CN115852002A - Primer composition for detecting mycobacterium tuberculosis drug-resistant gene and application thereof - Google Patents

Abstract

The application discloses a primer composition and application thereof. The primer composition can be used for detecting the full-length sequences of 45 drug resistance genes of 15 drugs for treating mycobacterium tuberculosis infection, can detect not only the currently known mutation, but also unknown mutation, and has wider detection coverage.

Description

Primer composition for detecting mycobacterium tuberculosis drug-resistant gene and application thereof

Technical Field

The application belongs to the technical field of gene detection, and particularly relates to a primer composition for detecting a drug-resistant gene of mycobacterium tuberculosis and application thereof.

Background

Tuberculosis is caused by Mycobacterium Tuberculosis (MTB), and mortality rates are the second place of infectious diseases. According to WHO's estimate, the world's latent tuberculosis infection population is close to 20 hundred million, and about 5-10% of M.tuberculosis infectors will develop active tuberculosis in their lifetime. Pulmonary infections account for 75% of active tuberculosis and are a major cause of high mortality from tuberculosis. The identification of mycobacterium tuberculosis infectors and the early appropriate treatment of patients with mycobacterium tuberculosis is undoubtedly the key to effective control of tuberculosis. However, the multidrug resistance of mycobacterium tuberculosis is the biggest difficult and serious challenge in the treatment process of modern tuberculosis. In recent 10 years, the drug resistance of mycobacterium tuberculosis to main clinical anti-mycobacterium tuberculosis drugs including isoniazid, rifampicin, ethambutol and the like has increased remarkably, resulting in prolonged illness. The selection of effective drugs and the acceleration of the diagnosis of drug resistance of tuberculosis drugs are problems which need to be solved urgently at present.

Since the isoniazid drug-resistant gene katG was discovered in the last 90s of the century, more than 40 drug-resistant genes of mycobacterium tuberculosis antituberculosis drugs including rifampicin drug-resistant gene rpoB have been found, and various mycobacterium tuberculosis drug-resistant gene detection methods based on real-time fluorescent quantitative PCR technology, isothermal (constant temperature) amplification technology, melting curve technology, nucleic acid mass spectrometry technology, liquid phase chip technology, gene sequencing technology and the like have also been established. The relevant cases are as follows:

patent CN201410815076.X discloses a method for detecting drug-resistant genes of mycobacterium tuberculosis based on multiplex real-time fluorescent PCR. The method comprises a group of probes capable of detecting the drug-resistant gene mutation of the mycobacterium tuberculosis in a sample, and is characterized in that the sample genome DNA is used as a template to carry out fluorescence quantitative PCR amplification to obtain an amplification curve, whether the gene has point mutation or not is judged through the amplification curve containing a certain fluorescence report group and a Ct value, the mutation conditions of 7 drug-resistant genes of the mycobacterium tuberculosis in the sample to be detected are detected in a short time, the result sensitivity is high, the specificity is good, and the analysis process is simple and convenient. The defects are that the detection range is limited, and only few known drug resistance genes and mutation sites can be detected. Meanwhile, the method has higher requirements on the primers, and the detection accuracy is influenced by the occurrence of the condition of nonspecific amplification.

Patent CN201911415507.2 discloses a tubercle bacillus drug-resistant gene detection method based on next-generation sequencing. The method comprises a series of sequencing primers aiming at drug resistance genes of the tubercle bacillus, the detection steps mainly comprise sample pretreatment, DNA extraction, target site PCR amplification, library construction and DNA sequencing, mutation detection can be simultaneously carried out on 48 sites of 17 drug resistance genes of common antituberculous drugs, and tuberculosis medication can be better guided. The method has the defects that the detection coverage is limited, only part of known drug-resistant genes and mutation sites are detected, more than 40 drug-resistant genes and thousands of drug-resistant mutation sites are reported at present, and the detection omission condition exists in the uncovered area.

Patent CN202110176138.7 discloses a method and a kit for detecting rifampicin and isoniazid resistance mutation of mycobacterium tuberculosis based on a fluorescence PCR melting curve method, which can rapidly and qualitatively detect rifampicin resistance gene rpoB and isoniazid resistance gene katG, inhA and ahpC resistance determinant zone resistance mutation in sputum culture samples positive to mycobacterium tuberculosis complex of tuberculosis patients in vitro. The disadvantage is that the detection coverage is limited, and only few and known drug-resistant genes and mutation sites can be detected. Meanwhile, the technology needs to distinguish samples with extremely small Tm value difference to identify the difference of single base, so the requirement on temperature resolution is quite high, and if the resolution of an instrument is not high, the detection cannot be carried out.

