A primer combination for detecting drug-resistant genes of Mycobacterium tuberculosis and its application

Abstract

The present application discloses a primer combination and its application. The primer combination of the present application can be used to detect the full-length sequences of 45 drug-resistant genes of 15 drugs for treating Mycobacterium tuberculosis infection, and can not only detect currently known mutations, but also be used to detect unknown mutations, with a wider detection coverage.

Description

Technical Field

[0001] The present application belongs to the technical field of gene detection, and specifically relates to a primer composition for detecting drug-resistant genes of Mycobacterium tuberculosis and its application. Background Art

[0002] Tuberculosis is caused by Mycobacterium tuberculosis (MTB). Identifying those infected with MTB and treating them appropriately as soon as possible is undoubtedly the key to effectively controlling tuberculosis. However, the multidrug resistance of MTB is the biggest difficulty and severe challenge faced in the treatment of modern tuberculosis. The resistance of MTB to clinical mainstream anti-tuberculosis drugs including isoniazid, rifampicin and ethambutol has increased significantly, resulting in delayed disease progression. Selecting effective drugs and accelerating the diagnosis of tuberculosis drug resistance are issues that need to be urgently addressed.

[0003] People have found more than 40 anti-tuberculosis drug resistance genes of Mycobacterium tuberculosis, including the rifampicin resistance gene rpoB, and have also created various Mycobacterium tuberculosis resistance gene detection methods based on real-time fluorescence quantitative PCR technology, isothermal (constant temperature) amplification technology, melting curve technology, nucleic acid mass spectrometry technology, liquid phase chip technology, gene sequencing technology, etc. Related cases are as follows:

[0004] Patent CN201410815076.X discloses a method for detecting drug-resistant genes of Mycobacterium tuberculosis based on multiplex real-time fluorescence PCR. The method includes a group of probes that can detect mutations in drug-resistant genes of Mycobacterium tuberculosis in samples, and uses sample genomic DNA as a template to perform fluorescence quantitative PCR amplification to obtain an amplification curve. The amplification curve and Ct value containing a certain fluorescent reporter group are used to determine whether the gene has a point mutation, and the mutation of 7 drug-resistant genes of Mycobacterium tuberculosis in the sample to be tested is detected in a relatively short period of time. The results are highly sensitive, specific, and the analysis process is simple. The disadvantage is that the detection range is limited, and only a few known drug-resistant genes and mutation sites can be detected. At the same time, the method has high requirements for primers, and non-specific amplification may occur, affecting the accuracy of detection.

[0005] Patent CN201911415507.2 discloses a method for detecting drug-resistant genes of Mycobacterium tuberculosis based on next-generation sequencing. The method includes a series of sequencing primers for drug-resistant genes of Mycobacterium tuberculosis. The detection steps mainly include sample pretreatment, DNA extraction, target site PCR amplification, library construction, and DNA sequencing. It can simultaneously detect mutations in 48 sites of 17 drug-resistant genes of common anti-tuberculosis drugs, which can better guide the use of tuberculosis drugs. The disadvantage is that the detection coverage is limited, and only some known drug-resistant genes and mutation sites are detected. At present, there are more than 40 drug-resistant genes and thousands of drug-resistant mutation sites reported, and there will be missed detections in uncovered areas.

[0006] Patent CN202110176138.7 discloses a method and kit for detecting rifampicin and isoniazid resistance mutations in Mycobacterium tuberculosis based on the fluorescent PCR melting curve method, which can quickly and qualitatively detect in vitro resistance mutations in the resistance-determining regions of rifampicin resistance gene rpoB and isoniazid resistance gene katG, inhA, and ahpC in sputum culture samples of tuberculosis patients that are positive for Mycobacterium tuberculosis complex. The disadvantage is that the detection coverage is limited, and only a few known resistance genes and mutation sites can be detected. At the same time, the technology needs to distinguish samples with very small differences in Tm values ​​to identify the differences in single bases. Therefore, the requirements for temperature resolution are quite high. If the resolution of the instrument is not high, it cannot be detected.

[0007] Patent CN202110752683.6 discloses a method for detecting Mycobacterium tuberculosis and drug-resistant gene mutation sites based on Luminex liquid phase chip. The method includes a series of detection primers and probes, which can detect whether Mycobacterium tuberculosis exists in the sample, and cover the drug-resistant mutation sites of drugs such as isoniazid, rifampicin, pyrazinamide, and streptomycin. The detection speed is fast, and it only takes 3.5 hours to complete a test, and up to 96 samples can be detected at the same time. The disadvantage is that due to the limitation of the type of microspheres, the number of detectable mutation sites is limited, and only known gene mutation sites can be detected, and unknown mutations cannot be detected. Luminex liquid phase chip technology can realize the detection of multiple sites at the same time, but it only plays its multi-throughput and high-efficiency characteristics in detection. In essence, it does not solve the problem of mismatch between many primers, probes, and templates in multiple amplification. In addition, since the primers need to be bound to the solid phase carrier, this limits the collision probability of the primers and templates to a certain extent and leads to a reduction in their amplification efficiency.

[0008] Patent CN202210006972.6 discloses a method for detecting drug-resistant genes of Mycobacterium tuberculosis based on nucleic acid mass spectrometry. This method combines single base extension reaction technology, determines the mutation sites to be tested of katG315, rpoB435 and embB306 genes, designs amplification primers and extension primers for each mutation site, and purifies the samples by PCR amplification, digestion treatment and extension reaction. The samples are prepared by nanoliter spotting and then tested by mass spectrometry. Finally, the site mutation information is determined by comparison, thereby determining the resistance of Mycobacterium tuberculosis to isoniazid, rifampicin and ethambutol. The test results can be widely used in clinical testing, disease prevention and other fields. The disadvantage is that unknown mutations cannot be detected, and there are certain requirements for the number of samples and sites tested each time. In addition, there may be an ion suppression effect. If the substance is impure and impurities are present, the sensitivity of the mass spectrometry will be reduced.

