CN113817850A - Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof - Google Patents
Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof Download PDFInfo
- Publication number
- CN113817850A CN113817850A CN202111044169.3A CN202111044169A CN113817850A CN 113817850 A CN113817850 A CN 113817850A CN 202111044169 A CN202111044169 A CN 202111044169A CN 113817850 A CN113817850 A CN 113817850A
- Authority
- CN
- China
- Prior art keywords
- gene
- sites
- artificial sequence
- dna
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000003814 drug Substances 0.000 title claims abstract description 186
- 229940079593 drug Drugs 0.000 title claims abstract description 168
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 152
- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 79
- 238000001514 detection method Methods 0.000 title claims abstract description 64
- 239000000203 mixture Substances 0.000 title claims abstract description 62
- 201000010099 disease Diseases 0.000 claims abstract description 13
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 408
- 206010059866 Drug resistance Diseases 0.000 claims description 64
- AEUTYOVWOVBAKS-UWVGGRQHSA-N ethambutol Chemical compound CC[C@@H](CO)NCCN[C@@H](CC)CO AEUTYOVWOVBAKS-UWVGGRQHSA-N 0.000 claims description 42
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 claims description 31
- 238000000034 method Methods 0.000 claims description 30
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 28
- 238000012163 sequencing technique Methods 0.000 claims description 24
- -1 A-292 Chemical compound 0.000 claims description 23
- 238000012360 testing method Methods 0.000 claims description 23
- 229960004821 amikacin Drugs 0.000 claims description 21
- LKCWBDHBTVXHDL-RMDFUYIESA-N amikacin Chemical compound O([C@@H]1[C@@H](N)C[C@H]([C@@H]([C@H]1O)O[C@@H]1[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O1)O)NC(=O)[C@@H](O)CCN)[C@H]1O[C@H](CN)[C@@H](O)[C@H](O)[C@H]1O LKCWBDHBTVXHDL-RMDFUYIESA-N 0.000 claims description 21
- 229960000285 ethambutol Drugs 0.000 claims description 21
- IPEHBUMCGVEMRF-UHFFFAOYSA-N pyrazinecarboxamide Chemical compound NC(=O)C1=CN=CC=N1 IPEHBUMCGVEMRF-UHFFFAOYSA-N 0.000 claims description 21
- 229960003702 moxifloxacin Drugs 0.000 claims description 20
- FABPRXSRWADJSP-MEDUHNTESA-N moxifloxacin Chemical compound COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 FABPRXSRWADJSP-MEDUHNTESA-N 0.000 claims description 20
- 229960005206 pyrazinamide Drugs 0.000 claims description 20
- WUBBRNOQWQTFEX-UHFFFAOYSA-N 4-aminosalicylic acid Chemical compound NC1=CC=C(C(O)=O)C(O)=C1 WUBBRNOQWQTFEX-UHFFFAOYSA-N 0.000 claims description 19
- 229960004909 aminosalicylic acid Drugs 0.000 claims description 19
- 229960001225 rifampicin Drugs 0.000 claims description 19
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 claims description 18
- 229960005322 streptomycin Drugs 0.000 claims description 14
- 101100383746 Chlamydomonas reinhardtii chlL gene Proteins 0.000 claims description 13
- 229930027917 kanamycin Natural products 0.000 claims description 13
- 229960000318 kanamycin Drugs 0.000 claims description 13
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 claims description 13
- 229930182823 kanamycin A Natural products 0.000 claims description 13
- 101150070825 rsmG gene Proteins 0.000 claims description 13
- 102220628501 Histone H1.1_D94H_mutation Human genes 0.000 claims description 12
- 101150005343 INHA gene Proteins 0.000 claims description 12
- ZWBTYMGEBZUQTK-PVLSIAFMSA-N [(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,32-tetrahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-1'-(2-methylpropyl)-6,23-dioxospiro[8,33-dioxa-24,27,29-triazapentacyclo[23.6.1.14,7.05,31.026,30]tritriaconta-1(32),2,4,9,19,21,24,26,30-nonaene-28,4'-piperidine]-13-yl] acetate Chemical compound CO[C@H]1\C=C\O[C@@]2(C)Oc3c(C2=O)c2c4NC5(CCN(CC(C)C)CC5)N=c4c(=NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@@H]1C)c(O)c2c(O)c3C ZWBTYMGEBZUQTK-PVLSIAFMSA-N 0.000 claims description 12
- AEOCXXJPGCBFJA-UHFFFAOYSA-N ethionamide Chemical compound CCC1=CC(C(N)=S)=CC=N1 AEOCXXJPGCBFJA-UHFFFAOYSA-N 0.000 claims description 12
- 229960002001 ethionamide Drugs 0.000 claims description 12
- 229960000885 rifabutin Drugs 0.000 claims description 12
- 101150090202 rpoB gene Proteins 0.000 claims description 11
- 108010065839 Capreomycin Proteins 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 10
- WDQPAMHFFCXSNU-BGABXYSRSA-N clofazimine Chemical compound C12=CC=CC=C2N=C2C=C(NC=3C=CC(Cl)=CC=3)C(=N/C(C)C)/C=C2N1C1=CC=C(Cl)C=C1 WDQPAMHFFCXSNU-BGABXYSRSA-N 0.000 claims description 10
- 229960004287 clofazimine Drugs 0.000 claims description 10
- VCOPTHOUUNAYKQ-WBTCAYNUSA-N (3s)-3,6-diamino-n-[[(2s,5s,8e,11s,15s)-15-amino-11-[(6r)-2-amino-1,4,5,6-tetrahydropyrimidin-6-yl]-8-[(carbamoylamino)methylidene]-2-(hydroxymethyl)-3,6,9,12,16-pentaoxo-1,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide;(3s)-3,6-diamino-n-[[(2s,5s,8 Chemical compound N1C(=O)\C(=C/NC(N)=O)NC(=O)[C@H](CNC(=O)C[C@@H](N)CCCN)NC(=O)[C@H](C)NC(=O)[C@@H](N)CNC(=O)[C@@H]1[C@@H]1NC(N)=NCC1.N1C(=O)\C(=C/NC(N)=O)NC(=O)[C@H](CNC(=O)C[C@@H](N)CCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CNC(=O)[C@@H]1[C@@H]1NC(N)=NCC1 VCOPTHOUUNAYKQ-WBTCAYNUSA-N 0.000 claims description 9
- 102220500062 Interferon-induced GTP-binding protein Mx2_V77A_mutation Human genes 0.000 claims description 9
- 102220473410 Photoreceptor ankyrin repeat protein_V138G_mutation Human genes 0.000 claims description 9
- 229960004602 capreomycin Drugs 0.000 claims description 9
- 229960003350 isoniazid Drugs 0.000 claims description 9
- 238000007403 mPCR Methods 0.000 claims description 9
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 9
- 102220330185 rs1364834319 Human genes 0.000 claims description 9
- 102220285261 rs1443234544 Human genes 0.000 claims description 9
- 102220005464 rs35203445 Human genes 0.000 claims description 9
- 102220057413 rs730881778 Human genes 0.000 claims description 9
- 230000003321 amplification Effects 0.000 claims description 8
- 101150070420 gyrA gene Proteins 0.000 claims description 8
- 101150013110 katG gene Proteins 0.000 claims description 8
- 238000003745 diagnosis Methods 0.000 claims description 7
- 101150013736 gyrB gene Proteins 0.000 claims description 7
- 229960003907 linezolid Drugs 0.000 claims description 7
- TYZROVQLWOKYKF-ZDUSSCGKSA-N linezolid Chemical compound O=C1O[C@@H](CNC(=O)C)CN1C(C=C1F)=CC=C1N1CCOCC1 TYZROVQLWOKYKF-ZDUSSCGKSA-N 0.000 claims description 7
- 101150071065 Ddn gene Proteins 0.000 claims description 6
- 102220628463 Histone H1.1_D94A_mutation Human genes 0.000 claims description 6
- 102220628523 Histone H1.1_D94N_mutation Human genes 0.000 claims description 6
- 102220628522 Histone H1.1_D94Y_mutation Human genes 0.000 claims description 6