Patent CN202110752683.6 discloses a method for detecting mycobacterium tuberculosis and drug-resistant gene mutation sites based on Luminex liquid phase chip. The method comprises a series of detection primers and probes, can detect whether the mycobacterium tuberculosis exists in a sample, covers drug-resistant mutation sites of drugs such as isoniazid, rifampicin, pyrazinamide, streptomycin and the like, has high detection speed, only needs 3.5 hours for completing one-time detection, and can simultaneously detect up to 96 samples at one time. The disadvantages are that the number of detectable mutation sites is limited by the kind of microspheres, and only known gene mutation sites can be detected, but unknown mutations cannot be detected. The Luminex liquid phase chip technology can realize the simultaneous detection of a plurality of sites, but the Luminex liquid phase chip technology only exerts the characteristics of multiple flux and high efficiency in the aspect of detection, and does not substantially solve the problem of mutual mismatching among a plurality of primers, probes and templates in multiple amplification. In addition, since it is necessary to bind the primer to the solid support, this limits the probability of collision of the primer with the template to some extent and leads to a decrease in amplification efficiency thereof.

Patent CN202210006972.6 discloses a detection method of mycobacterium tuberculosis drug-resistant gene based on nucleic acid mass spectrum, which combines a single base extension reaction technology, determines mutation sites to be detected of katG315, rpoB435 and embB306 genes, designs an amplification primer and extension primers of each mutation site, uses resin for purification after PCR amplification, digestion treatment and extension reaction, nano-liter sample application to prepare a sample and perform mass spectrum detection, and finally determines site mutation information through comparison, thereby judging the drug resistance of mycobacterium tuberculosis to isoniazid, rifampicin and ethambutol. The detection result can be widely applied to the fields of clinical examination, disease prevention and the like. The disadvantages are that unknown mutation can not be detected, and the number of samples and sites detected each time has certain requirements. In addition, there may be ion suppression effects, which can reduce the sensitivity of the mass spectrometer if the material is impure or has impurities present.

In summary, the current technical scheme aims at the detection of drug-resistant genes of tuberculosis, or the detection range is small, and the drug-resistant genes and mutation sites cannot be covered in a large range, so that the drug-resistant sites cannot be accurately detected; or, although there is a large coverage, it can only be detected for known genes and specific target mutations (usually known sites).

Disclosure of Invention

Aiming at 15 anti-tubercle bacillus drugs, common 45 drug-resistant genes are collected, the position information of the related genes on a genome is obtained in an NCBI database, the full-length information of the genes is obtained, the primer composition and the detection method are designed by using a multiple PCR targeted capture technology and a high-throughput second-generation sequencing technology, and the full-length sequence of all 45 drug-resistant genes can be detected, so that the mutation of any site in the genes can be detected, namely, not only can the common 45 drug-resistant genes of mycobacterium tuberculosis be covered, but also thousands of known drug-resistant related mutation sites can be detected, and the new mutation on the drug-resistant genes can be detected, so that the clinical drug-resistant tuberculosis can be more accurately and more comprehensively guided.

In a first aspect, the present application provides a primer composition comprising 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.

In a second aspect, the present application provides a kit for detecting drug-resistant genes of mycobacterium tuberculosis, which comprises the primer composition provided in the first aspect of the present application.

In a third aspect, the present application provides the 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-resistant gene of mycobacterium tuberculosis and/or any mutation thereof.

In a fourth aspect of the present application, there is provided a method for detecting a drug-resistant gene of mycobacterium tuberculosis and/or any mutation thereof in a sample, comprising:

1) Extracting DNA in the sample;

2) Performing multiplex PCR amplification using the primer composition provided in the first aspect of the present application or the kit provided in the second aspect of the present application, using the DNA as a template;

3) Sequencing and sequence alignment are carried out on the obtained amplification products so as to obtain the information of the drug tolerance gene of the mycobacterium tuberculosis and/or the mutation of the drug tolerance gene.

The beneficial effect of this application: the primer composition provided by the application can be used for amplifying target genes at one time, covers the full-length sequences of all known 45 drug-resistant genes of 15 drugs for treating mycobacterium tuberculosis infection, can be used for detecting the currently known mutation, and more importantly, can also be used for detecting unknown mutation, and has wider detection coverage. In addition, the Tm value, GC content, hairpin structure, primer dimer, non-specific amplification and other aspects of the primers are fully considered and verified through reasonable primer design, and the high specificity and sensitivity of amplification can be still ensured under the condition that hundreds of pairs of primers exist.

Drawings

FIG. 1 shows 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

In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only one embodiment of the present application, and other embodiments can be obtained by those skilled in the art according to the drawings.

Definition of

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

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.

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

In some embodiments, the primer in the first primer combination and the primer in the second primer combination are each independently present in the primer composition, i.e., the primer in the first primer combination is not in contact with the primer in the second primer combination.

In some embodiments, in order to improve the specificity of the multiplex PCR reaction, the primers in the first primer combination can be used in the multiplex PCR reaction in the first reaction system, the primers in the second primer combination can be used in the multiplex PCR reaction in the second reaction system, and after the reaction is finished, the amplification products of the two reaction systems are combined to be used as the amplification product of the multiplex PCR reaction using the primer composition of the present application.

In a second aspect, the present application provides a kit for detecting drug-resistant genes of mycobacterium tuberculosis, which comprises the primer composition provided in the first aspect of the present application.