[0009] In summary, the current technical solutions for the detection of tuberculosis drug-resistant genes either have a small detection range and cannot cover a large range of drug-resistant genes and mutation sites, resulting in the inability to accurately detect drug-resistant sites; or although they have a large coverage range, they can only detect known genes and specific target mutations (usually known sites). Summary of the invention

[0010] The present application targets 15 anti-tuberculosis drugs, collects 45 common drug-resistant genes, obtains the location information of related genes on the genome in the NCBI database, and obtains the full-length information of the genes. By using multiplex PCR targeted capture technology and high-throughput second-generation sequencing technology, the primer composition and detection method of the present application are designed, and the full-length sequence of all 45 drug-resistant genes can be detected, so that mutations at any site in these genes can be detected, that is, not only the 45 common drug-resistant genes of Mycobacterium tuberculosis can be covered, thousands of known drug-resistant-related mutation sites can be detected, but also new mutations on drug-resistant genes can be detected, which can guide clinical tuberculosis medication more accurately and comprehensively.

[0011] In a first aspect, the present application provides a primer composition, which comprises a first primer combination and a second primer combination, wherein the first primer combination comprises primers shown in SEQ ID NO.1~392, and the second primer combination comprises primers shown in SEQ ID NO.393-784.

[0012] The second aspect of the present application provides a Mycobacterium tuberculosis drug resistance gene detection kit, which includes the primer composition provided in the first aspect of the present application.

[0013] The third aspect of the present application provides use of the primer composition provided in the first aspect of the present application or the kit provided in the second aspect of the present application for detecting a drug resistance gene of Mycobacterium tuberculosis and / or any mutation thereof.

[0014] The fourth aspect of the present application provides a method for detecting a drug resistance gene and / or any mutation thereof of Mycobacterium tuberculosis in a sample, comprising:

[0015] 1) extracting DNA from the sample;

[0016] 2) using the primer combination provided in the first aspect of the present application or the kit provided in the second aspect of the present application to perform multiplex PCR amplification using the DNA as a template;

[0017] 3) Sequencing and sequence alignment of the obtained amplified products to obtain information on the drug resistance gene and / or its mutation of Mycobacterium tuberculosis.

[0018] Beneficial effects of the present application: The target gene that the primer composition provided by the present application can amplify at one time covers the full-length sequence of 45 drug-resistant genes of all 15 known drugs that can be used to treat Mycobacterium tuberculosis infection. It can not only detect currently known mutations, but more importantly, it can also be used to detect unknown mutations, with a wider detection coverage. In addition, through reasonable primer design, the Tm value, GC content, hairpin structure, primer dimer and non-specific amplification of the primers are fully considered and verified, and the high specificity and sensitivity of amplification can still be guaranteed in the presence of hundreds of primer pairs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the comparison result between the ponA1 gene sequence detected in Example 2 of the present application and the ponA1 gene sequence in the CARD drug resistance gene database. DETAILED DESCRIPTION

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present application, and for ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0021] definition

[0022] As used herein, the terms "a" and "an" and "the" and similar referents refer to both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0023] As used herein, the terms "about," "substantially," and "similar to" mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range may depend in part on the manner in which the value is measured or determined or on the limitations of the measurement system.

[0024] In a first aspect, the present application provides a primer composition, which comprises a first primer combination and a second primer combination, wherein the first primer combination comprises primers shown in SEQ ID NO.1~392, and the second primer combination comprises primers shown in SEQ ID NO.393-784.

[0025] In some embodiments, the primers in the first primer combination and the primers in the second primer combination are each independently present in the primer composition, that is, the primers in the first primer combination do not contact the primers in the second primer combination.

[0026] In some embodiments, in order to improve the specificity of the multiplex PCR reaction, a two-tube reaction can be performed, in which the primers in the first primer combination are used in the multiplex PCR reaction in the first reaction system, and the primers in the second primer combination are used in the multiplex PCR reaction in the second reaction system. After the reaction is completed, the amplification products of the two reaction systems are combined as the amplification products of the multiplex PCR reaction using the primer combination of the present application.

[0027] The second aspect of the present application provides a Mycobacterium tuberculosis drug resistance gene detection kit, which includes the primer composition provided in the first aspect of the present application.

[0028] In some embodiments, the kit may further include other PCR reagents besides primers, such as a polymerase mixture, dNTPs, a PCR enhancement buffer, and MgCl2.

[0029] In some embodiments, the kit may further include reagents for connecting adapters, such as illumina / MGISEQ 5'-end Index sequencing adapter and illumina / MGISEQ 3'-end Index sequencing adapter.

[0030] In some embodiments, the concentration of each primer in the primer composition is 0.2-0.8 μM, preferably 0.5 μM.

[0031] In some embodiments, the kit may further include instructions for use.

[0032] The third aspect of the present application provides use of the primer composition provided in the first aspect of the present application or the kit provided in the second aspect of the present application for detecting a drug resistance gene of Mycobacterium tuberculosis and / or any mutation thereof.

[0033] In some embodiments, the Mycobacterium tuberculosis drugs described in the present application cover 15 mainstream first-line and second-line anti-tuberculosis drugs, including at least one of rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, levofloxacin / moxifloxacin, bedaquiline, linezolid, clofazimine, cycloserine, delamanid, amikacin, capreomycin, ethionamide, and para-aminosalicylic acid.

[0034] The drug resistance of Mycobacterium tuberculosis can be analyzed more comprehensively and accurately.

