gene is less than 4.

[0128] Further, the type of drug resistance of Mycobacterium tuberculosis can be determined according to the type of drug resistance gene with mutation. For example, after judging whether the rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene are mutated respectively: a) if there is a mutation in the rpoB gene, the drug resistance type is rifampicin (RFP) resistance; b) if there is a mutation in the inhA, katG and / or ahpC gene, the drug resistance type is isohexadecanidine (INH) resistance; c) if there is a mutation in the gyrA and / or gyrB gene, the drug resistance type is fluoroquinolone (FLQ) drug resistance. In certain embodiments, if there are any two or all three mutations in a), b), and c), the drug resistance type is cross-resistance to the corresponding two or all three groups of tuberculosis drugs.

[0129] Advantages of the present invention include:

[0130] (1) The primer and probe combination of the present invention detects multiple sites of multiple genes, thereby accurately distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria and accurately determining the drug resistance category of Mycobacterium tuberculosis.

[0131] (2) The combination of primers and probes of the present invention, in conjunction with a nucleic acid detection analyzer, can achieve automation and integration from nucleic acid extraction to test result output. It has a high degree of automation, no risk of cross contamination, no need for professional operation, simple steps and short time consumption. The entire process from sample pretreatment to result output can be completed within 90 minutes.

[0132] (3) The combination of primers and probes of the present invention is divided into combination 1 and combination 2. Combination 1 and combination 2 are not mixed in the same reaction system, thereby avoiding mutual interference between different primers and probes and improving the accuracy of the detection result.

[0133] (4) The primer and probe combination of the present invention, in combination with the nucleic acid extraction method, can further improve the efficiency of nucleic acid extraction and detection. For example, during the detection process, using a filter membrane to filter food residues can reduce interference with experimental results; using ultrasonic lysis and heating can promote the release of nucleic acids and improve the sensitivity of detection.

[0134] (5) The combination of primers and probes of the present invention can be stored in a kit in a lyophilized form, which is convenient for the storage and transportation of the reagents.

[0135] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0136] Example 1: Design of primer probe

[0137] The primer-probe combination was designed by analyzing and studying the 16s rRNA of mycobacteria, Mycobacterium tuberculosis (IS6110 gene fragment and IS1081 gene fragment) and its drug resistance genes (rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene). The nucleotide sequences of the primers and probes in the primer-probe combination for PCR amplification are shown in Table 1.

[0138] Table 1. Nucleotide sequences of primers and probes in the primer-probe combination for PCR amplification

[0139]

[0140]

[0141] Note: F represents the upstream primer, R represents the downstream primer, P represents the probe, and the sequence shown is considered as the positive strand.

[0142] Example 2: Design of primer-probe combination

[0143] In this example, the primer and probe combinations were designed using the primers and probes in Table 1. The combinations of primers and probes in the test examples and comparative examples are shown in Table 2.

[0144] Table 2. Combinations of primers and probes in test examples and comparative examples

[0145]

[0146]

[0147] Note: In Table 2, the internal reference gene is the RNase P gene, the external reference gene is the 16s rRNA gene of Bacillus subtilis, and the discriminant gene is the conserved region outside the resistance determining region RRDR of the rpoB gene.

[0148] In the test example, reaction tube A includes 1 pair of primers and 8 probes for rpoB gene, 1 pair of primers and 2 probes for katG gene, 1 pair of primers and 3 probes for inhA gene, 1 pair of primers and 2 probes for ahpC gene, 1 pair of primers and 1 probe for IS6110 gene, 1 pair of primers and 1 probe for IS1081 gene, 1 pair of primers and 1 probe for Mycobacterium 16s rRNA gene, and 1 pair of primers and 1 probe for external reference; reaction tube B includes 1 pair of primers and 3 probes for rpoB gene, 1 pair of primers and 1 probe for discrimination gene, 1 pair of primers and 5 probes for gyrA gene, 1 pair of primers and 2 probes for gyrB gene, 1 pair of primers and 1 probe for internal reference (RNase P gene), and 1 pair of primers and 1 probe for external reference. The concentration of the primer probe in the PCR reaction system in the kit is 100nM-300nM, and the amplification systems in the A / B tubes are all freeze-dried balls for easy storage.