In some embodiments, the kit may further comprise other PCR reagents other than primers, such as polymerase mix, dntps, PCR enhancement buffer, and MgCl ₂ And the like.

In some embodiments, the kit may further comprise reagents for ligating linkers, such as an illumina/MGISEQ 5 'Index sequencing linker, and an illumina/MGISEQ 3' Index sequencing linker.

In some embodiments, the primer composition of each primer is used in a concentration of 0.2-0.8. Mu.M, preferably 0.5. Mu.M.

In some embodiments, instructions for use may also be included in the kit.

In a third aspect, the present application provides the 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-resistant gene of mycobacterium tuberculosis and/or any mutation thereof.

In some embodiments, the mycobacterium tuberculosis drug described herein covers 15 mainstream first-line, second-line antitubercular drugs including at least one of rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, levofloxacin/moxifloxacin, bedaquiline, linezolid, clofazimine, cycloserine, delamanid, amikacin, capreomycin, ethionamide, para-aminosalicylic acid.

Can analyze the drug resistance of the mycobacterium tuberculosis more comprehensively and more accurately.

In some embodiments, the mycobacterium tuberculosis drug resistance gene includes 45 common drug resistance genes for the 15 drugs described above, such as at least one of 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, whilb 7, and the like.

In some embodiments, the primers provided herein can be used to detect all of the 45 M.tuberculosis drug resistance genes.

In some embodiments, the amplification products using the primer compositions provided herein can cover the full-length sequence of the 45 M.tuberculosis drug-resistant genes.

The primer provided by the application has high coverage of the drug-resistant gene sequence, so that the primer provided by the application can also be used for detecting the mutation at any position in the 45 drug-resistant genes of the mycobacterium tuberculosis, the mutation is not limited to the known mutation site, and the primer can also be used for detecting new mutation, thereby being more beneficial to the diagnosis of the drug resistance of tuberculosis.

In some embodiments, the primer compositions of the present application can be used to detect any known mutation in the 45 drug resistance genes of the 15 drugs described above.

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

(1) 108 polymorphic sites of 4 drug resistance genes against rifampicin, including:

1) rpoB gene 82 sites: G95F, V170F, P206R, A286V, Y314C, H323Y, V359A, T400A, F424L, F424V, T427I, Q429H, Q429V, L430P, S431G, S431T, Q432A, Q432E, Q432K, Q432L, Q432P, M434I, D435A, D435H, D435F, D435G, D435N, D435V, D435Y, N437S, N438P, S441L, S441Q, S441P, S441T, S441X, L443F, T444S, T444I, H445A, H445C, H445D, H F, H445G, H445L, H445P, H445R, H445S, H445Y, H445X, H445Q, H445N, K446Q, K446R, R448X, L449Q, S450C, S450F, S450L, S450Q, S450W, S450Y, S450P, S450X, A451G, A451V, L452P, P454L, I480V, I491F, R511L, T526S, D545E, H Q, T676P, E761D, G981D,1292-/GCC,1296-/TTC, AAC-, 1328-/GAC,1396-/ATTC;

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;

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

4) ponA1 gene 4 sites: G363D, T658A, G363D, Q365H;

(2) 122 polymorphic sites of 2 drug resistance genes aiming at isoniazid, comprising:

1) 104 sites of the 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, S175X, T180K, G182R, W191G, W191R, P232R, M257I, D259E, H270Q, H270G, K274X, T275P, G297V, W300C, W300S, S302R, S315G, S315I, S315N, S315T, S315R, W321G, W321F, T326M, W326L, I335V, Y337C, W352R 341, Q352X, 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, D A,22-/T,54C/-,356GCG/-,367G/-,591-/T,971C/-,1022-/G,1284GCC/-,1364T/-,1431GGC/-,1572-/T,1668A/-,1682A/-,1899-/C, 1955/AAT, AAT 2101, AAT-;

2) 18 sites 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;

(3) 76 polymorphic loci of 7 drug resistance genes for ethambutol comprising:

1) The embB gene has 38 sites: 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;

2) The embA gene has 8 sites: -32G/-, -16C/G, -16C/T, -16C/a, -12C/T, -12C/a, -11C/a, D4N;

3) 3 sites of the embC gene: T270I, D329G, N394D;

4) 2 sites of the embR gene: P49A, P243S;

5) rpoC gene 1 site: G332R;

6) ubiA gene 23 sites: L31P, a35E, a35S, a38V, V55G, V55M, V148A, G165C, S173A, K174R, W175G, F176L, I179T, M180V, V188A, V229G, L235P, a237C, a237V, R240C, S244T, a249G, a278V;

7) 1 site of the aftA gene: L198L

(4) 190 polymorphic sites of 3 drug resistance genes aiming at pyrazinamide, comprising:

1) The panD gene has 14 sites: H21R, I49V, I115T, M117I, E126X, a128S, E130G, P134S, L136R, V138A, V138E, V138G, M171I;