- 102220628506 Histone H1.1_G88A_mutation Human genes 0.000 claims description 6
- 102220628505 Histone H1.1_G88C_mutation Human genes 0.000 claims description 6
- 102220628464 Histone H1.1_S91P_mutation Human genes 0.000 claims description 6
- 102220521681 Insulin-induced gene 1 protein_S94A_mutation Human genes 0.000 claims description 6
- 102220493392 Sodium/calcium exchanger 3_I21T_mutation Human genes 0.000 claims description 6
- 101150014588 ethA gene Proteins 0.000 claims description 6
- 101150088356 fbiA gene Proteins 0.000 claims description 6
- 101150075154 fbiC gene Proteins 0.000 claims description 6
- 101150041999 fgd1 gene Proteins 0.000 claims description 6
- 101150068528 mabA gene Proteins 0.000 claims description 6
- 101150017363 pepQ gene Proteins 0.000 claims description 6
- 101150042391 rpoC gene Proteins 0.000 claims description 6
- 102200026998 rs104894205 Human genes 0.000 claims description 6
- 102200083538 rs530109812 Human genes 0.000 claims description 6
- 102220090408 rs876657989 Human genes 0.000 claims description 6
- 101150072314 thyA gene Proteins 0.000 claims description 6
- 101150060849 whiB7 gene Proteins 0.000 claims description 6
- 108091093088 Amplicon Proteins 0.000 claims description 5
- 101150062801 embB gene Proteins 0.000 claims description 5
- 101150022921 pncA gene Proteins 0.000 claims description 5
- 101150098466 rpsL gene Proteins 0.000 claims description 5
- GSDSWSVVBLHKDQ-UHFFFAOYSA-N 9-fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-2,3-dihydro-7H-[1,4]oxazino[2,3,4-ij]quinoline-6-carboxylic acid Chemical compound FC1=CC(C(C(C(O)=O)=C2)=O)=C3N2C(C)COC3=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-UHFFFAOYSA-N 0.000 claims description 4
- DYDCUQKUCUHJBH-UWTATZPHSA-N D-Cycloserine Chemical compound N[C@@H]1CONC1=O DYDCUQKUCUHJBH-UWTATZPHSA-N 0.000 claims description 4
- DYDCUQKUCUHJBH-UHFFFAOYSA-N D-Cycloserine Natural products NC1CONC1=O DYDCUQKUCUHJBH-UHFFFAOYSA-N 0.000 claims description 4
- 229960000508 bedaquiline Drugs 0.000 claims description 4
- QUIJNHUBAXPXFS-XLJNKUFUSA-N bedaquiline Chemical compound C1([C@H](C2=CC3=CC(Br)=CC=C3N=C2OC)[C@@](O)(CCN(C)C)C=2C3=CC=CC=C3C=CC=2)=CC=CC=C1 QUIJNHUBAXPXFS-XLJNKUFUSA-N 0.000 claims description 4
- 229960003077 cycloserine Drugs 0.000 claims description 4
- 238000001647 drug administration Methods 0.000 claims description 4
- 229960001699 ofloxacin Drugs 0.000 claims description 4
- 101150077293 rplC gene Proteins 0.000 claims description 4
- 101150008822 rpsA gene Proteins 0.000 claims description 4
- 101150007324 ubiA gene Proteins 0.000 claims description 4
- OWNWYCOLFIFTLK-UHFFFAOYSA-N 4-[2-(tert-butylamino)-1-hydroxyethyl]-2-(hydroxymethyl)phenol;hydron;chloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 OWNWYCOLFIFTLK-UHFFFAOYSA-N 0.000 claims description 3
- 102220615425 40S ribosomal protein S13_K43R_mutation Human genes 0.000 claims description 3
- 102220615489 40S ribosomal protein S13_K43T_mutation Human genes 0.000 claims description 3
- 102220615430 40S ribosomal protein S13_K88Q_mutation Human genes 0.000 claims description 3
- 102220615571 40S ribosomal protein S13_K88R_mutation Human genes 0.000 claims description 3
- 102220581393 ATP synthase subunit d, mitochondrial_W83C_mutation Human genes 0.000 claims description 3
- 102100022524 Alpha-1-antichymotrypsin Human genes 0.000 claims description 3
- 102220557088 Alstrom syndrome protein 1_I21V_mutation Human genes 0.000 claims description 3
- 101100397068 Bacillus subtilis (strain 168) iolD gene Proteins 0.000 claims description 3
- 102220583485 Cellular tumor antigen p53_D49Y_mutation Human genes 0.000 claims description 3
- 102220584161 Cellular tumor antigen p53_P75L_mutation Human genes 0.000 claims description 3
- 102220584160 Cellular tumor antigen p53_P75R_mutation Human genes 0.000 claims description 3
- 102220584230 Cellular tumor antigen p53_P75S_mutation Human genes 0.000 claims description 3
- 102220519288 Conserved oligomeric Golgi complex subunit 3_H75N_mutation Human genes 0.000 claims description 3
- 102220473254 Emerin_S49A_mutation Human genes 0.000 claims description 3
- 102220499828 Epiphycan_S150C_mutation Human genes 0.000 claims description 3
- 102220557165 Epithelial membrane protein 1_D1024N_mutation Human genes 0.000 claims description 3
- 102220529131 Eukaryotic translation initiation factor 2 subunit 1_S53P_mutation Human genes 0.000 claims description 3
- 102220635973 Exopolyphosphatase PRUNE1_D28A_mutation Human genes 0.000 claims description 3
- 101150002627 F12L gene Proteins 0.000 claims description 3
- 102220566358 Folate receptor alpha_E40A_mutation Human genes 0.000 claims description 3
- 102220566357 Folate receptor alpha_E40G_mutation Human genes 0.000 claims description 3
- 102220566362 Folate receptor alpha_F152S_mutation Human genes 0.000 claims description 3
- 102220566356 Folate receptor alpha_I43A_mutation Human genes 0.000 claims description 3
- 102220566355 Folate receptor alpha_I43T_mutation Human genes 0.000 claims description 3
- 102220566354 Folate receptor alpha_N73S_mutation Human genes 0.000 claims description 3
- 102220566363 Folate receptor alpha_S150G_mutation Human genes 0.000 claims description 3
- 102220643640 G-protein coupled receptor 42_V256A_mutation Human genes 0.000 claims description 3
- 102220577803 Hepatocyte nuclear factor 3-alpha_C20S_mutation Human genes 0.000 claims description 3
- 102220577755 Hepatocyte nuclear factor 3-alpha_Q88E_mutation Human genes 0.000 claims description 3
- 102220628525 Histone H1.1_T80A_mutation Human genes 0.000 claims description 3
- 101000678026 Homo sapiens Alpha-1-antichymotrypsin Proteins 0.000 claims description 3
- 102220510412 Matrix-remodeling-associated protein 5_V54A_mutation Human genes 0.000 claims description 3
- 102220473411 Photoreceptor ankyrin repeat protein_H21R_mutation Human genes 0.000 claims description 3
- 102220473413 Photoreceptor ankyrin repeat protein_I49V_mutation Human genes 0.000 claims description 3
- 102220473409 Photoreceptor ankyrin repeat protein_L136R_mutation Human genes 0.000 claims description 3
- 102220473366 Photoreceptor ankyrin repeat protein_M117I_mutation Human genes 0.000 claims description 3
- 102220467674 Podocan-like protein 1_H48Q_mutation Human genes 0.000 claims description 3
- 102220636543 Prostate and breast cancer overexpressed gene 1 protein_I73T_mutation Human genes 0.000 claims description 3
- 102220472513 Protein ENL_H48N_mutation Human genes 0.000 claims description 3
- 102220480119 Protein FAM3A_I21M_mutation Human genes 0.000 claims description 3
- 102220516121 Protein KASH5_D66N_mutation Human genes 0.000 claims description 3
- 102220465408 Protein arginine N-methyltransferase 1_K88M_mutation Human genes 0.000 claims description 3
- 102220513889 Protein stum homolog_D111A_mutation Human genes 0.000 claims description 3