[0149] In the comparative example, the difference from the test example is that the primer-probe combination of reaction tube A is different from that of reaction tube B. In the comparative example, reaction tube A includes 1 pair of primers and 11 probes for rpoB gene, 1 pair of primers and 1 probe for IS6110 gene, 1 pair of primers and 1 probe for IS1081 gene, 1 pair of primers and 1 probe for mycobacterium 16s rRNA gene, and 1 pair of primers and 1 probe for external reference; reaction tube B includes 1 pair of primers and 1 probe for discriminant gene, 1 pair of primers and 2 probes for katG gene, 1 pair of primers and 3 probes for inhA gene, 1 pair of primers and 2 probes for ahpC gene, 1 pair of primers and 5 probes for gyrA gene, 1 pair of primers and 2 probes for gyrB gene, 1 pair of primers and 1 probe for internal reference, and 1 pair of primers and 1 probe for external reference. The concentration of the primer probe in the PCR reaction system in the kit is 100nM-300nM, and the amplification systems in the A / B tubes are all freeze-dried balls for easy storage.

[0150] Example 3: Design of a kit for PCR detection

[0151] In this embodiment, a kit is designed, and the composition of the reagents stored in each chamber of the kit is as follows:

[0152] 1) The neutralization and inactivation solution includes 30% isopropanol, 10% Triton X-100, and 60% glacial acetic acid;

[0153] 2) The lysis and binding solution includes 100% sodium perchlorate and 10 μL magnetic bead solution;

[0154] 3) Washing solution 1, washing solution 2 and washing solution 3 are the same, including 80% ethanol;

[0155] 4) The eluent includes 0.01 M Tris-HCl;

[0156] 5) The sample chamber stores freeze-dried external references;

[0157] 6) The liquefied liquid is stored separately outside the card box and includes 40% sodium hydroxide;

[0158] 7) Amplification tubes A and B are respectively provided with an amplification system in the form of freeze-dried pellets, the amplification system comprising the primer-probe combination shown in Table 2, wherein the concentration of the primer-probe is 100 nM to 300 nM. The amplification system also comprises an enzyme (e.g., a polymerase or a nicking enzyme), a buffer (e.g., an amplification buffer, dNTPs, a Mg-containing 2+ solutions, etc.) and lyophilized protective agents, all in the form of lyophilized pellets.

[0159] Example 4: Sputum sample processing and detection

[0160] In this embodiment, taking sputum sample detection as an example, the detection was performed using the kit described in Example 3:

[0161] 1. Sample liquefaction: Mix the sputum sample with the liquefaction solution in a ratio of 5:1, place at room temperature for 15 minutes, and set aside after thorough liquefaction;

[0162] 2. Sample addition: Before using the kit, gently mix the reagents in each chamber to mix evenly and wash the magnetic beads stuck to the wall into the liquid. After mixing, open the lid of the kit, tear off the sealing film, and add 300μL of liquefied sputum.

[0163] 3. Sample testing:

[0164] 1) Use the F-MP100 instrument of Fosun Diagnostics (Shanghai) Co., Ltd. and operate the machine according to the instrument instructions. Turn on the power of the instrument and wait for it to be used after the self-check is completed.

[0165] 2) Open the instrument door, align the QR code on the test kit with the code scanning port, scan successfully, automatically identify and display the name of the test kit, and the instrument automatically matches the corresponding test program; put the test kit into the instrument reagent tank and close the door. Sample ID and operator ID: optional as needed.

[0166] 3) Click the "Start Detection" button to start the detection.

[0167] 4. Data Analysis:

[0168] After the test is completed, the instrument will directly analyze the results and automatically determine the qualitative test results of the sample to be tested. After the test program is completed, the instrument will automatically report the test results.

[0169] 5. Result analysis:

[0170] After testing, the detection results of target genes (A / B-FAM / A-HEX / B-ROX), external reference genes (A / B-CY5.5), internal reference genes (B-CY5), mycobacterium genes (A-CY5), Mycobacterium tuberculosis complex genes (A-ROX), and discriminant genes (B-HEX) were obtained.