[0035] In some embodiments, the Mycobacterium tuberculosis drug resistance gene includes 45 common drug resistance genes for the above 15 drugs, such as ald, alr, atpE, cycA, ddl, ddn, dfrA, eis, embA, embB, embC, embR, ethA, ethR, fbiA, fbiB, fbiC, fgd1, folC, gid, gyrA, gyrB, inhA, katG, panD, pepQ, pncA, ponA1, ribD, rplC, rpoA, rpoB, rpoC, rpsA, rpsL, rrl, rrs, Rv0678, Rv1979c, aftA, thyA, thyX, tlyA, ubiA, whiB7, etc.

[0036] In some embodiments, the primers provided in the present application can be used to detect all of the 45 Mycobacterium tuberculosis drug resistance genes.

[0037] In some embodiments, the amplification product using the primer combination provided in the present application can cover the full-length sequence of the 45 Mycobacterium tuberculosis drug resistance genes.

[0038] Since the primers provided in the present application have a high coverage of drug-resistant gene sequences, the primers provided in the present application can also be used to detect mutations at any position in the 45 Mycobacterium tuberculosis drug resistance genes. The mutations are not limited to known mutation sites and can also be used to detect new mutations, which is more conducive to the diagnosis of tuberculosis drug resistance.

[0039] In some embodiments, the primer composition of the present application can be used to detect any known mutations in the 45 drug-resistant genes of the above 15 drugs.

[0040] In some embodiments, the known mutations include but are not limited to:

[0041] (1) 108 polymorphic sites of four rifampicin resistance genes, including:

[0042] 1) 82 sites of rpoB gene: G95F, V170F, P206R, A286V, Y314C, H323Y, V359A, T400A, F424L, F424V, T427I, Q429H, Q429V, L430P, S431G, S431T, Q432A, Q432E, Q432K, Q432 L, Q432P, M434I, D435A, D435H, D435F, D435G, D435N, D435V, D435Y, N437S, N438 P, S441L, S441Q, S441P, S441T, S441X, L443F, T444S, T444I, H445A, H445C, H445 D, H445F, H445G, H445L, H445P, H445R, H445S, H445Y, H445X, H445Q, H445N, K44 6Q, K446R, R448X, L449Q, S450C, S450F, S450L, S450Q, S450W, S450Y, S450P, S45 0X, A451G, A451V, L452P, P454L, I480V, I491F, R511L, T526S, D545E, H674Q, T67 6P, E761D, G981D, 1292- / GCC, 1296- / TTC, 1312AAC / -, 1328- / GAC, 1396- / ATTC;

[0043] 2) 19 sites of rpoC gene: A172V, G332R, N416S, N416T, P434T, F452L, F452S, V483A, D485N, I491T, L516P, L527V, G594E, P601L, N689S, D747A, D747G, N826K, I885V;

[0044] 3) 3 sites of rpoA gene: R186C, T187PA, E319K;

[0045] 4) 4 sites of ponA1 gene: G363D, T658A, G363D, Q365H;

[0046] (2) 122 polymorphic sites of two isoniazid resistance genes, including:

[0047] 1) 104 loci of katG gene: V1A, C20S, Q88E, W90R, W90X, R104L, R104Q, A106V, H108Q, H108G, A109V, A110V, G125D, Q127P, N138S, N138H, A139P, A139V, S140N, L141F, D142G, L148R, L148A, W149X, Y155C, L159P, S1 75X, T180K, G182R, W191G, W191R, P232R, M257I, D259E, H270Q, H270G, K274X, T275P, G297V, W300C, W 300S, S302R, S315G, S315I, S315N, S315T, S315R, W321G, W321F, T326M, W328L, I335V, Y337C, W341R, Q 352X, L378P, T380I, D381G, L398P, Y413H, D419Y, D419H, M420T, Q434X, Q461P, R463L, Q471R, V473F, W477X, S481L, W505X, D542H, S575X, L587M, L587P, R595X, A614E, L619P, L627P, R632C, V633A, L634F, S671X, D695A, S700P, L704S, D735A, 22- / T, 54C / -, 356GCG / -, 367G / -, 591- / T, 971C / -, 1022- / G, 128 4GCC / -, 1364T / -, 1431GGC / -, 1572- / T, 1668A / -, 1682A / -, 1899- / C, 1955- / G, 2002- / T, 2101- / AACT;

[0048] 2) 18 loci of inhA gene: -59C / G, -34G / C, -34G / T, -24C / T, -17A / T, -16C / G, -15A / T, -9A / T, -8T / A, -8T / C, -8T / G, -5C / G, I21T, I21M, I21V, G90P, S94A, I194T;

[0049] (3) 76 polymorphic sites of 7 ethambutol resistance genes, including:

[0050] 1) 38 loci of the embB gene: L74R, F285L, S297A, M306I, M306L, M306V, Y319N, Y319S, Y319C, D328Y, D328G, F330V, Y334H, S347T, S347I, D354A, E378A, P397T, E405D, G406A, G406C, G406D, G406S, G406N, M423T, Q445R, Q497K, Q497P, Q497R, Q497H, E504D, Q853P, A630I, M1000R, H1002R, D1024T, D1024N, N1033K;

[0051] 2) 8 loci of the embA gene: -32G / -, -16C / G, -16C / T, -16C / A, -12C / T, -12C / A, -11C / A, D4N;

[0052] 3) Three sites of the embC gene: T270I, D329G, and N394D;

[0053] 4) 2 sites of embR gene: P49A, P243S;

[0054] 5) 1 site of rpoC gene: G332R;

[0055] 6) 23 sites of ubiA gene: L31P, A35E, A35S, A38V, V55G, V55M, V148A, G165C, S173A, K174R, W175G, F176L, I179T, M180V, V188A, V229G, L235P, A237C, A237V, R240C, S244T, A249G, A278V;