2) pncA gene 172 sites: -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,410 TGT-' -,414-/CATT, 418-/CACG/, 419-/G, 422/, 423-/AG,455-/C, 457-/ATGGGC-/494, 471-/C-, 523/A-, 523/GCA-, 4/L-, 4-CT-, 4/GA, 525/A-, 525/T-, 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, 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;

3) rpsA gene 4 sites: T5S, E67D, D123A,1313CCG/-;

(5) 77 polymorphic sites of 3 drug resistance genes against streptomycin, comprising:

1) 7 sites of the rpsL gene: T40I, K43R, K43T, R86G, K88Q, K88R, K88M;

2) 13 sites 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;

3) 57 sites of 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, A200E, 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/-;

(6) 13 polymorphic sites of 2 drug resistance genes aiming at levofloxacin/moxifloxacin, comprising:

1) 11 sites of the gyrA gene: G88C, G88A, a90V, a90G, S91P, D94A, D94G, D94H, D94N, D94Y, D94V;

2) 2 sites of the gyrB gene: D461N, E501D;

(7) 62 polymorphic sites of 5 drug resistance genes aiming at bedaquiline, comprising:

1) atpE gene 13 sites: -72T/C, -53G/a, D28G, D28N, D28V, E61D, a63P, a63V,83A/G,83A/T,183G/T,187G/C;

2) mmpL5 gene 1 site: S602P;

3) pepQ Gene 1 site: L44P;

4) 46 sites 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, R135G,136-/G, L136P,138-/GA,138-/G, E138G,138-/G,141-/C,141-/C, M146T,185-/CAG,189C/A,192-/G, 193G-, -200T/G, 202A/G,214C/T,259-/G, 345-, -292-, -403C/G;

5) 1 site of Rv1979c gene: M245L;

(8) 10 polymorphic sites of 2 drug resistance genes aiming at linezolid, comprising:

1) 3 sites of the rplC gene: C154N, C154R, H155D;

2) rrl gene 7 sites: 2061G/T,2270G/C,2270G/T,2576G/T,2576G/C,2746G/A,2814G/T;

(9) 81 polymorphic sites aiming at 3 drug-resistant genes of clofazimine comprise:

1) pepQ gene 7 sites: L44P,207C/-,265G/T,346-/A,478C/-,486CCT/-,833C/-;

2) 73 sites 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, V120M, 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/-;

3) 1 site of Rv1979c gene: V351A;

(10) 7 polymorphic sites of 4 drug resistance genes aiming at cycloserine, comprising:

1) 3 sites of alr gene: -26G/T, L113R, D344N;

2) 2 sites of the cycA gene: T236A, V301A;

3) ddl gene 1 site: L372R;

4) 1 site of the ald gene: 32T/C;

(11) 20 polymorphic sites of 5 drug resistance genes against delamanib, including:

1) ddn gene 5 sites: L49P, G53D, R72W, E83D, W88X;

2) fbiA gene 6 sites: D49T, D49Y, Q120R, R175H, L250X, T302M;

3) fbiB gene 3 sites: F220L, L447R, L448R;

4) fbiC gene 3 sites: T273A, R536L, T681I;

5) 3 sites of the fgd1 gene: G104S, L270M, L296E;

(12) 23 polymorphic sites of 4 drug resistance genes against amikacin, comprising:

1) rrs gene 5 sites: 513C/T,514A/C,1401A/G,1402C/T,1484G/T;

2) 6 sites of whiB7 gene: 86C/-,124C/-,128G/-,133C/-,133-/C,179G/-;

3) gid gene 9 sites: 102G/-,104T/G,230T/C,254A/G,286C/T, L35R, V77A, D85G, R96C;

4) eis gene 3 sites: -14C/T, -10G/C, -12C/T

(13) 33 polymorphic sites of 4 drug resistance genes of the capreomycin, comprising:

1) rrs gene 5 sites: 513C/T,514A/C,1401A/G,1402C/T,1484G/T;

2) the thyA gene has 18 sites: 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;

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

4) tlyA gene 1 site: R14W;

(14) 63 polymorphic loci of 3 drug resistance genes aiming at ethionamide, comprising:

1) 56 sites of the ethA gene: -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/-,341A/-, a341V, D357Y, S375Y, P378L,382-/G, S390F, V398L, L405X, Y438X,441-/T, G450S, P454L, W455/R, L-/G, 672-/G, 703T-,/753-/G, 869-/a, 892-/a-, 908-/G-, 1233-T-, -, gl 3/G, gl, 478-, gl 3 a-;

2) 1 site of ethR gene: A95T;

3) 6 sites of inhA gene: -17G/T, -15C/T, -8T/a, I21T, S94A, I194T;

(15) 72 polymorphic sites of 5 drug resistance genes against aminosalicylic acid, including:

1) 3 sites of the dfrA gene: V54A, S66C, C110R;

2) folC gene 30 sites: 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;

3) 1 site of the ribD gene: G8R;

4) thyA gene 37 sites: 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, Y X, a259P, V261G, V263I, X264R.