[0171] 1) Judgment of qualified test:

[0172] The results of the external reference gene (A / B-CY5.5) should show a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), and Ct≤35, which is considered to be a qualified test.

[0173] The results of the internal reference gene (B-CY5) should show a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), and Ct≤35, which is considered to be a qualified test.

[0174] If one of the internal or external references fails the test, the test is considered invalid; conversely, if both the internal and external references pass the test, the test is considered valid and the next step is judged.

[0175] 2) Determination of negative / positive test for Mycobacterium tuberculosis:

[0176] If only the result of the mycobacterium 16s rRNA gene (A-CY5) presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase) and Ct≤35, the sample to be tested is considered to be a sample of non-tuberculous mycobacteria.

[0177] If the 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence meet the following conditions: the Mycobacterium 16s rRNA gene (A-CY5) result presents a typical S-shaped amplification curve (including an S-shaped curve that has not reached the plateau phase) and Ct≤35, and the IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve and Ct≤30, then the sample to be tested is considered to be a sample positive for Mycobacterium tuberculosis.

[0178] If the 16s rRNA, IS6110 insertion sequence and IS1081 insertion sequence do not meet the following conditions: the Mycobacterium 16s rRNA gene (A-CY5) result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase) and Ct≤35, and the IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve and Ct≤30, then the sample to be tested is considered to be a Mycobacterium-negative sample.

[0179] 3) Determination of whether the drug resistance gene has mutated:

[0180] The results of 16s rRNA gene (A-CY5) should show a typical S-type amplification curve (including the S curve that has not reached the plateau phase), and Ct≤35; the results of IS6110 and IS1081 should show a typical S-type amplification curve (including the S curve that has not reached the plateau phase), and Ct≤30; the results of discriminant gene (B-HEX) should show a typical S-type amplification curve (including the S curve that has not reached the plateau phase), and Ct≤35; if this condition is met, it can be determined whether the drug-resistant gene is mutated. If this condition is not met, the detection of the drug-resistant gene mutation is considered invalid.

[0181] Determine whether the drug resistance genes rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene are mutated:

[0182] If the rpoB gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the rpoB gene has a mutation; otherwise, the rpoB gene does not have a mutation.

[0183] If the katG gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the katG gene has a mutation; otherwise, the katG gene does not have a mutation.

[0184] If the inhA gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the inhA gene has a mutation; otherwise, the inhA gene does not have a mutation.

[0185] If the ahpC gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the ahpC gene has a mutation; otherwise, the ahpC gene does not have a mutation.

[0186] If the gyrA gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the gyrA gene has a mutation; otherwise, the gyrA gene does not have a mutation.

[0187] If the gyrB gene detection result presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase), Ct≤35, and the difference between its Ct value and the Ct value of the discriminant gene is <4, then it is judged that the gyrB gene has a mutation; otherwise, the gyrB gene does not have a mutation.

[0188] After judging whether the above-mentioned drug-resistant genes are mutated, if there is a mutation in the rpoB gene, the drug resistance type is rifampicin (RFP) resistance; if there is a mutation in the inhA, katG and / or ahpC gene, the drug resistance type is isohexamic acid (INH) resistance; if there is a mutation in the gyrA and / or gyrB gene, the drug resistance type is fluoroquinolone (FLQ) drug resistance.

[0189] Example 5: Test results of test examples and comparative examples

[0190] In order to verify the accuracy of the kit in Example 3 of the present application for detecting drug-resistant genes of mycobacteria and Mycobacterium tuberculosis, the primer and probe combination in Table 2 was used and the detection method in Example 4 was applied to detect 20 strain samples.

[0191] The test results of the test example are shown in Table 3, and the test results of the comparative example are shown in Table 4.

[0192] Table 3. Test results of the test cases

[0193]

[0194]

[0195] Note: - indicates not detected.

[0196] Table 4. Test results of comparative examples

[0197]

[0198]

[0199]

[0200] Note: - indicates not detected.

[0201] Example 6: Accuracy analysis of test results

[0202] In this example, all 20 strain samples were subjected to drug sensitivity testing and sequencing identification to determine all sample information, as shown in Table 5.