[0056] 7) 1 site of aftA gene: L198L

[0057] (4) 190 polymorphic sites of three pyrazinamide resistance genes, including:

[0058] 1) 14 loci of panD gene: H21R, I49V, I115T, M117T, M117I, E126X, A128S, E130G, P134S, L136R, V138A, V138E, V138G, M171I;

[0059] 2) 172 sites of the pncA gene: -12T / C, -11A / G, -11A / C, 7G / -, 53C / -, 67- / T, 171CCGGCA / -, 182- / A, 184C / -, 185C / -, 187- / A, 193- / A, 194- / GGACTAT, 194- / A, 206- / C, 206- / CCA, 232- / A, 235- / G, 242- / TTCCA, 248- / C, 251- / C, 251G / -, 291T / -, 292- / AT, 376- / GA, 394- / GGT, 394- / C, 395- / T, 406G / -, 408- / A, 410- / T, 410TGT / -, 414- / CATT, 418- / GCGCCAGACG, 419- / G, 422A / -, 423- / AG, 455- / C, 457- / ATGGCTTGGC, 458- / C, 471- / A, 480A / -, 494- / C, 523- / A, 525- / A, 525CCGCCA / -, 570- / CT, A3E, L4W, L4S, I5T, I6T, V7L, V7G, D8N, V9G, Q10P, D12A, C14G, S18C, G23W, I31S, Y34D, Y41X, K48E, K48T, D49G, H51D, H51Q, H51P, H51R, P54L, P54Q, H57R, H57P, H57D, H57Y, S59P, S59F, P62S, P62T, P62L, D63H, D63A, D63G, Y64X, S66P, S67P, S67W, W68C, W68R, W68G, W68S, W68X, P69L, P69R, H71Q, H71Y, H71R, C72R, T76P, G78C, G78V, H82R, L85P, L85R, I90S, F94C, F94S, Y95T, K96R, G97C, G97R, G97S, G97D, G97A, G97V, Y99X, A102V, Y103C, Y103X, S104G, S104R, G108R, L116P, W119G, L120R, R123P, D129Y, G132D, G132S, G132A, I133T, I133S, A134V, T135P, D136N, D136Y, D136A, H137R, C138R, V139M, V139A, V139G, V139L, R140P, Q141P, Q141X, T142A, T142P, T142M, T142K, A143G, A143D, A146P, A146T, A146V, A146E, L151S, T167P, T168S, T168P,V169A, L172P, L172R, M175V, M175R, M175T, V180F, V180G, L182S, X187W;,

[0060] 3) 4 loci of rpsA gene: T5S, E67D, D123A, 1313CCG / -;

[0061] (5) 77 polymorphic sites of three streptomycin resistance genes, including:

[0062] 1) 7 loci of rpsL gene: T40I, K43R, K43T, R86G, K88Q, K88R, K88M;

[0063] 2) 13 loci of rrs gene: 419C / T, 462C / T, 492C / T, 513C / T, 514A / C, 514A / T, 516C / T, 517C / T, 878G / A, 905C / A, 905C / T, 906A / G, 907A / C;

[0064] 3) 57 loci of the gid gene: F12L, G34V, G34E, R47W, H48N, H48Q, R64W, V65G, G69D, S70R, G71E, G71X, G73A, P75L, P75R, P75S, L79S, A80P, R83P, P84L, P93L, G117V, R118S, R118L, A134E, S136X, R137P, R137W, A138T, A138V, Y195X, A2 00E, V203L, 40C / -, 61C / -, 98- / G, 98G / -, 102G / -, 107T / -, 112C / -, 115C / -, 202G / -, 202- / GC, 211G / -, 237- / G , 238G / C, 297- / A, 347- / G, 347G / -, 351G / -, 366CT / -, 384G / -, 404T / -, 451C / -, 456GA / -, 503A / -, 749ATTC / -;

[0065] (6) 13 polymorphic sites of two resistance genes for levofloxacin / moxifloxacin, including:

[0066] 1) 11 loci of the gyrA gene: G88C, G88A, A90V, A90G, S91P, D94A, D94G, D94H, D94N, D94Y, D94V;

[0067] 2) Two loci of the gyrB gene: D461N, E501D;

[0068] (7) 62 polymorphic sites of 5 bedaquiline resistance genes, including:

[0069] 1) 13 loci of atpE gene: -72T / C, -53G / A, D28A, D28G, D28N, D28V, E61D, A63P, A63V, 83A / G, 83A / T, 183G / T, 187G / C;

[0070] 2) 1 site of mmpL5 gene: S602P;

[0071] 3) 1 site of pepQ gene: L44P;

[0072] 4) 46 loci of the Rv0678 gene: V1A, 2T / C, S2I, V20G, E21D, Q22L, T33A, A36T, L39A, C46R, S53L, S53P, S63R, G66V, S68G, R72W, L74P, L83P, Y92*, F93G, R94Q, 97A / G, A102P, L117R, R134*, R 135G, 136- / G, L136P, 138- / GA, 138- / G, E138G, 138- / G, 141- / C, 141- / C, M146T, 185- / C AG, 189C / A, 192- / G, 193G / -, 200T / G, 202A / G, 214C / T, 259- / G, 292A / -, 345G / -, 403C / G;

[0073] 5) 1 site of Rv1979c gene: M245L;

[0074] (8) 10 polymorphic sites of two linezolid resistance genes, including:

[0075] 1) Three sites of rplC gene: C154N, C154R, H155D;

[0076] 2) 7 loci of rrl gene: 2061G / T, 2270G / C, 2270G / T, 2576G / T, 2576G / C, 2746G / A, 2814G / T;

[0077] (9) 81 polymorphic sites of three clofazimine resistance genes, including:

[0078] 1) 7 loci of pepQ gene: L44P, 207C / -, 265G / T, 346- / A, 478C / -, 486CCT / -, 833C / -;