5) thyX gene 1 site: -16C/T.

In some embodiments, the primer compositions of the present application can be used to detect any unknown mutation in the 45 drug resistance genes of the 15 drugs mentioned above.

The unknown mutation referred to in the present application is understood as a mutation related to the drug resistance of Mycobacterium tuberculosis which has not been reported so far.

In a fourth aspect of the present application, there is provided a method for detecting a drug-resistant gene of mycobacterium tuberculosis and/or any mutation thereof in a sample, comprising:

1) Extracting DNA in the sample;

2) Performing multiplex PCR amplification using the primer composition provided in the first aspect of the present application or the kit provided in the second aspect of the present application, using the DNA as a template;

3) Sequencing and sequence alignment are carried out on the obtained amplification products so as to obtain the information of the drug tolerance gene of the mycobacterium tuberculosis and/or the mutation of the drug tolerance gene.

In some embodiments, the sample is selected from at least one of sputum, alveolar lavage fluid, blood, tissue (e.g., lung tissue, kidney tissue, intestinal tissue, etc.), bacterial culture, and the like.

In some embodiments, the sample may be cultured without microorganisms, such as sputum, alveolar lavage, blood, and tissue, and the DNA extraction may be performed directly using a commercially available kit, which is not limited herein.

In other embodiments, the bacteria culture may be obtained by culturing the sputum, alveolar lavage fluid, blood, or tissue with a microorganism, and then extracting DNA from the obtained bacteria culture using a commercially available kit, which is not limited herein.

In the multiplex PCR amplification of the present application, the reagents other than the template and the primer are conventional in the art, and can be selected by those skilled in the art according to the specific situation, and the present application is not limited herein.

In the present application, the conditions for multiplex PCR amplification can be specifically selected by those skilled in the art according to the actual conditions, such as the volume of the reaction system, the state of the apparatus, and the selection of the enzyme, and the present application is not limited thereto. In some embodiments, the reaction temperature conditions for the multiplex PCR amplification are, in order:

(1) Keeping the temperature at 93-97 ℃ for 3-4 mins;

(2) Keeping the temperature of 97-99 ℃ for 15-25s, keeping the temperature of 58-62 ℃ for 3-7 min, and sequentially carrying out 18-22 cycles;

(3) Keeping the temperature of 71-73 ℃ for 4-6 min.

In some embodiments, the conditions for multiplex PCR amplification may further comprise: the temperature of the heating lid was given 104-106 ℃ before adjusting the reaction temperature.

In some embodiments, wherein in step 2), the multiplex PCR amplification comprises performing multiplex PCR amplification using the first primer set and the second primer set of the primer composition with the DNA as a template, respectively, and combining the amplification products. The amplification mode can be understood as two-tube amplification, and the two-tube amplification is favorable for further improving the specificity of the multiplex PCR reaction.

In some embodiments, the amplification conditions for multiplex PCR amplification with the first primer set (T1) and multiplex PCR amplification with the second primer set (T2) may be different.

In some embodiments, the reaction temperature and time for multiplex PCR amplification of T1 are, in order:

(1) Keeping the temperature at 93-97 ℃ for 3-4 mins;

(2) Keeping the temperature of 97-99 ℃ for 15-25s, keeping the temperature of 58-62 ℃ for 5-7 min, and sequentially carrying out 18-22 cycles;

(3) Keeping the temperature of 71-73 ℃ for 4-6 min.

In some embodiments, the reaction temperature and time for multiplex PCR amplification of T2 are, in order:

(1) Keeping the temperature of 93-97 ℃ for 3-4 mins;

(2) Keeping the temperature of 97-99 ℃ for 15-25s, keeping the temperature of 58-62 ℃ for 3-5 min, and sequentially carrying out 18-22 cycles;

(3) Keeping the temperature of 71-73 ℃ for 4-6 min.

In some embodiments, before the sequencing in step 3), a step of purifying the obtained amplification product is further included.

In some embodiments, when two-tube amplification is used, the two-tube amplification products can be combined first and then purified, so as to reduce the number of purification operations and save cost.

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

In some embodiments, the sequencing is next-generation sequencing. The second-generation sequencing has the characteristic of high throughput, and is favorable for quickly obtaining a sequencing result.

In some embodiments, the second generation sequencing may be implemented using the existing MGISEQ (chinese wisdom) sequencing platform, illumina (because of memna) sequencing platform, and the application is not limited thereto.

In some embodiments, the sequence alignment comprises aligning the sequence information obtained by sequencing with genomic sequences of mycobacterium tuberculosis in a known database and combining with a database of drug-resistant genes to obtain the full-length sequence of the drug-resistant genes of mycobacterium tuberculosis and information of all mutations contained in each gene. The mutations include both known mutations and unknown mutations that have not been reported.

In some embodiments, the known database may be the NCBI database, numbered NC — 000962.3.

In some embodiments, the drug-resistant gene database can be a CARD drug-resistant gene database.