[0203] Table 5. Sample information

[0204] Sample No. Sample name result Drug resistance Gene Mutation type 1 Mycobacterium tuberculosis Positive RFP rpoB 511CTG>CCG 2 Mycobacterium tuberculosis Positive RFP rpoB 516GAC>GTC 3 Mycobacterium tuberculosis Positive RFP rpoB 526CAC>GAC 4 Mycobacterium tuberculosis Positive RFP rpoB 531TCG>TTG 5 Mycobacterium tuberculosis Positive RFP rpoB 531TCG>TGG 6 Mycobacterium tuberculosis Positive INH iA -15C>T 7 Mycobacterium tuberculosis Positive INH iA -8T>A 8 Mycobacterium tuberculosis Positive INH katG 315AGC>ACC 9 Mycobacterium tuberculosis Positive INH katG 315AGC>ACA 10 Mycobacterium tuberculosis Positive FQ yrA 90GCG>GTG 11 Mycobacterium tuberculosis Positive FQ yrA 94GAC>GGC 12 Mycobacterium tuberculosis Positive FLQ ikB 551GGG>AGG 13 Mycobacterium tuberculosis Positive FQ ikB 540GAA>GAC 14 Mycobacterium tuberculosis Positive / / / 15 Mycobacterium tuberculosis Positive / / / 16 Mycobacterium kanssa Negative / / / 17 Mycobacterium fortuitum Negative / / / 18 Mycobacterium chelonae Negative / / / 19 Mycobacterium intracellulare Negative / / / 20 Mycobacterium abscessus Negative / / /

[0205] The information of the actual samples in Table 5 was compared with the test results in Example 5 to analyze the accuracy of the test examples and comparative examples.

[0206] By comparing Table 3 with Table 5, it can be seen that the 20 strain samples were tested using the primer and probe combination of the test example, the kit formula of Example 3 and the detection method in Example 4, and 13 tuberculosis-resistant strains, 2 tuberculosis non-resistant strains, and 5 non-tuberculosis strains were identified. The results are completely consistent with the actual results of the samples in Table 5, the test results are in line with expectations, and the test has good accuracy and sensitivity.

[0207] By comparing Table 4 with Table 5, it can be seen that the primer and probe composition of the comparative example, the kit formulation of Example 3 and the detection method in Example 4 were used to detect 20 strain samples, and 11 tuberculosis-resistant strains, 4 tuberculosis non-resistant strains, and 5 non-tuberculosis strains were identified. This result differed from the actual results of the samples in Table 5 by 2 samples, and the drug resistance of sample 2 and sample 4 was not detected, resulting in a false negative result.

[0208] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. A combination of primers and probes for detecting Mycobacterium tuberculosis and its drug resistance, characterized in that: The genes targeted by the primers and probes include: IS6110 insertion sequence, IS1081 insertion sequence, 16s rRNA sequence, rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene.

2. The composition according to claim 1, characterized in that The primers and probes are used to detect mutations in genes associated with drug resistance for treating tuberculosis, and include or consist of mutations selected from the following group: mutations in the rpoB gene 516GAC>GTC, 531TCG>TTG, 533CTG>CCG, 511CTG>CCG, 513CAA>AAA, 522TCG>CAG, 526CAC>GAC, 526CAC>CTC, 526CAC>CGC, 526CAC>TAC, and 531TCG>TGG; mutations of 315AGC>ACC and 315AGC>ACA in the katG gene; mutations of -15C>T, -8T>C, and -8T>A in the inhA gene; mutations of -32G>A and -10C>A in the ahpC gene; mutations of 88GGC>TGC, 90GCG>GTG, 94GAC>GGC, 94GAC>AAC, and 94GAC>GCC in the gyrA gene; mutations 551GGG>AGG and 540GAA>GAC in the gyrB gene; Preferably, mutations in the rpoB gene are associated with rifampicin resistance, mutations in the katG gene, inhA gene and ahpC gene are associated with isoniazid resistance; and mutations in the gyrA gene and gyrB gene are associated with fluoroquinolone resistance.