[0079] 2) 73 loci of the Rv0678 gene: 1- / T, 2T / C, V3I, N4T, D5G, E13A, V20F, 29- / T, T33N, A36V, G41A, W42X, L43R, C46Y, Q51R, S53L, 58G / T, A59V, S63N, S63R, G65E, G66V, S68N, S68G, Q76X, A84E, V85A, G87R, R89L, R90C, R90P, A102V, A102T, R105G, A110V, L114P, L117R, A118P, D119E, V 120M, G121R, L122P, R123T, 125G / A, G126S, G126R, G126A, D127A, A128P, P130Q, R134X, R135W, M139T, 141- / GA, D141H, M146T, N148H , V149I, L154P, R156X, G162E, D165N, 193G / -, 193- / G, 198G / -, 199- / G, 202A / G, 292A / -, 364- / C, 422- / G, 438- / AT, 444CG / -, 453C / -;

[0080] 3) 1 site of Rv1979c gene: V351A;

[0081] (10) Seven polymorphic sites of four cycloserine resistance genes, including:

[0082] 1) Three loci of the alr gene: -26G / T, L113R, D344N;

[0083] 2) Two sites in the cycA gene: T236A, V301A;

[0084] 3) 1 locus of ddl gene: L372R;

[0085] 4) 1 site of ald gene: 32T / C;

[0086] (11) 20 polymorphic sites of 5 resistance genes to Delamanid, including:

[0087] 1) 5 loci of ddn gene: L49P, G53D, R72W, E83D, W88X;

[0088] 2) 6 loci of fbiA gene: D49T, D49Y, Q120R, R175H, L250X, T302M;

[0089] 3) 3 loci of fbiB gene: F220L, L447R, L448R;

[0090] 4) 3 sites of fbiC gene: T273A, R536L, T681I;

[0091] 5) 3 loci of fgd1 gene: G104S, L270M, L296E;

[0092] (12) 23 polymorphic sites of 4 amikacin resistance genes, including:

[0093] 1) 5 loci of rrs gene: 513C / T, 514A / C, 1401A / G, 1402C / T, 1484G / T;

[0094] 2) 6 loci of whiB7 gene: 86C / -, 124C / -, 128G / -, 133C / -, 133- / C, 179G / -;

[0095] 3) 9 loci of gid gene: 102G / -, 104T / G, 230T / C, 254A / G, 286C / T, L35R, V77A, D85G, R96C;

[0096] 4) Three sites of the eis gene: -14C / T, -10G / C, -12C / T

[0097] (13) 33 polymorphic sites of 4 capreomycin resistance genes, including:

[0098] 1) 5 loci of rrs gene: 513C / T, 514A / C, 1401A / G, 1402C / T, 1484G / T;

[0099] 2) 18 loci of thyA gene: R3X, Q22X, D57H, H68R, E75X, G196E, N236K, A253W, 7C / T, 64C / T, 90- / G, 202- / GC, 203- / C, 203- / GC, 220T / C, 223G / T, 708T / G, 755- / GT;

[0100] 3) 9 loci of gidB gene: 102G / -, 104T / G, 230T / C, 254A / G, 286C / T, L35R, V77A, D85G, R96C;

[0101] 4) 1 locus of tlyA gene: R14W;

[0102] (14) 63 polymorphic sites of three ethionamide resistance genes, including:

[0103] 1) ethA gene 56 loci: -11A / G, -7T / C, M1R, M1T, V10M, G11V, H22Q, Q24X, 30G / -, Y32D, G43C, W45G, S57Y, T61K, T88I, 107A / -, 110A / -, 157- / T, Q165P, M204V, L205P, Q206X, R207G, Y211S, Q246R, W256C, S266R, P334A, 338A / -, 341 A / -, A341V, D357Y, S375Y, P378L, 382- / G, S390F, V398L, L405X, Y438X, 441- / T, G450S, P454L, W455R, L478R, 6 72- / G, 703T / -, 753- / G, 869- / A, 892A / -, 908G / -, 980T / -, 1175- / CG, 1190- / TGG, 1239T / -, 1242T / -, 1393- / GA;

[0104] 2) ethR gene 1 site: A95T;

[0105] 3) 6 loci of inhA gene: -17G / T, -15C / T, -8T / A, I21T, S94A, I194T;

[0106] (15) 72 polymorphic sites of 5 resistance genes to para-aminosalicylic acid, including:

[0107] 1) Three sites of dfrA gene: V54A, S66C, C110R;

[0108] 2) 30 loci of folC gene: E40A, E40G, E40K, E40Q, I43A, I43F, I43S, I43T, I43V, R49P, R49W, L56V, N73S, R91W, D111A, G112S, D135A, S150C, S150G, S150R, F152L, F152S, E153A, E153G, V256A, S335I, R410W, E434Q, A457V, A457X;

[0109] 3) ribD gene 1 site: G8R;

[0110] 4) 37 sites of thyA gene: G15R, T22I, T22A, Y36C, H75N, G76X, V77F, W83C, W83X, G91E, G91R, W98X, S105P, Q111X, R126Q, R127L, N134K, L143P, L146R, H147N, F152V, C161Y, L172P, A182P, L183V, Q191R, H207R, I211V, R222G, P224L, R235P, Y251X, A259P, V261G, V263G, V263I, X264R.

[0111] 5) thyX gene 1 site: −16 C / T.

[0112] In some embodiments, the primer combination of the present application can be used to detect any unknown mutation in the 45 drug-resistant genes of the above 15 drugs.

[0113] The unknown mutations mentioned in the present application can be understood as mutations related to the drug resistance of Mycobacterium tuberculosis that have not been reported so far.