- 102220547630 Protocadherin-10_L79S_mutation Human genes 0.000 claims description 3
- 102220474379 Retinoic acid receptor RXR-alpha_E434A_mutation Human genes 0.000 claims description 3
- 102220512389 Ribonuclease kappa_D135A_mutation Human genes 0.000 claims description 3
- 102100026757 Serine/threonine-protein kinase 19 Human genes 0.000 claims description 3
- 102220493406 Sodium/calcium exchanger 3_I43F_mutation Human genes 0.000 claims description 3
- 102220501733 Sodium/calcium exchanger 3_I43V_mutation Human genes 0.000 claims description 3
- 102220506618 Sphingosine-1-phosphate phosphatase 1_S335I_mutation Human genes 0.000 claims description 3
- 102220565024 Survival motor neuron protein_L39A_mutation Human genes 0.000 claims description 3
- 102220484140 T cell receptor alpha variable 34_S63R_mutation Human genes 0.000 claims description 3
- 102220641563 Transcription factor MafB_S66C_mutation Human genes 0.000 claims description 3
- 102220575110 UDP-glucuronosyltransferase 1A10_F93G_mutation Human genes 0.000 claims description 3
- 101100389989 Vaccinia virus (strain Ankara) MVA042L gene Proteins 0.000 claims description 3
- 101100389992 Vaccinia virus (strain Western Reserve) VACWR051 gene Proteins 0.000 claims description 3
- 101100389993 Variola virus (isolate Human/India/Ind3/1967) C16L gene Proteins 0.000 claims description 3
- 102220567135 Vasopressin V2 receptor_R64W_mutation Human genes 0.000 claims description 3
- 101150024831 ahpC gene Proteins 0.000 claims description 3
- 101150070828 alr gene Proteins 0.000 claims description 3
- 101150099875 atpE gene Proteins 0.000 claims description 3
- 102220424078 c.145C>G Human genes 0.000 claims description 3
- 102220351489 c.146G>C Human genes 0.000 claims description 3
- 102220355303 c.221T>G Human genes 0.000 claims description 3
- 102220412685 c.248T>C Human genes 0.000 claims description 3
- 102220405661 c.249G>C Human genes 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 101150052102 cycA gene Proteins 0.000 claims description 3
- 101150109034 ddlA gene Proteins 0.000 claims description 3
- 101150020935 dfrA gene Proteins 0.000 claims description 3
- 101150016486 efpA gene Proteins 0.000 claims description 3
- 101150111189 eis gene Proteins 0.000 claims description 3
- 101150005689 embA gene Proteins 0.000 claims description 3
- 101150011764 embC gene Proteins 0.000 claims description 3
- 101150104409 embR gene Proteins 0.000 claims description 3
- 101150023883 ethR gene Proteins 0.000 claims description 3
- 101150024406 fabD gene Proteins 0.000 claims description 3
- 101150018736 fbiB gene Proteins 0.000 claims description 3
- 101150073342 folC gene Proteins 0.000 claims description 3
- 101150028709 furA gene Proteins 0.000 claims description 3
- 101150028828 iniA gene Proteins 0.000 claims description 3
- 101150092878 iniB gene Proteins 0.000 claims description 3
- 101150034439 iniC gene Proteins 0.000 claims description 3
- 101150075271 kasA gene Proteins 0.000 claims description 3
- 101150114749 mmpL5 gene Proteins 0.000 claims description 3
- 101150087123 nat gene Proteins 0.000 claims description 3
- 101150106215 ndh gene Proteins 0.000 claims description 3
- 101150076071 panD gene Proteins 0.000 claims description 3
- 239000008194 pharmaceutical composition Substances 0.000 claims description 3
- 101150022503 phoR gene Proteins 0.000 claims description 3
- 101150003625 ribD gene Proteins 0.000 claims description 3
- 101150037064 rpoA gene Proteins 0.000 claims description 3
- 101150038244 rpoZ gene Proteins 0.000 claims description 3
- 102200052551 rs104894166 Human genes 0.000 claims description 3
- 102220233169 rs104894501 Human genes 0.000 claims description 3
- 102200099206 rs104894519 Human genes 0.000 claims description 3
- 102200075063 rs104894696 Human genes 0.000 claims description 3
- 102220198041 rs1057519837 Human genes 0.000 claims description 3
- 102200140795 rs1057520039 Human genes 0.000 claims description 3
- 102220214691 rs1060501895 Human genes 0.000 claims description 3
- 102220226097 rs1064793802 Human genes 0.000 claims description 3
- 102220009428 rs111033636 Human genes 0.000 claims description 3
- 102200012564 rs111033663 Human genes 0.000 claims description 3
- 102200076676 rs116262672 Human genes 0.000 claims description 3
- 102200140928 rs121908340 Human genes 0.000 claims description 3
- 102200108190 rs121912669 Human genes 0.000 claims description 3
- 102220198035 rs121913396 Human genes 0.000 claims description 3
- 102200110904 rs121917881 Human genes 0.000 claims description 3
- 102200115883 rs121918076 Human genes 0.000 claims description 3
- 102220266857 rs1290433318 Human genes 0.000 claims description 3
- 102220262410 rs1306740707 Human genes 0.000 claims description 3
- 102220267412 rs1316316435 Human genes 0.000 claims description 3
- 102200018951 rs137852608 Human genes 0.000 claims description 3
- 102200096359 rs140076803 Human genes 0.000 claims description 3
- 102220060025 rs141586345 Human genes 0.000 claims description 3
- 102220191196 rs142422937 Human genes 0.000 claims description 3
- 102220101985 rs143626223 Human genes 0.000 claims description 3
- 102200089608 rs1553619431 Human genes 0.000 claims description 3
- 102220251125 rs1555263137 Human genes 0.000 claims description 3
- 102220283477 rs1555734937 Human genes 0.000 claims description 3
- 102220024847 rs199473037 Human genes 0.000 claims description 3
- 102220000821 rs237025 Human genes 0.000 claims description 3
- 102200051957 rs2472553 Human genes 0.000 claims description 3
- 102200030294 rs267600971 Human genes 0.000 claims description 3
- 102200110982 rs28931568 Human genes 0.000 claims description 3
- 102200044877 rs28931589 Human genes 0.000 claims description 3
- 102200044940 rs28931589 Human genes 0.000 claims description 3
- 102200095460 rs28940291 Human genes 0.000 claims description 3
- 102200082819 rs33952850 Human genes 0.000 claims description 3
- 102220202059 rs368077581 Human genes 0.000 claims description 3
- 102220036790 rs371011390 Human genes 0.000 claims description 3
- 102200050844 rs386833793 Human genes 0.000 claims description 3
- 102200050852 rs386833816 Human genes 0.000 claims description 3
- 102200062348 rs397514725 Human genes 0.000 claims description 3
- 102220026083 rs397518427 Human genes 0.000 claims description 3
- 102220085613 rs537362211 Human genes 0.000 claims description 3
- 102220056410 rs560874115 Human genes 0.000 claims description 3
- 102220163625 rs566738926 Human genes 0.000 claims description 3
- 102200124760 rs587777729 Human genes 0.000 claims description 3
- 102220039363 rs587778174 Human genes 0.000 claims description 3
- 102220040539 rs587778365 Human genes 0.000 claims description 3
- 102200159389 rs58999456 Human genes 0.000 claims description 3
- 102200100725 rs61752871 Human genes 0.000 claims description 3