3. The composition according to claim 1 or 2, characterized in that The primer sequences for the rpoB gene are shown in SEQ ID NOs: 1-2, the primer sequences for the katG gene are shown in SEQ ID NOs: 14-15, the primer sequences for the inhA gene are shown in SEQ ID NOs: 18-19, the primer sequences for the ahpC gene are shown in SEQ ID NOs: 23-24, the primer sequences for the gyrA gene are shown in SEQ ID NOs: 27-28, the primer sequences for the gyrB gene are shown in SEQ ID NOs: 34-35, the primer sequences for the IS6110 insertion sequence are shown in SEQ ID NOs: 38-39, the primer sequences for the IS1081 insertion sequence are shown in SEQ ID NOs: 41-42, and / or the primer sequences for 16s rRNA are shown in SEQ ID NOs: 44-45; Preferably, the sequences of the upstream primer and the downstream primer for the rpoB gene are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; the sequences of the upstream primer and the downstream primer for the katG gene are shown in SEQ ID NO:14 and SEQ ID NO:15, respectively; the sequences of the upstream primer and the downstream primer for the inhA gene are shown in SEQ ID NO:18 and SEQ ID NO:19, respectively; the sequences of the upstream primer and the downstream primer for the ahpC gene are shown in SEQ ID NO:23 and SEQ ID NO:24, respectively; the sequences of the upstream primer and the downstream primer for the gyrA gene are shown in SEQ ID NO:27 and SEQ ID NO:28, respectively; the sequences of the upstream primer and the downstream primer for the gyrB gene are shown in SEQ ID NO:34 and SEQ ID NO:35, respectively; the sequences of the upstream primer and the downstream primer for the IS6110 insertion sequence are shown in SEQ ID NO:38 and SEQ ID NO:39, respectively; the sequences of the upstream primer and the downstream primer for the IS1081 insertion sequence are shown in SEQ ID NO:39 and SEQ ID NO:31, respectively. NO:41 and SEQ ID NO:42; the sequences of the upstream primer and downstream primer for 16s rRNA are shown in SEQ ID NO:44 and SEQ ID NO:45, respectively.

4. The composition according to any one of claims 1 to 3, characterized in that The probe sequence for the rpoB gene is shown in SEQ ID NOs: 3-13, the probe sequence for the katG gene is shown in SEQ ID NOs: 16-17, the probe sequence for the inhA gene is shown in SEQ ID NOs: 20-22, the probe sequence for the ahpC gene is shown in SEQ ID NOs: 25-26, the probe sequence for the gyrA gene is shown in SEQ ID NOs: 29-33, the probe sequence for the gyrB gene is shown in SEQ ID NOs: 36-37, the probe sequence for the IS6110 insertion sequence is shown in SEQ ID NO: 40, the probe sequence for the IS1081 insertion sequence is shown in SEQ ID NO: 43, and the probe sequence for 16s rRNA is shown in SEQ ID NO: 46; Preferably, the 5' end and 3' end of the probe are modified with a fluorescent group and a quenching group, respectively; more preferably, the fluorescent group includes: FAM, HEX, ROX, CY5 and / or CY5.5, and the quenching group includes: BHQ1, BHQ2 and / or MGB; More preferably, the probe for rpoB gene is labeled with FAM fluorescent marker, the probes for katG gene, inhA gene and ahpC gene are labeled with HEX fluorescent marker, the probes for gyrA gene and gyrB gene are labeled with ROX fluorescent marker, the probes for IS6110 insertion sequence and IS1081 insertion sequence are labeled with ROX fluorescent marker, and the probe for 16s rRNA is labeled with CY5 fluorescent marker.

5. The composition according to any one of claims 1 to 4, characterized in that The composition also includes primers and probes for internal reference genes, external reference genes and / or discriminant genes; Preferably, the internal reference gene includes: ribonuclease P gene, and / or the external reference gene includes: Bacillus subtilis 16s rRNA gene, and / or the discriminant gene: the conserved region outside the resistance determining region RRDR of the rpoB gene; More preferably, the primer sequence for the internal reference gene is as shown in SEQ ID NO:47-48, more preferably including the upstream primer shown in SEQ ID NO:47 and the downstream primer shown in SEQ ID NO:48; the probe sequence for the internal reference gene is as shown in SEQ ID NO:49; The primer sequences for the external reference gene are shown in SEQ ID NOs: 50-51, more preferably including the upstream primer shown in SEQ ID NO: 50 and the downstream primer shown in SEQ ID NO: 51; the probe sequence for the external reference gene is shown in SEQ ID NO: 52; The primer sequence for the discriminant gene is shown in SEQ ID NO:53-54, and more preferably includes the upstream primer shown in SEQ ID NO:53 and the downstream primer shown in SEQ ID NO:54; the probe sequence for the external reference gene is shown in SEQ ID NO:

55.