The skilled person may also select from other known databases regarding mycobacterium tuberculosis genomes or drug resistance genes, which is not limited herein.

In some embodiments, performing the second-generation sequencing further comprises a step of linking the multiplex PCR amplification products to an adaptor sequence, which is a routine operation of the second-generation sequencing, and a person skilled in the art can select an appropriate adaptor sequence and linking mode according to a sequencing platform, which is not limited herein.

Illustratively, when using the Illumina (neminer) sequencing platform, one can select the Illumina5 'end Index sequencing linker and the Illumina3' end Index sequencing linker that match it; when the MGISEQ (Chinesota chinensis) sequencing platform is adopted, the matched MGISEQ5 'end Index sequencing joint and MGISEQ 3' end Index sequencing joint can be adopted; this is a common technique for second generation sequencing and is not limited herein.

In some embodiments, the linker sequence can be added by PCR amplification or by sticky end ligation, which is a routine procedure in the art and is not limited herein.

The multiplex PCR reaction conditions for connecting the adaptor sequence to the multiplex PCR amplification product of the present application are reaction conditions commonly used in the art, and the present application is not limited herein, and in some embodiments, the multiplex PCR reaction temperature and time for connecting the adaptor sequence are:

(1) Keeping the temperature of 93-97 ℃ for 2-4 mins;

(2) Keeping the temperature at 97-99 ℃ for 15-25s, keeping the temperature at 56-60 ℃ for 45-90s, keeping the temperature at 70-73 ℃ for 20-40 s, and sequentially carrying out 8-12 cycles;

(3) Keeping the temperature of 71-73 ℃ for 4-6 min.

Amplification products to which linker sequences are attached are commonly referred to in the art as libraries.

In some embodiments, next generation sequencing may also include a step of library concentration determination. In some preferred embodiments, the library concentration may be 10-50 ng/. Mu.L.

In some embodiments, secondary sequencing may also include library fragment length and purity measurements. In some embodiments, the library target fragment distribution interval is between 280bp and 420 bp.

In some embodiments, the library may be sequenced using a MGISEQ sequencer, with the sequencing strategy being PE150.

In some embodiments, the amount of raw sequencing data per sequencing sample is greater than 300Mb bases.

In some embodiments, the method further comprises a step of performing data filtering on the sequencing data to remove low-quality data before performing the sequence alignment, which is a conventional technique in the art, and the skilled person can implement the method as required or by using a conventional data filtering method, which is not limited herein.

The primer composition and the use thereof of the present application will be described below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 genomic target region determination and primer design

1) By consulting literature data, the information of the related drug resistance genes of the mycobacterium tuberculosis is collected, and finally 45 reported drug resistance related genes of the tuberculosis are determined.

2) And taking the genome of the mycobacterium tuberculosis H37Rv as a reference genome, and acquiring the position information of the related gene on the genome and the full-length information of the gene in an NCBI database according to the searched gene name.

The 45 drug 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, inhA, katG, panD, pepQ, pnCA, ponA1, ribD, rplC, rpoA, rpoB, rpoC, rpsA, rpsL, rrl, rrs, rv0678, rv1979c, aftA, thyA, thyX, tlyA, ubiA, whiB7

The 15 tuberculosis treating medicines include: rifampin, isoniazid, ethambutol, pyrazinamide, streptomycin, levofloxacin/moxifloxacin, bedaquiline, linezolid, clofazimine, cycloserine, delamanid, amikacin, capreomycin, ethionamide and para-aminosalicylic acid.

3) Multiplex PCR primers are designed for a target gene region using primer design software such as AIdesign, primerPlex, and the like. After the primer is preliminarily designed, the primer is subjected to quality control of a standard process by using primer quality control software, the Tm value, GC content, hairpin structure, primer dimer, non-specific amplification and other aspects of the primer are scored, and the amplification effect of the primer is preliminarily evaluated before an experiment.

The primer sequence is shown as SEQ ID NO. 1-784, wherein SEQ ID NO. 1-196 is a forward primer in the first primer combination, and SEQ ID NO. 197-392 is a reverse primer sequentially paired with the forward primer; SEQ ID nos. 393 to 588 are forward primers in the second primer combination, and SEQ ID nos. 589 to 784 are reverse primers sequentially paired with the forward primers.

4) After primer design is complete, commercial primer synthesis is performed to obtain the primer composition of the present application.

Example 2 detection of Mycobacterium tuberculosis drug-resistant Gene, i.e., mutation thereof

1. Sample pretreatment and DNA extraction

The test sample referred to in this application may comprise a variety of common sample types, such as sputum, alveolar lavage, blood, tissue, bacterial culture, and the like. In this example, the sample is obtained from Mycobacterium tuberculosis culture sample of Shenzhen hospital, the conventional commercial DNA extraction kit (DNA extraction kit of Tiangen Biochemical technology Co., ltd., product number NG 550) is used, the total DNA is extracted from the sample according to the corresponding method, and then the total DNA is utilized

3.0Fluorometer (Qubit dsDNA HS Assay Kit, cat # Q32851) detects the DNA concentration, and the quality is qualified (85 ng total DNA, 1.7 ng/. Mu.l concentration) for the next multiplex PCR amplification.