- 102220027906 rs63751107 Human genes 0.000 claims description 3
- 102220010994 rs727503110 Human genes 0.000 claims description 3
- 102220054485 rs727505160 Human genes 0.000 claims description 3
- 102200031651 rs730880031 Human genes 0.000 claims description 3
- 102220064848 rs747251132 Human genes 0.000 claims description 3
- 102200081272 rs768933093 Human genes 0.000 claims description 3
- 102220217782 rs772108759 Human genes 0.000 claims description 3
- 102220184357 rs777228104 Human genes 0.000 claims description 3
- 102220285547 rs777948908 Human genes 0.000 claims description 3
- 102220286163 rs781670952 Human genes 0.000 claims description 3
- 102200047293 rs794727236 Human genes 0.000 claims description 3
- 102220085887 rs864622014 Human genes 0.000 claims description 3
- 102220090215 rs875989892 Human genes 0.000 claims description 3
- 102220096725 rs876659333 Human genes 0.000 claims description 3
- 102220285772 rs876659692 Human genes 0.000 claims description 3
- 102220098091 rs876661320 Human genes 0.000 claims description 3
- 102220101146 rs878853645 Human genes 0.000 claims description 3
- 102220102818 rs878855160 Human genes 0.000 claims description 3
- 238000012165 high-throughput sequencing Methods 0.000 claims description 2
- 102220339301 rs193922247 Human genes 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 38
- 201000008827 tuberculosis Diseases 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 4
- 239000000284 extract Substances 0.000 abstract description 3
- 238000011835 investigation Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 48
- 238000003752 polymerase chain reaction Methods 0.000 description 31
- 238000006243 chemical reaction Methods 0.000 description 18
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 17
- 229960003376 levofloxacin Drugs 0.000 description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 230000035772 mutation Effects 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- MNHVNIJQQRJYDH-UHFFFAOYSA-N 2-[2-(1-chlorocyclopropyl)-3-(2-chlorophenyl)-2-hydroxypropyl]-1,2-dihydro-1,2,4-triazole-3-thione Chemical compound N1=CNC(=S)N1CC(C1(Cl)CC1)(O)CC1=CC=CC=C1Cl MNHVNIJQQRJYDH-UHFFFAOYSA-N 0.000 description 8
- 241000193830 Bacillus <bacterium> Species 0.000 description 8
- 239000005825 Prothioconazole Substances 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000012795 verification Methods 0.000 description 8
- 239000011324 bead Substances 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 238000011156 evaluation Methods 0.000 description 7
- SMWDFEZZVXVKRB-UHFFFAOYSA-N Quinoline Chemical compound N1=CC=CC2=CC=CC=C21 SMWDFEZZVXVKRB-UHFFFAOYSA-N 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- NWXMGUDVXFXRIG-WESIUVDSSA-N (4s,4as,5as,6s,12ar)-4-(dimethylamino)-1,6,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4,4a,5,5a-tetrahydrotetracene-2-carboxamide Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(=O)C(C(N)=O)=C(O)[C@@]4(O)C(=O)C3=C(O)C2=C1O NWXMGUDVXFXRIG-WESIUVDSSA-N 0.000 description 5
- 206010036790 Productive cough Diseases 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 210000003802 sputum Anatomy 0.000 description 5
- 208000024794 sputum Diseases 0.000 description 5
- 238000007400 DNA extraction Methods 0.000 description 4
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 description 4
- 101100509674 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) katG3 gene Proteins 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 101150110946 gatC gene Proteins 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 201000009671 multidrug-resistant tuberculosis Diseases 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 101150085857 rpo2 gene Proteins 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 238000012070 whole genome sequencing analysis Methods 0.000 description 4
- LWEAHXKXKDCSIE-UHFFFAOYSA-M 2,3-di(propan-2-yl)naphthalene-1-sulfonate Chemical compound C1=CC=C2C(S([O-])(=O)=O)=C(C(C)C)C(C(C)C)=CC2=C1 LWEAHXKXKDCSIE-UHFFFAOYSA-M 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- ZLHZLMOSPGACSZ-NSHDSACASA-N (6s)-2-nitro-6-[[4-(trifluoromethoxy)phenyl]methoxy]-6,7-dihydro-5h-imidazo[2,1-b][1,3]oxazine Chemical compound O([C@H]1CN2C=C(N=C2OC1)[N+](=O)[O-])CC1=CC=C(OC(F)(F)F)C=C1 ZLHZLMOSPGACSZ-NSHDSACASA-N 0.000 description 2
- 101100490139 Mus musculus Acer3 gene Proteins 0.000 description 2
- 241001302239 Mycobacterium tuberculosis complex Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 2
- KGTSLTYUUFWZNW-PPJQWWMSSA-N [(7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-26-[(E)-(4-methylpiperazin-1-yl)iminomethyl]-6,23-dioxo-8,30-dioxa-24-azatetracyclo[23.3.1.14,7.05,28]triaconta-1(29),2,4,9,19,21,25,27-octaen-13-yl] acetate pyridine-4-carbohydrazide Chemical compound NNC(=O)c1ccncc1.CO[C@H]1\C=C\O[C@@]2(C)Oc3c(C2=O)c2c(O)c(\C=N\N4CCN(C)CC4)c(NC(=O)\C(C)=C/C=C/[C@H](C)[C@H](O)[C@@H](C)[C@@H](O)[C@@H](C)[C@H](OC(C)=O)[C@@H]1C)c(O)c2c(O)c3C KGTSLTYUUFWZNW-PPJQWWMSSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- MAEBCGDGGATMSC-OSHGGGOQSA-N cyclamine Chemical compound O([C@H]1CO[C@H]([C@@H]([C@H]1O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O[C@@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@H]1CC[C@]2(C)[C@H]3CCC45OCC6([C@@H](C[C@@]4(C)[C@]3(C)CCC2C1(C)C)O)CC[C@@](C[C@@H]65)(C)C=O)[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O MAEBCGDGGATMSC-OSHGGGOQSA-N 0.000 description 2
- 125000004122 cyclic group Chemical group 0.000 description 2
- 229960003496 delamanid Drugs 0.000 description 2
- XDAOLTSRNUSPPH-XMMPIXPASA-N delamanid Chemical compound C([C@]1(C)OC2=NC(=CN2C1)[N+]([O-])=O)OC(C=C1)=CC=C1N(CC1)CCC1OC1=CC=C(OC(F)(F)F)C=C1 XDAOLTSRNUSPPH-XMMPIXPASA-N 0.000 description 2
- 229940072185 drug for treatment of tuberculosis Drugs 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000010801 machine learning Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229950008905 pretomanid Drugs 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 102200062436 rs118203347 Human genes 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- VCOPTHOUUNAYKQ-DAOVIKHBSA-N 3,6-diamino-n-[[(2s,5s,8z,11s,15s)-15-amino-11-(2-amino-1,4,5,6-tetrahydropyrimidin-6-yl)-8-[(carbamoylamino)methylidene]-2-(hydroxymethyl)-3,6,9,12,16-pentaoxo-1,4,7,10,13-pentazacyclohexadec-5-yl]methyl]hexanamide;3,6-diamino-n-[[(2s,5s,8z,11s,15s)-15-a Chemical compound N1C(=O)\C(=C\NC(N)=O)NC(=O)[C@H](CNC(=O)CC(N)CCCN)NC(=O)[C@H](C)NC(=O)[C@@H](N)CNC(=O)[C@@H]1C1NC(=N)NCC1.N1C(=O)\C(=C\NC(N)=O)NC(=O)[C@H](CNC(=O)CC(N)CCCN)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CNC(=O)[C@@H]1C1NC(=N)NCC1 VCOPTHOUUNAYKQ-DAOVIKHBSA-N 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 102220539398 ATP-sensitive inward rectifier potassium channel 11_W91R_mutation Human genes 0.000 description 1