[0114] The fourth aspect of the present application provides a method for detecting a drug resistance gene and / or any mutation thereof of Mycobacterium tuberculosis in a sample, comprising:

[0115] 1) extracting DNA from the sample;

[0116] 2) using the primer combination provided in the first aspect of the present application or the kit provided in the second aspect of the present application to perform multiplex PCR amplification using the DNA as a template;

[0117] 3) Sequencing and sequence alignment of the obtained amplified products to obtain information on the drug resistance gene and / or its mutation of Mycobacterium tuberculosis.

[0118] In some embodiments, the sample is selected from at least one of sputum, alveolar lavage fluid, blood, tissue (such as lung tissue, kidney tissue, intestinal tissue, etc.), bacterial culture, etc.

[0119] In some embodiments, the sample may not have been cultured for microorganisms, such as sputum, alveolar lavage fluid, blood, or tissue, and DNA may be directly extracted using commercially available kits, which is not limited in this application.

[0120] In other embodiments, sputum, alveolar lavage fluid, blood, and tissue may be used for microbial culture to obtain bacterial culture, and then DNA may be extracted from the obtained bacterial culture using a commercially available kit, which is not limited in the present application.

[0121] In the multiplex PCR amplification of the present application, other reagents except template and primer are conventional reagents in the art, and those skilled in the art can select them according to specific circumstances, and the present application does not limit them here.

[0122] In the present application, the conditions for multiplex PCR amplification can be specifically selected by those skilled in the art according to actual conditions, such as the volume of the reaction system, the state of the instrument, the selection of the enzyme, etc., and the present application does not limit them here. In some embodiments, the reaction temperature conditions for the multiplex PCR amplification are:

[0123] (1) 93℃~97℃ for 3~4mins;

[0124] (2) 97°C-99°C for 15-25 seconds, 58°C-62°C for 3-7 minutes, and repeat for 18-22 cycles;

[0125] (3) Maintain at 71℃~73℃ for 4~6 minutes.

[0126] In some embodiments, the conditions for the multiplex PCR amplification may further include: providing a heating cover temperature of 104-106° C. before adjusting the reaction temperature.

[0127] In some embodiments, in step 2), the multiplex PCR amplification includes using the first primer set and the second primer set in the primer composition to perform multiplex PCR amplification using the DNA as a template, respectively, and includes combining the amplification products. The above amplification method can be understood as a two-tube amplification, and the use of a two-tube amplification is conducive to further improving the specificity of the multiplex PCR reaction.

[0128] In some embodiments, the amplification conditions of the multiplex PCR amplification (T1) performed using the first primer set and the multiplex PCR amplification (T2) performed using the second primer set may be different.

[0129] In some embodiments, the reaction temperature and time of the multiplex PCR amplification of T1 are:

[0130] (1) 93℃~97℃ for 3~4mins;

[0131] (2) 97°C-99°C for 15-25 seconds, 58°C-62°C for 5-7 minutes, and repeat for 18-22 cycles;

[0132] (3) Maintain at 71℃~73℃ for 4~6 minutes.

[0133] In some embodiments, the reaction temperature and time of the multiplex PCR amplification of T2 are:

[0134] (1) 93℃~97℃ for 3~4mins;

[0135] (2) 97°C-99°C for 15-25 seconds, 58°C-62°C for 3-5 minutes, and repeat for 18-22 cycles;

[0136] (3) Maintain at 71℃~73℃ for 4~6 minutes.

[0137] In some embodiments, step 3) further includes a step of purifying the obtained amplified product before sequencing.

[0138] In some embodiments, when two-tube amplification is used, the amplification products of the two tubes can be combined first, and then the products can be purified to reduce the number of purification operations and save costs.

[0139] In some embodiments, the purification is magnetic bead purification or column purification.

[0140] In some embodiments, the sequencing is second generation sequencing. Second generation sequencing has the characteristics of high throughput, which is conducive to quickly obtaining sequencing results.

[0141] In some embodiments, the second-generation sequencing can be implemented using the existing MGISEQ (BGI) sequencing platform or Illumina (Illumina) sequencing platform, and this application is not limited thereto.

[0142] In some embodiments, the sequence comparison includes comparing the sequence information obtained by sequencing with the genome sequence of Mycobacterium tuberculosis in a known database, and combining it with a drug resistance gene database, so as to obtain the full-length sequence of the drug resistance gene of Mycobacterium tuberculosis and all mutation information contained in each gene. The mutations include both known mutations and unknown mutations that have not been reported.

[0143] In some embodiments, the known database may be a NCBI database with a number of NC_000962.3.

[0144] In some embodiments, the drug resistance gene database may be a CARD drug resistance gene database.

[0145] Those skilled in the art may also select from other known databases on the genome or drug-resistant gene of Mycobacterium tuberculosis, and this application is not limited thereto.

[0146] In some embodiments, the second-generation sequencing also includes the step of connecting the multiple PCR amplification products to the adapter sequence. This is a routine operation of the second-generation sequencing. Those skilled in the art can select the appropriate adapter sequence and connection method according to the sequencing platform, and this application is not limited thereto.

[0147] Exemplarily, when using the Illumina sequencing platform, the matching illumina5'-end Index sequencing adapter and illumina3'-end Index sequencing adapter can be selected; when using the MGISEQ (MGI) sequencing platform, the matching MGISEQ 5'-end Index sequencing adapter and MGISEQ 3'-end Index sequencing adapter can be used; this is a common technology for second-generation sequencing, and this application is not limited here.

[0148] In some embodiments, the linker sequence can be added by PCR amplification or sticky end ligation, which is a routine operation in the art and is not limited in this application.