6. The composition according to any one of claims 1 to 5, characterized in that The composition of the primer and the probe comprises: Composition 1, comprising or consisting of the following probes: Probes for the rpoB gene as shown in SEQ ID NOs:6-13, probes for the katG gene as shown in SEQ ID NOs:16-17, probes for the inhA gene as shown in SEQ ID NOs:20-22, probes for the ahpC gene as shown in SEQ ID NOs:25-26, probes for the IS6110 insertion sequence as shown in SEQ ID NO:40, probes for the IS1081 insertion sequence as shown in SEQ ID NO:43, and probes for 16s rRNA as shown in SEQ ID NO:46; Composition 1 also includes or consists of the following primers: Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 14-15 for the katG gene, primers as shown in SEQ ID NOs: 18-19 for the inhA gene, primers as shown in SEQ ID NOs: 23-24 for the ahpC gene, primers as shown in SEQ ID NOs: 38-39 for the IS6110 insertion sequence, primers as shown in SEQ ID NOs: 41-42 for the IS1081 insertion sequence, and primers as shown in SEQ ID NOs: 44-45 for 16s rRNA; Composition 2, comprising or consisting of the following probes: Probes for the rpoB gene as shown in SEQ ID NOs: 3-5, probes for the gyrA gene as shown in SEQ ID NOs: 29-33, and probes for the gyrB gene as shown in SEQ ID NOs: 36-37; Composition 2 also includes or consists of the following primers: Primers as shown in SEQ ID NOs: 1-2 for the rpoB gene, primers as shown in SEQ ID NOs: 27-28 for the gyrA gene, and primers as shown in SEQ ID NOs: 34-35 for the gyrB gene; Wherein, composition 1 and composition 2 are not mixed in the same reaction system; Preferably, the internal reference gene is set in composition 2, and / or the discriminant gene is set in composition 2, and / or the external reference gene is set in composition 1 and composition 2 simultaneously, separately or sequentially.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a detection reagent, a kit or a detection device for detecting Mycobacterium tuberculosis and its drug resistance; Preferably, the concentration of the combination of primers and probes is 100 nM to 300 nM, more preferably 100 to 150 nM, 100 to 200 nM, 100 to 250 nM, 150 to 200 nM, 150 to 250 nM, 200 to 250 nM, 150 to 300 nM or 200 to 300 nM; Preferably, the detection reagents further include one, more or all of the following reagents: The liquefied liquid more preferably comprises sodium hydroxide, more preferably 30% to 50% sodium hydroxide; A neutralization inactivation solution, more preferably comprising isopropanol, Triton X-100 and glacial acetic acid, more preferably 10% to 40% isopropanol, 2% to 20% Triton X-100 and 50% to 80% glacial acetic acid; Lysis and binding solution, more preferably comprising sodium perchlorate and magnetic bead solution, more preferably 100% sodium perchlorate and 10 μL magnetic bead solution; The washing solution more preferably comprises ethanol, more preferably 80% ethanol; The eluent more preferably comprises Tris-HCl, more preferably 0.0001 to 1 M Tris-HCl; Reagents for PCR reactions; and / or, Preferably, the kit includes the detection reagent, more preferably, the combination of primers and probes is present in the kit in a lyophilized form, more preferably, composition 1 and composition 2 are two lyophilized reagents placed in different chambers or containers; and / or, Preferably, the detection device further comprises: A nucleic acid analysis instrument, more preferably a PCR instrument.