- 101100380241 Caenorhabditis elegans arx-2 gene Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 1
- 101100176848 Escherichia phage N15 gene 15 gene Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 208000037357 HIV infectious disease Diseases 0.000 description 1
- 206010065048 Latent tuberculosis Diseases 0.000 description 1
- 206010048723 Multiple-drug resistance Diseases 0.000 description 1
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 1
- 101000856404 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) Carboxylesterase Culp1 Proteins 0.000 description 1
- 101100022063 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) lysG gene Proteins 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 101100209986 Rattus norvegicus Slc18a1 gene Proteins 0.000 description 1
- 101150071661 SLC25A20 gene Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 101150092805 actc1 gene Proteins 0.000 description 1
- 208000036981 active tuberculosis Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 238000007846 asymmetric PCR Methods 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 101150102633 cact gene Proteins 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 208000015355 drug-resistant tuberculosis Diseases 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 101150079015 esxB gene Proteins 0.000 description 1
- 238000013210 evaluation model Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 208000033353 latent tuberculosis infection Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000002418 meninge Anatomy 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 101150012629 parE gene Proteins 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000007637 random forest analysis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 208000036347 rifampicin-resistant tuberculosis Diseases 0.000 description 1
- 229940116736 romycin Drugs 0.000 description 1
- 102220259626 rs1553875105 Human genes 0.000 description 1
- 102220335514 rs762212949 Human genes 0.000 description 1
- 102200151021 rs863225227 Human genes 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 101150083559 tlyA gene Proteins 0.000 description 1
- 229940043263 traditional drug Drugs 0.000 description 1
- 229960001005 tuberculin Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a primer composition for drug-resistant gene detection of mycobacterium tuberculosis and a kit thereof, belonging to the technical field of gene detection. The primer composition or the kit can be used for detecting 1200 sequence variation sites in 58 drug-resistant genes related to 20 drugs for clinically treating mycobacterium tuberculosis, can directly identify the mycobacterium tuberculosis for clinical extracts without culture, improves the detection number and the detection coverage of the sequence variation sites, reduces the omission factor of the sequence variation sites, has strong detection specificity and high sensitivity, can perform parallel detection on a large number of samples, and reduces the detection cost. The primer composition and the kit thereof can be widely applied to scientific research, research on drug curative effect, investigation on tuberculosis epidemic disease and guidance on tuberculosis clinical medication.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer composition for drug resistance gene detection of a mycobacterium tuberculosis drug and application thereof in guiding drug resistance detection of the mycobacterium tuberculosis drug.
Background
Tuberculosis is a chronic infectious disease caused by infection with mycobacterium tuberculosis. Mycobacterium tuberculosis can invade various organs of the whole body of a human body, 80 percent of the Mycobacterium tuberculosis occurs in the lung and is called as pulmonary tuberculosis, and other parts such as cervical lymph, meninges, peritoneum, intestines, skin, bones and the like can also be infected secondarily. Mycobacterium tuberculosis (Mycobacterium tuberculosis), commonly known as tubercle bacillus, is a pathogen causing tuberculosis, is an obligate aerobic bacterium, is acid-fast staining positive, has no flagella, has pili, has microcapsules but does not form spores, has no teichoic acid of gram-positive bacteria and no lipopolysaccharide of gram-negative bacteria on the bacterial wall, has higher lipid content of the cell wall, is easy to influence the absorption of nutrient substances, and therefore, grows slowly. Tuberculosis is one of ten leading causes of death worldwide, and is still an important disease threatening human health to date.
In recent years, along with high mobility of population, unstable economic living conditions, poor patient compliance, bad living habits such as HIV infection, smoking, diabetes and the like, the drug resistance of tuberculosis patients to drugs is gradually enhanced, the number of drug resistant people is continuously increased, and drug resistant tuberculosis is more difficult to treat in recent years. In 2019, nearly 50 million people worldwide suffer from rifampicin-resistant tuberculosis, of which 78% suffer from multi-drug resistant tuberculosis. Drug resistant patients often develop resistance to first-line drugs such as rifampicin or isoniazid, resulting in prolonged illness. The treatment of multi-drug resistant tuberculosis is a treatment scheme which requires the simultaneous use of first-line and second-line drugs. The low detection rate of multi-drug resistant tuberculosis drug resistance is due in part to the lack of rapid and accurate diagnosis. The selection of effective drugs and the acceleration of the diagnosis of drug resistance of drugs are the problems which are urgently needed to be solved at present. With the development of molecular biology technology, the drug resistance mechanism and the molecular basis of drug resistance of mycobacterium tuberculosis have been mostly elucidated. The current methods for detecting drug-resistant genes include: direct sequencing of DNA: the method is complicated to operate, high in cost and limited in clinical popularization, and other detection methods are evaluated by the method; 2. polymerase chain reaction-single strand conformation polymorphism analysis RCR-SSCP: the method is influenced by the length of detected DNA, gel concentration, buffer solution ionic strength and electrophoresis temperature, cannot give specific mutation positions and mutation properties, and can only be used for screening gene mutation; 3. polymerase chain reaction-restriction length polymorphism analysis PCR-RFLP: the method can analyze whether a point mutation exists in a known sequence, but the nature of the mutation is unknown; 4. probe hybridization method: the operation is complicated; 5. gene chip technology; 6. real-time fluorescent quantitative PCR method; 7. a multiplex PCR method; 8. metagenomic sequencing.