[0149] The multiplex PCR reaction conditions for connecting the multiplex PCR amplification products of the present application to the adapter sequence are the commonly used reaction conditions in the art, and the present application is not limited thereto. In some embodiments, the multiplex PCR reaction temperature and time for connecting the adapter sequence are:

[0150] (1) 93℃~97℃ for 2~4mins;

[0151] (2) 97℃~99℃ for 15~25s, 56℃~60℃ for 45-90s, 70℃~73℃ for 20~40s, and repeat for 8~12 cycles;

[0152] (3) Maintain at 71℃~73℃ for 4~6 minutes.

[0153] In the art, the amplification product connected with the adapter sequence is usually called a library.

[0154] In some embodiments, the second generation sequencing may further include a step of determining the library concentration. In some preferred embodiments, the library concentration may be 10-50 ng / μL.

[0155] In some embodiments, the second generation sequencing can also include library fragment length and purity measurement. In some embodiments, the library target fragment distribution interval is between 280bp-420bp.

[0156] In some embodiments, the library can be sequenced using an MGISEQ sequencer, and the sequencing strategy is PE150.

[0157] In some embodiments, the amount of raw sequencing data of each sequencing sample is greater than 300 Mb bases.

[0158] In some embodiments, before performing sequence alignment, the step of filtering the sequencing data to remove low-quality data is also included. This is a routine technique in the art, and those skilled in the art can implement it as needed or by using conventional data filtering methods, and this application is not limited thereto.

[0159] The primer combination and its use of the present application are described below by specific examples. The following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this area or the product instructions are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0160] Example 1 Determination of genomic target region and primer design

[0161] 1) By consulting literature, we collected information on Mycobacterium tuberculosis-related drug resistance genes and ultimately identified 45 reported tuberculosis-related drug resistance genes.

[0162] 2) Using the genome of Mycobacterium tuberculosis H37Rv as the reference genome, the location information of the relevant genes on the genome and the full-length information of the genes were obtained in the NCBI database according to the gene names found.

[0163] The 45 resistance-related genes include: ald, alr, atpE, cycA, ddl, ddn, dfrA, eis, embA, embB, embC, embR, ethA, ethR, fbiA, fbiB, fbiC, fgd1, folC, gid, gyrA, gyrB, i nhA, katG, panD, pepQ, pncA, ponA1, ribD, rplC, rpoA, rpoB, rpoC, rpsA, rpsL, rrl, rrs, Rv0678, Rv1979c, aftA, thyA, thyX, tlyA, ubiA, whiB7

[0164] The 15 tuberculosis treatment drugs involved include: rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, levofloxacin / moxifloxacin, bedaquiline, linezolid, clofazimine, cycloserine, delamanid, amikacin, capreomycin, ethionamide and para-aminosalicylic acid.

[0165] 3) Use primer design software such as AIdesign, PrimerPlex, etc. to design multiple PCR primers for the target gene region. After the primers are initially designed, the primers are quality controlled according to the standard process through primer quality control software, mainly scoring the primers' Tm value, GC content, hairpin structure, primer dimer, and non-specific amplification, and preliminarily evaluating the amplification effect of the primers before the experiment.

[0166] The primer sequences are shown in SEQ ID NO.1~784, wherein SEQ ID NO.1~196 are forward primers in the first primer combination, and SEQ ID NO.197~392 are reverse primers paired sequentially with the forward primers; SEQ ID NO.393~588 are forward primers in the second primer combination, and SEQ ID NO.589~784 are reverse primers paired sequentially with the forward primers.

[0167] 4) After the primers are designed, commercial primer synthesis is performed to obtain the primer combination of the present application.

[0168] Example 2 Detection of drug resistance genes and mutations of Mycobacterium tuberculosis

[0169] 1. Sample pretreatment and DNA extraction

[0170] The test samples involved in this application may include a variety of common sample types, such as sputum, alveolar lavage fluid, blood, tissue, bacterial culture, etc. In this embodiment, the sample is from a Mycobacterium tuberculosis culture sample from a hospital in Shenzhen. A conventional commercial DNA extraction kit (DNA extraction kit of Tiangen Biochemical Technology Co., Ltd., catalog number NG550) is used to extract total DNA from the sample according to the corresponding method, and then the DNA concentration is detected using Qubit® 3.0 Fluorometer (Qubit dsDNAHS Assay Kit, catalog number Q32851). After passing the quality inspection (total DNA amount is 85 ng, concentration is 1.7 ng / μl), it is used for the next multiplex PCR amplification.

[0171] 2. Multiplex PCR amplification and library construction

[0172] Multiplex PCR amplification was performed using the primer combination obtained in Example 1 and the extracted DNA.

[0173] 2.1 First round of multiplex PCR reaction

[0174] The first round of multiplex PCR reaction was divided into two reaction tubes, one tube used the first primer combination Primer pool T1 containing the primers shown in SEQ ID NO.1~392, and the other tube used the second primer combination Primer pool T2 containing the primers shown in SEQ ID NO.393~784. The concentration of each primer in Primer pool T1 and Primer pool T2 was 0.5μM. The other reagents in the two reaction tubes were the same. The multiplex PCR reaction system is shown in Table 1 below:

[0175] Table 1 Multiplex PCR reaction system

[0176]

[0177] Note: IGT-EM808 polymerase mixture, Enhancer buffer NB (1N), and Enhancerbuffer M were purchased from Agilent Biotech Co., Ltd. with the catalog number M60012.

[0178] The multiplex PCR reaction conditions were as follows:

[0179]

[0180] After the first round of PCR reaction was completed, 15 μL of PCR from each of the two reaction tubes was combined.

[0181] 2.2 Magnetic bead purification of combined products

[0182] 1). Prepare 80% ethanol with anhydrous ethanol and nuclease-free water in advance and keep it at room temperature for later use. Please try to use freshly prepared 80% ethanol for magnetic bead purification.