2. Multiplex PCR amplification and library construction

Multiplex PCR amplification was performed using the primer composition obtained in example 1 and the extracted DNA.

2.1 round 1 multiplex PCR reaction

The 1 st round of multiplex PCR reaction is divided into 2 reaction tubes, wherein a first Primer combination Primer pool T1 consisting of primers shown in SEQ ID NO. 1-392 is adopted in one tube, and a second Primer combination Primer pool T2 consisting of primers shown in SEQ ID NO.393-784 is adopted in the other tube. Each Primer concentration in Primer pool T1 and Primer pool T2 was 0.5. Mu.M. The other reagents in the 2 reaction tubes were the same. The multiplex PCR reaction system is shown in Table 1 below:

TABLE 1 multiplex PCR reaction System

Reagent	Volume (μ L)
		ddH 2 O	3.2
Enhancer buffer NB(1N)	3.5
		Enhancer buffer M	2.5
Primer pool T1/T2	5
		DNA	5.8
IGT-EM808polymerase mixture	10
		Total of	30

Note: IGT-EM808 polymerase mix, enhancer buffer NB (1N), enhancer buffer M are all available from Aijiekang Biotech under the trade designation M60012.

The multiplex PCR reaction conditions were as follows:

after the 1 st round of PCR reaction, 15. Mu.L of each PCR of 2 reaction tubes was combined.

2.2 magnetic bead purification of pooled products

1) Prepare 80% ethanol with anhydrous ethanol and nuclease-free water in advance, and place at room temperature for standby, please use freshly prepared 80% ethanol for magnetic bead purification as much as possible.

2) Taking out purified magnetic beads (Agencourt AMPure XP kit, purchased from Beckmann Coulter, inc., with the product number of A63880) from a refrigerator at 4 ℃ in advance, mixing uniformly, and placing at room temperature for balancing for 30min; the purified beads, which had equilibrated to room temperature, were vortexed and mixed until ready for use.

3) Add 0.9 volume of magnetic beads (27. Mu.L) to 30. Mu.L of the PCR pool, pipette or vortex and mix well, and let stand at room temperature for 5min.

4) Instantaneous centrifugation, place the PCR tube on a magnetic rack for 3min until the solution is clear.

5) The supernatant was removed completely, the PCR tube was removed from the magnetic stand, 50. Mu.L of YF buffer B (purchased from Aijitacon Biotech, inc., cat # M60022) was added to the tube, pipetted and mixed well, and allowed to stand at room temperature for 5min.

6) Transient centrifugation, place PCR tube on DynaMag-96 Side magnetic frame for 3min.

7) The PCR tube was held on the magnetic stand, the supernatant was carefully removed, 180. Mu.L of 80% ethanol solution was added to the PCR tube, and the tube was allowed to stand for 30 seconds.

8) The PCR tube was held on the magnetic stand, the supernatant was removed, 180. Mu.L of 80% ethanol solution was again added to the PCR tube, and the tube was allowed to stand for 30 seconds, and the supernatant was removed.

9) Cover the tube cap, centrifuge instantaneously, centrifuge the residual ethanol to the bottom of the tube, place the PCR tube on a magnetic rack, carefully use a 10. Mu.L pipette to remove the residual ethanol at the bottom, and take care not to attract the beads.

10 Keeping the PCR tube on a magnetic frame, standing for 3-5 min at room temperature, and airing the magnetic beads to completely volatilize the residual ethanol.

11 Adding 24 μ L of nuclease-free water, taking down the PCR tube from the magnetic frame, sucking or vortexing, mixing, and standing at room temperature for 2min.

12 Instant centrifugation, placing the PCR tube on a magnetic frame for 2min until the solution is clarified.

13 Pipette 13.5. Mu.L of supernatant, and transfer to a new PCR tube, the tube supernatant being the combined multiplex PCR product.

2.3 multiplex PCR reactions with 2 nd round linker sequences

TABLE 2 linker sequence reaction systems

Reagent	Volume (mu L)
		PCR product	13.5
Enhancer buffer M	2.5
		ddH2O	2
UDI Index(5μM)	2
		IGT-EM808polymerase mixture	10

Note: enhancer buffer M, IGT-EM808 polymerase mix, was purchased from Aigetaikang Biotech under the trade designation M60022, and UDI Index (5. Mu.M) was purchased from Aigetaikang Biotech under the trade designation M70142.

The multiplex PCR reaction system is as follows:

heating the cover to 105 ℃;

95℃3min；

2s at 98 ℃, 1min at 58 ℃, 30s at 72 ℃ and 9 cycles;

72℃5min。

2.4 round 2 magnetic bead purification

And purifying the product of the 2 nd round joint sequence multiplex PCR reaction by adopting the same magnetic bead purification step as the previous step to obtain a multiplex PCR library.