For example, chinese patent 201210184401.8 discloses a non-fluorescent DNA microarray detection method and kit for multi-drug resistant mycobacterium tuberculosis, which comprises PCR forward and reverse amplification primers and oligonucleotide probe sequences for main drug resistant gene nucleic acid fragments of mycobacterium tuberculosis rifampin and isoniazid, and can detect mutations at rpoB gene 511, 516, 526, 531 and 533 sites, katG gene 315 site, and inhA gene-15 site, and can rapidly detect the mutation of main drug resistant gene of mycobacterium tuberculosis rifampin and isoniazid.
Chinese patent 201410093113.0 discloses a method for extracting DNA of tubercle bacillus and a kit for detecting multiple drug resistance thereof, which comprises the steps of extracting DNA to be detected from clinical samples, determining infection of tubercle bacillus, drug resistance and infection of drug-resistant strains by using fluorescent real-time quantitative PCR, determining the infection quantity of pathogenic bacteria, detecting 29 drug-resistant gene loci of rifampicin, isoniazid and ethambutol, and having simple, convenient and quick application and accurate and reliable results.
Chinese patent 201610657425.9 discloses a kit for detecting drug-resistant genes of mycobacterium tuberculosis and related applications, wherein the kit establishes multiple real-time fluorescent PCR (polymerase chain reaction) technologies of multiple sites on mycobacterium tuberculosis drug-resistant genes rpoB (sites 531, 526, 516, 511), katG (site S315), inhA (site-15C), aphC (sites-10C-T, -9G-A), rpsL (sites 43, 88), embB (site 306), gyrA (sites 90, 94) and rrs (sites 1401, 1402, 1484), and realizes the purpose of short-time efficient detection of the drug-resistant genes of mycobacterium tuberculosis by using fluorescent probes doubly labeled by Minor Groove Binders (MGB) and Locked Nucleic Acids (LNA).
Chinese patent 201880058656.5 discloses a kit for tuberculosis diagnosis and a method for diagnosing tuberculosis by using the kit, comprising (a) primers capable of simultaneously and specifically amplifying a mycobacterium tuberculosis-specific gene and a gene ubiquitous in mycobacteria; and (b) a probe capable of identifying the amplified product; and to a method for simultaneously diagnosing Mycobacterium tuberculosis and nontuberculous mycobacteria using the kit.
Chinese patent 201811043058.9 discloses a tuberculosis infection diagnostic kit, a screening system and the application of the kit, wherein the kit comprises mitogen and antigen stimulant; at least three of the antigen stimulator tuberculin pure protein or its derivatives ESAT6, CFP10, TB7.7, MPT6, CFP21, EsxV, Rv1985c and Rv 0222; a novel skin test for screening latent tuberculosis infection and active tuberculosis is established, and the skin test has the advantages of strong specificity, high sensitivity, small influence of individual basic immunity difference and the like.
Chinese patent 201910656477.8 discloses a mutant gene for drug-resistant detection of Mycobacterium tuberculosis, a detection method and a kit thereof, and discloses mutant sites of drug-resistant genes of Mycobacterium tuberculosis (katG genes: Q88P, M257V, W91R, A122D, Q127P, A312E, S315T/S315N, D419Y; rpoB genes: 1291gcc _ in, L430P, S450L/S450F/S450W, L452P, H445D/H445L/H36445, D435 445Y/D435Y/D Y; gidB genes: 102g _ del, 386g _ in, 351g _ del, pncA genes: T-11c, 134a _ del, 281a _ in, 399a _ in, 528g _ del, 85g _ 3628, 119D _ del, Y D72, Y D Y, Y/Y D Y, Y D Y/Y, K, Y, K, Y, K, Y, Rifampicin, pyrazinamide, levofloxacin, streptomycin, kanamycin resistance, and a high-throughput drug resistance test method and kit for mycobacterium tuberculosis are used for drug resistance test of mycobacterium tuberculosis, and contribute to shortening the waiting time of patients and giving patients an accurate treatment scheme.
Chinese patent 201911020331.0 discloses a kit and a method for detecting mycobacterium tuberculosis isoniazid drug-resistant mutant genes, wherein the kit establishes a multiple PCR amplification system of three target genes katG, inhA and aphC of mycobacterium tuberculosis isoniazid drug-resistant mutation, can distinguish wild types from different mutant types by a high-resolution melting curve method, can realize multiple asymmetric PCR reaction by combining a molecular beacon probe, can quickly and accurately detect the mutant sites of the tuberculosis resistant strains, and can perform multi-site detection at one time.
Chinese patent 201911415507.2 discloses a tubercle bacillus drug resistance detection kit and a tubercle bacillus drug resistance detection method, which comprises sequencing primers (one or more of rpoB, katG, inhA-promoter, inhA-structure, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes) of tubercle bacillus drug resistance genes; tubercle bacillus nucleic acid detection reagents (primer pair 1 for IS6110, primer pair 2 for IS6110 and probe primer for IS 6110); and (5) drug resistance detection. The kit has good sensitivity, specificity and accuracy, can simultaneously carry out mutation detection on 48 sites of 17 drug-resistant genes of common antituberculosis drugs and deletion detection of a gene spacer segment, and can more accurately and comprehensively guide tuberculosis medication.
Chinese patent 201911422142.6 discloses a sample treatment reagent for detecting mycobacterium tuberculosis nucleic acid, a kit and a nucleic acid amplification method for mycobacterium tuberculosis, which can extract mycobacterium tuberculosis DNA from a sputum sample more quickly and effectively, effectively remove interference of human DNA, completely inactivate mycobacterium tuberculosis, reduce the risk of personnel infection, and are safer and more efficient.
Chinese patent 202010104800.3 discloses a Mycobacterium tuberculosis complex rapid detection kit and a preparation method thereof, which comprises an identification reagent (comprising ten pairs of primers), a preparation method of the rapid detection kit (comprising primers and probes, a kit program, a specificity and sensitivity experiment, a repetition experiment, a target gene pool and a specificity detection kit); and detecting and identifying the tubercle bacillus.
Although the above patents have performed sputum sample treatment, mycobacterium tuberculosis identification and drug-resistant gene locus detection of partial drugs, none of them has reached the level of replacing the traditional drug sensitivity test.
Therefore, it is highly desirable to provide a primer composition or a kit for drug-resistant gene detection of mycobacterium tuberculosis with high detection accuracy, sensitivity and specificity.