[0183] 2). Take out the purified magnetic beads (Agencourt AMPure XP kit, purchased from Beckman Coulter, Inc., catalog number A63880) from the 4°C refrigerator in advance, mix well and place at room temperature for equilibration for 30 min; vortex the purified magnetic beads that have been equilibrated to room temperature and set aside.

[0184] 3). Add 0.9 times volume of magnetic beads (27 μL) to 30 μL of PCR product, pipette or vortex to mix, and let stand at room temperature for 5 min.

[0185] 4). Centrifuge briefly and place the PCR tube on a magnetic rack for 3 min until the solution is clear.

[0186] 5). Completely remove the supernatant, remove the PCR tube from the magnetic stand, add 50 μL YF buffer B (purchased from Agilent Biotech, catalog number M60022) into the tube, pipette to mix, and let stand at room temperature for 5 min.

[0187] 6). Centrifuge briefly and place the PCR tube on the DynaMag-96 Side magnetic stand for 3 min.

[0188] 7). Keep the PCR tube on the magnetic rack, carefully remove the supernatant, add 180μL 80% ethanol solution into the PCR tube, and let it stand for 30s.

[0189] 8). Keep the PCR tube on the magnetic rack, discard the supernatant, add 180μL 80% ethanol solution into the PCR tube again, let it stand for 30s, and discard the supernatant.

[0190] 9). Cover the tube cap and centrifuge briefly to remove the remaining ethanol to the bottom of the tube. Place the PCR tube on the magnetic rack and carefully use a 10μL pipette to remove the remaining ethanol at the bottom, being careful not to absorb the magnetic beads.

[0191] 10). Keep the PCR tube on the magnetic rack and let it stand at room temperature for 3 to 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0192] 11). Add 24 μL of nuclease-free water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let stand at room temperature for 2 min.

[0193] 12). Centrifuge briefly and place the PCR tube on a magnetic rack for 2 min to wait for the solution to clarify.

[0194] 13). Use a pipette to aspirate 13.5 μL of supernatant and transfer it to a new PCR tube. The supernatant in the tube is the combined multiple PCR product.

[0195] 2.3 Second round of multiplex PCR reaction of adapter sequences

[0196] Table 2 Linker sequence reaction system

[0197]

[0198] Note: Enhancer buffer M and IGT-EM808 polymerase mixture were purchased from Agilent Biotech Co., Ltd. with the catalog number M60022, and UDI Index (5μM) was purchased from Agilent Biotech Co., Ltd. with the catalog number M70142.

[0199] The multiplex PCR reaction system is as follows:

[0200] Heating cover 105℃;

[0201] 95℃ 3min;

[0202] 98°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles;

[0203] 72℃ 5min.

[0204] 2.4 Second round of magnetic bead purification

[0205] The same magnetic bead purification steps as above were used to purify the products of the second round of adapter sequence multiplex PCR reaction to obtain a multiplex PCR library.

[0206] 2.5 Library quantification

[0207] Take 1 μL of library and use Qubit® 3.0 Fluorometer (Qubit dsDNA HS Assay Kit) to measure the library concentration and record the library concentration. The optimal concentration is about 50 ng / μL.

[0208] 2.6 Library quality control

[0209] Take 1 μL of library sample and use Qsep100 fully automatic nucleic acid protein analysis system to measure the length and purity of library fragments. The target fragment distribution range of normal library is between 280bp and 420bp.

[0210] 3. Sequencing

[0211] The constructed sequencing library was sequenced on the MGISEQ sequencer with a sequencing strategy of PE150, and sequencing data was generated. The amount of raw sequencing data for each sample was greater than 300Mb bases.

[0212] 4. Bioinformatics analysis

[0213] The raw sequencing data were first filtered to remove low-quality data, and then sequence alignment was performed. The reference genome was provided by the NCBI database, numbered NC_000962.3, and the coverage of drug-resistant gene detection and drug-resistant mutation sites were analyzed.

[0214] 4.1 Analysis of drug resistance gene detection coverage

[0215] The following Table 3 is the statistical information of the strain-related drug resistance gene detection coverage obtained in the example. The results show that the coverage of 45 genes can reach 100%, indicating that the primer composition and detection method of the present application can cover the full-length sequence of the relevant genes.

[0216] Table 3 Analysis of drug resistance gene detection coverage

[0217]

[0218] 4.2 Analysis of drug resistance gene mutation sites

[0219] By performing sequence analysis on the obtained strain-related drug-resistant genes and combining them with the CARD drug-resistant gene database, we can determine whether the relevant genes have drug-resistant mutations. Taking the ponA1 gene as an example, the length of this gene is 2037bp. By performing mutation analysis on the sequence of this gene and comparing it with the sequence in the CARD drug-resistant gene database, the results are as follows: Figure 1 shown. Figure 1 Sequence 1 is the ponA1 gene sequence and known drug-resistant mutation sites recorded in the CARD drug-resistant gene database (bases enlarged in font in the sequence), and sequence 2 is the ponA1 gene sequence and mutation sites obtained by analyzing the sample to be tested in this embodiment, wherein the sites marked with boxes are newly discovered mutation sites. The results show that the primer composition of the present application and the method using the same can display the full-length sequence information of the drug-resistant gene of the sample to be tested. In addition to detecting known mutation sites, unknown mutations can also be detected and analyzed, thereby discovering gene mutations at any site of the gene.

[0220] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A primer composition comprising a first primer combination and a second primer combination, wherein: The first primer combination includes primers shown in SEQ ID NOs. 1 to 392, and the second primer combination includes primers shown in SEQ ID NOs. 393 to 784.

2. A Mycobacterium tuberculosis drug resistance gene detection kit, comprising the primer combination according to claim 1.

3. Use of the primer composition according to claim 1 or the kit according to claim 2 in preparing a product for detecting drug resistance genes of Mycobacterium tuberculosis.

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