8. A detection reagent, kit or detection device for detecting Mycobacterium tuberculosis and its drug resistance, characterized in that: The detection reagent, kit or detection device comprises the composition according to any one of claims 1 to 6; Preferably, the detection reagent, kit or detection device is as described in claim 7.

9. A method for detecting Mycobacterium tuberculosis and its drug resistance, characterized in that: The method comprises: 1) contacting a sample to be tested with a detection reagent, a kit or a detection device comprising the primer and probe combination according to any one of claims 1 to 6; 2) obtaining the test results of the sample to be tested in 1); 3) analyzing whether Mycobacterium tuberculosis is positive based on the test results in 2), and analyzing the type of drug resistance of Mycobacterium tuberculosis; Preferably, the sample to be tested includes: plasma, serum, sputum, urine, feces, body fluid; more preferably, the sample to be tested is a sputum sample; and / or, Preferably, the detection reagent, kit or detection device is as described in claim 7 or 8; and / or, Preferably, step 2) of the method further includes: analyzing the detection results of internal references and / or external references: if the detection results of the internal references and / or external references meet the requirements, performing step 3); if the detection results of the internal references and / or external references do not meet the requirements, step 3) is not performed; more preferably, the requirements for the detection results of the internal references and / or external references are: the internal references and / or external references present a typical S-shaped amplification curve and the Ct value of the internal references and / or external references is ≤35; more preferably, the typical S-shaped amplification curve includes an S curve that has not reached the plateau phase.

10. The method according to claim 9, characterized in that Whether Mycobacterium tuberculosis is positive is analyzed based on the test results of 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence. The type of drug resistance of Mycobacterium tuberculosis is analyzed based on the test results of rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene, gyrB gene and discriminant gene. Preferably, the test results of 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence are analyzed separately to see whether they meet the requirements. If only 16s rRNA meets the requirements, the sample to be tested is considered to be a sample of non-tuberculous mycobacteria; if 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence all meet the requirements, the sample to be tested is considered to be a sample positive for Mycobacterium tuberculosis; if 16s rRNA, IS6110 insertion sequence, and IS1081 insertion sequence all do not meet the requirements, the sample to be tested is considered to be a sample negative for Mycobacterium; More preferably, the requirements for the test results of 16s rRNA analysis to meet are: 16s rRNA presents a typical S-shaped amplification curve (including an S curve that has not reached the plateau phase) and Ct≤35; for 16s The detection results of rRNA, IS6110 insertion sequence and IS1081 insertion sequence all meet the requirements that: Mycobacterium 16srRNA presents a typical S-shaped amplification curve with Ct≤35, IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve with Ct≤30; more preferably, the typical S-shaped amplification curve includes an S curve that has not reached the plateau phase; and / or, Preferably, whether there is a mutation is analyzed based on the detection results of the rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene, gyrB gene and the discriminant gene, and the type of drug resistance of Mycobacterium tuberculosis is determined according to the type of the mutated gene. More preferably, if there is an rpoB gene mutation, the drug resistance type is rifampicin (RFP) resistance; if there is an inhA, katG and / or ahpC gene mutation, the drug resistance type is isohexamidine (INH) resistance; if there is a gyrA and / or gyrB gene mutation, the drug resistance type is fluoroquinolone (FLQ) drug resistance; More preferably, the detection results of the drug resistance gene rpoB gene, katG gene, inhA gene, ahpC gene, gyrA gene and gyrB gene and the discriminant gene are analyzed respectively to see whether they meet the requirements. If they meet the requirements, it is considered that the drug resistance gene has a mutation. If they do not meet the requirements, it is considered that the drug resistance gene does not have a mutation. More preferably, the requirements for the detection results of the drug-resistant gene are: Mycobacterium 16s rRNA presents a typical S-shaped amplification curve and Ct≤35, IS6110 insertion sequence and IS1081 insertion sequence present a typical S-shaped amplification curve and Ct≤30, the discriminant gene presents a typical S-shaped amplification curve and Ct≤35, the drug-resistant gene presents a typical S-shaped amplification curve and Ct value≤35, and the Ct difference between the corresponding drug-resistant gene mutant detection target and the discriminant gene is <4; more preferably, the typical S-shaped amplification curve includes an S curve that has not reached the plateau phase.

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