Disclosure of Invention
Aiming at the defects, in order to avoid the culture of the mycobacterium tuberculosis, improve the detection accuracy of drug-resistant gene loci, increase the drug-resistant drug species of the mycobacterium tuberculosis and improve the sensitivity and specificity of drug-resistant detection, the invention provides a primer composition or a kit for quickly detecting the drug-resistant gene of the mycobacterium tuberculosis, and the drug-resistant gene of the mycobacterium tuberculosis is detected by using a multiple PCR targeted capture technology and a high-throughput second-generation sequencing technology. The method comprises the steps of simultaneously amplifying a plurality of target regions on genome DNA by utilizing a multiplex PCR technology to obtain amplicons, then adding second-generation sequencing joints to two sides of the amplicons in a PCR mode to obtain an amplicon library, carrying out second-generation sequencing to obtain sequence information of the target regions, and carrying out drug resistance identification. On the premise of ensuring amplification uniformity, after rapid targeted linear amplification is carried out on hundreds of SNP and InDel sites, a mainstream sequencing platform (illumina) is used for carrying out parallel detection on a large number of samples and deep analysis on data based on a bioinformatics method. Based on the design of multi-parameter primers, the capture efficiency is greatly improved, the omission factor is reduced, bacterial culture is not needed, the selected drugs are more comprehensive, and the range of clinically selectable drugs is enlarged. The primer composition can be used for carrying out drug resistance detection on first-line and second-line common drugs for clinically treating mycobacterium tuberculosis, and specifically comprises the following components: rifampin (rifampicin, RIF), Isoniazid (INH), Ethambutol (EMB), streptomycin (streptomycin, Sm), Moxifloxacin (MFX), Levofloxacin (LFX), Amikacin (AK), kanamycin (Km), capreomycin (Cm), pyrazinamide (pyrazinamide, PZA), rifabutin (rifabutin, RFB), ethionamide (ethionamide, Eto), clofazimine (clofazimin, Cfz), aminosalicylic acid (paranasal acid, PAS), Prothioconazole (PTO), cycloserine (cyclamine, cyclamenine), mannich (quinoline, mannich), mannich (23), mannich (58, 25, and so as to obtain a low cost assay.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides a primer composition for drug-resistant gene detection of mycobacterium tuberculosis, wherein the primer composition is shown in table 1 below.
TABLE 1 primer compositions
Specifically, the mycobacterium tuberculosis-related drugs include rifampicin (rifampicin, RIF), Isoniazid (INH), ethambutol (ethambutol, EMB), streptomycin (streptomycin, Sm), moxifloxacin (moxifloxacin, MFX), Levofloxacin (Levofloxacin, LFX), amikacin (amikacin, AK), kanamycin (kanamycin, Km), capreomycin (c.romycin, Cm), pyrazinamide (pyrazinamide, p), rifabutin (rifabutin, RFB), ethionamide (ethionamide, Eto), clofazimine (clozimin, Cfz), para-aminosalicylic acid (parasalicylic acid, swinium), Prothioconazole (PAS), cyclamine (cyclomycin ), mannide (bendazamide, mannide), mannich (quinoline (Bdq), and mannich (lazide).
Specifically, the primer composition is used for detecting the following mycobacterium tuberculosis drug-resistant gene detection sites:
(1) rifampicin comprises 105 polymorphic loci of 4 drug-resistant genes, which are respectively:
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, D435A, N36437, N A, S441A, L A, T36444, T444, H A, H13272, S441A, S3678, S A, L36445, S A, L A, S A, L36445, L A, S A, L A, S A, S A, 3678, A, 36445H A, S A, S3678, A, L A, S A, L36445H A, L A, L A, tc A, L A;
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) rpoZ gene 1 site: I55X;
(2) isoniazid, including 207 polymorphic sites of 18 drug-resistant genes, which are respectively:
1) 104 sites of the katG gene: V1A, C20S, Q88E, W90R, W90X, R104L, R104Q, A106V, H108Q, H108G, A109V, A110V, G125D, Q127D, N138D, N138D, A139D, A139D, S140D, L141D, D142D, L148D, L148D, W149D, Y155D, L159D, S175D, T180D, G182D, W191D, W191D, P232D, M257D, D259D, H270D, K274D, T D, G297D, G300, W300, S302, S315, H270D, H270D, K274D, S300, S3673, S315, S D, L D, S315, S300, S D, S315, L D, S D, L300, S D, S315, S D, L D, S315, S D, L300, S D, S315, S D, S315, L D, S315, S D, L D, S315, S D, S315, L D, S D, L D, S3673D D, S315, S D, S3673D D, S D, L D, S3673D D, S D, L D, S3673D D, S D, L D, S1-S3673, S3673D 3673, S3673D D, S3673D 3673, S315, L3673, S1-S D, S3673, S D, S3673, S1-S D, S315, S D, L D, S1-S315, S D, S315, S1-S D, L D, S D, 1668A/-, 1682A/-, 1899-/C, 1955-/G, 2002-/T, 2101-/AACT;
2) 4 sites of furA gene: S5P, -7G/A, -10A/C, -12G/A;
3) 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;
4) ahpC gene 25 sites: -6G/A, -9G/A, -15C/T, -30C/T, -34T/G, -39C/T, -46-/T, -46A/T, -46A/G, -48G/A, -49T/G, -49T/C, -51G/A, -52C/A, -52C/G, -52C/T, -54C/T, -57C/T, -72C/T, -74G/A, -76T/C, -81C/T, -82-/AT, -86T/A, -88G/A;
5) nat gene 3 sites: T175A, D188H, G207R;
6) ndh gene 8 sites: R13C, L104F, L104P, T110A, G225D, R268H, E360L, E360K;
7) the iniA gene has 4 sites: P3A, H481G, H481Q, R537H;
8) the iniB gene has 11 sites: 180C/-, 198G/-, 222T/-, 686G/-, 725-/T, 750C/-, 878T/-, 1019-/C, 1038G/-, 1056A/-, A222T;
9) the iniC gene has 3 sites: 79-/T, 98-/A, W83G;
10) kasA gene 6 sites: D66N, R121K, G269S, G312S, G387D, F413L;
11) 13 sites of fabG1 gene: 609G/A (L203L), -8T/A, -8T/C, -8T/G, -15C/A, -15C/G, -15C/T, -16A/C, -16A/G, -16A/T, -17G/A, -17G/C, -17G/T;
12) rv0340 gene 1 site: V163I;
13) fadE24 gene 1 site: Q85R;
14) rv1592c gene 1 site: P42L;
15) rv1772 gene 1 site: T4A;
16) fabD gene 1 site: A3T;
17) phoR gene 1 site: P186L;
18) 2 sites of the efpA gene: I73T, E520V;
(3) ethambutol, comprising 75 polymorphic sites of 6 drug-resistant genes, which are respectively:
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, M423N, Q445N, Q497N, E504N, Q853N, a 630N, M1000N, H1002N, D361024, D1024N, N1033N;
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, a 278V;
(4) streptomycin, which comprises 77 polymorphic sites of 3 drug-resistant genes, and is respectively:
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 the gidB 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 '-, G202/-202, 202-/351, G' -, G/-347C '-, C/-347, G/-TCG/-20421, G/-TCG/-347, G/-TCG/-S, G/-347, G/-TCG/-20421, G/-347, C' -, C/-347, G/-347, C/-TCG/-347, C/-102, C/-TCG, C/-347, C/-102, C/-TCG/-347, C/-TCG, C/-237, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, C/-347, C/-2, C/-347, C/-102, C/-2, C/-347, C/-102, C/-347, C/-102, C/-2, C/-102;