2.5 library quantification

1 μ L of the library was used

3.0Fluorometer (Qubit dsDNA HS Assay Kit) to determine the library concentration, record the library concentration. The optimal concentration is about 50 ng/. Mu.L.

2.6 library quality testing

Taking 1 mu L of library sample, and using a Qsep100 full-automatic nucleic acid protein analysis system to measure the length and purity of the library fragment, wherein the distribution interval of the target fragment of the normal library is between 280bp and 420 bp.

3. Sequencing on machine

And (3) carrying out on-machine sequencing on the established sequencing library on a MGISEQ sequencer, wherein the sequencing strategy is PE150, and generating sequencing data. The amount of raw sequencing data per sample was greater than 300Mb bases.

4. Biological information analysis

And (3) firstly carrying out data filtration on off-line original sequencing data, removing low-quality data, then carrying out sequence comparison, wherein a reference genome is provided by an NCBI database and is numbered as NC-000962.3, and the detection coverage of a drug-resistant gene and a drug-resistant mutation site are analyzed.

4.1 drug resistance Gene detection coverage analysis

The following table 3 shows statistical information of the coverage of detection of the drug-resistant genes related to the strains obtained in the example, and the results show that the coverage of 45 genes can reach 100%, which indicates that the primer composition and the detection method of the present application can cover the full-length sequences of the related genes.

TABLE 3 drug resistance Gene detection coverage analysis

/>

4.2 analysis of drug-resistant Gene mutation sites

And (3) carrying out sequence analysis on the obtained related drug-resistant genes of the strains, and judging whether the related genes have drug-resistant mutation or not by combining a CARD drug-resistant gene database. Taking the ponA1 gene as an example, the length of the gene is 2037bp, and the results are shown in FIG. 1 by performing mutation analysis on the sequence of the gene and performing alignment analysis on the sequence in the CARD drug-resistant gene database. In fig. 1, sequence 1 is a ponA1 gene sequence and a known drug-resistant mutation site (base with enlarged font in the sequence) recorded in a CARD drug-resistant gene database, and sequence 2 is a ponA1 gene sequence and a mutation site obtained by analyzing a sample to be detected in this embodiment, wherein a site marked in a box is a newly found mutation site, and the result shows that the primer composition and the method using the same can display the full-length sequence information of the drug-resistant gene of the sample to be detected, and can detect and analyze unknown mutation in addition to the known mutation site, thereby finding out the gene mutation at any site of the gene.

The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (10)

1. A primer composition comprising a first primer combination and a second primer combination, wherein the first primer combination comprises: the primers shown in SEQ ID NO. 1-392, and the second primer combination comprises the primers shown in SEQ ID NO. 393-784.

2. A kit for detecting drug-resistant gene of Mycobacterium tuberculosis, comprising the primer composition of claim 1.

3. Use of the primer composition of claim 1 or the kit of claim 2 for the detection of a drug-resistant gene of mycobacterium tuberculosis and/or any mutation thereof.

4. The use of claim 3, wherein the Mycobacterium tuberculosis drug comprises at least one of rifampin, isoniazid, ethambutol, pyrazinamide, streptomycin, levofloxacin/moxifloxacin, bedaquiline, linezolid, clofazimine, cycloserine, delamasil, amikacin, capreomycin, ethionamide, para-aminosalicylic acid.

5. The use of claim 3, wherein the Mycobacterium tuberculosis drug resistance gene comprises at least one of the 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, thybX, tlyA, whiiA, whiiB 7 genes; preferably all of said genes are included.

6. A method of detecting a mycobacterium tuberculosis drug resistance gene and/or any mutation thereof in a sample, comprising:

1) Extracting DNA in the sample;

2) Performing multiplex PCR amplification using the primer composition according to claim 1 or the kit according to claim 2, using the DNA as a template;

3) Sequencing and sequence comparison are carried out on the obtained amplification products so as to obtain the information of the drug tolerance gene of the mycobacterium tuberculosis and/or the mutation of the drug tolerance gene;

preferably, the sequencing is next generation sequencing.

7. The method of claim 6, wherein the sample is selected from at least one of sputum, alveolar lavage, blood, tissue, bacterial culture.

8. The method according to claim 6, wherein in step 2), the multiplex PCR amplification comprises performing multiplex PCR amplification using the first primer set and the second primer set of the primer composition with the DNA as a template, respectively, and combining the amplification products.

9. The method of claim 6, wherein the temperature conditions for the multiplex PCR amplification reaction are, in order:

(1) Keeping the temperature at 93-97 ℃ for 3-4 mins;

(2) Keeping the temperature of 97-99 ℃ for 15-25s, keeping the temperature of 58-62 ℃ for 3-7 min, and sequentially carrying out 18-22 cycles;

(3) Keeping the temperature of 71-73 ℃ for 4-6 min.

10. The method according to claim 6, wherein prior to the sequencing in step 3), further comprising the step of purifying the obtained amplification product; preferably, the purification is magnetic bead purification.

Images