(5) the moxifloxacin comprises 13 polymorphic sites of 2 drug resistance genes, and the polymorphic sites are respectively as follows:
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;
(6) the ofloxacin comprises 39 polymorphic sites of 2 drug-resistant genes, which are respectively:
1) 16 sites of the gyrA gene: a74S, T80A, N83V, G88C, G88A, D89N, a90V, a90G, S91P, D94A, D94G, D94H, D94N, D94Y, D94V, G668D;
2) the gyrB gene has 23 sites: M291I, R446C, S447Y, D461A, D461H, D461N, G470A, I485C, I486L, D494A, N499D, T500N, E501V, a504T, T511N, Q538D, Q538H, Q538T, Q538K, H539N, I540D, I540V, E592S;
(7) amikacin, which comprises 20 polymorphic sites of 3 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
3) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(8) kanamycin comprises 30 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) 10 loci of the eis gene: -6G/T, -8C/T, -8C/A, -10G/A, -10G/C, -12C/T, -14C/T, -15C/G, -37G/T, -43A/T;
3) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
4) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(9) the capreomycin comprises 32 polymorphic sites of 3 drug-resistant genes, which are respectively:
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;
(10) pyrazinamide, including 190 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) the panD gene has 14 sites: H21R, I49V, I115T, M117T, 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, 410TGT/-, in a preferred embodiment, the composition is formulated as a pharmaceutical composition, for use in a medicament for treating a disease or condition, for example, a disease or a condition, for example, or a disease or a in a or a condition, for the like, or a condition, or a, or a condition, or a, or, 414-/CATT, 418-/, 419-/G, 422A/-, 423-/AG, 455-/C, 457-/, 458-/C, 471-/A, 480A/-, 494-/C, 523-/A, 525-/A, 525CCGCCA/-, 570-/CT, A3, L4, L4, I5, I6, V7, V7, D8, V9, Q10, D12, C14, S18, G23, I31, Y34, Y41, K48, K48, D49, H51, H51, H51, H51, P54, P54, H57, H57, H57, S59, S59, P62, P62, P62, D63, D63, D63, Y64, S66, S67, W68, W68, W68, W68, W69, P62, P96, G97, G94, G85, G97, G96, G85, G97, G85, G95, and G95, g97, Y99, a102, Y103, S104, G108, L116, W119, L120, R123, D129, G132, I133, a134, T135, D136, H137, C138, V139, R140, Q141, T142, a143, a146, L151, T167, T168, V169, L172, M175, V180, L182, X187;
3) rpsA gene 4 sites: T5S, E67D, D123A, 1313 CCG/-;
(11) the rifabutin comprises 15 polymorphic loci of 1 drug-resistant gene, which are respectively:
1) rpoB gene 15 sites: G95F, P206R, Y314C, H323Y, Q429H, Q432A, Q432E, Q432K, Q432P, H445D, H445R, H445Y, S450F, S450L, S450W;
(12) the ethionamide comprises 66 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 56 sites of the ethA gene: -11A/G, -7T/C, M1, V10, G11, H22, Q24, 30G/-, Y32, G43, W45, S57, T61, T88, 107A/-, 110A/-, 157-/T, Q165, M204, L205, Q206, R207, Y211, Q246, W256, S266, P334, 338A/-, 341A/-, a341, D357, S375, P378, 382-/G, S390, V398, L405, Y438, 441-/T, G450, P454, W455, L478, 672-/G, 703/-, 753-/G, 869-/a, 892A/-, G/-, 980T '-, 1175-/CG, 1190-/TGG, 1239T/-, 1242T' -, 1393/-;
2) 1 site of ethR gene: A95T;
3) 3 sites of fabG1 gene: -15C/T, -8T/a, 609G/a;
4) inhA gene 6 sites: -17G/T, -15C/T, -8T/a, I21T, S94A, I194T;
(13) the clofazimine comprises 81 polymorphic sites of 3 drug-resistant genes, and is respectively as follows:
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, V3, N4, D5, E13, V20, 29-/T, T33, A36, G41, W42, L43, C46, Q51, S53, 58G/T, A59, S63, S63, G65, G66, S68, S68, Q76, A84, V85, G87, R89, R90, R90, A102, A102, R105, A110, L114, L117, A118, D119, V120, G121, L122, R123, 125G/A, G126, G126, D127, A128, P130, R134, R135, M139, 141-/GA, D141, M146, N148, V149, L154, R156, G162, D165, 193G/, 193-/G, 198G-/G-202, A-292, G-202, G-C-364, CG/, CG-444;
3) rv1979c gene 1 site: V351A;
(14) the p-aminosalicylic acid comprises 71 polymorphic sites of 4 drug-resistant genes, wherein the polymorphic sites are respectively as follows:
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, a 457X;
3) 1 site of 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, I211R, R222R, P224R, R235R, Y251R, a 259R, V261R, V263R, X263R;
(15) the albuterol hydrochloride comprises 62 polymorphic sites of 2 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 58 sites of the ethA gene: 11-/A, 21-/C, Q24, 30G/-, 65A/-, 107A/-, W109, 129-/C, 136GATT/-, 138T/-, Y147, 157-/T, Q165, S197, 202-/CC, Q206, 229G/-, 243-/A, Q254, Y276, 298T/-, K309, 312-/G, 338A/-, 382-/G, W391, L405, 412-/A, Y438, 440-/AC, 596C/, 672-/G, 703T '-, 752-/G, 752-/GC, 755-/C, 767G/-, GG 7/-, 773G/, 822G, 767G' -, 752-C, 767G/-, and G, 18, 822-/G, 849-/C, 859A/-, 869-/A, 892A/-, 895GG/-, 908G/-, 925A/-, 980T/-, 1121T/-, 1135A/-, 1175-/CG, 1190-/TGG, 1201T/-, 1239T/-, 1304-/C, 1386A/-, 1404-/C;
2) inhA gene 4 sites: -15A/T, -15C/T, S49A, I194T;
(16) the cycloserine comprises 6 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) 3 sites of alr gene: -26G/T, L113R, D344N;
2) 2 sites of the cycA gene: T236A, V301A;
3) ddlA gene 1 site: L372R;
(17) the bedaquiline comprises 62 polymorphic sites of 5 drug-resistant genes, which are respectively:
1) atpE gene 13 sites: -72T/C, -53G/a, D28A, 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-/G193, 193-, -200T/G, 202A/G, 214C/T, 259-/G-, 345-G, 292-G/;
5) rv1979c gene 1 site: M245L;
(18) linezolid, comprising 10 polymorphic sites of 2 drug-resistant genes, which are respectively:
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;
(19) the delamannich comprises 20 polymorphic sites of 5 drug-resistant genes, which are respectively:
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) fgd1 gene 3 sites: G104S, L270M, L296E;
(20) the PrinMannich total comprises 19 polymorphic sites of 4 drug-resistant genes, which are respectively:
1) ddn gene 7 sites: S11X, W20X, Q58X, W88R, Y136X, Q137X, W139X;
2) fbiA gene 1 site: T146A;
3) fbiC gene 9 sites: G310X, W435X, Q436X, W444X, E637X, K644X, W652X, M709I, S715R;
4) fgd1 gene 2 sites: G71D, E230K.
On the other hand, the invention provides the application of the primer composition in preparing a kit for detecting drug resistance of the mycobacterium tuberculosis and/or a kit for guiding the drug administration of the mycobacterium tuberculosis.
In another aspect, the invention provides a kit for detecting drug resistance of mycobacterium tuberculosis and/or a kit for guiding drug administration of mycobacterium tuberculosis, wherein the kit comprises the primer composition.
Specifically, the concentration of each primer in the primer composition is 0.2-0.8. mu.M, preferably 0.5. mu.M.
Specifically, the kit further comprises Polymerase mix (high fidelity PCR Polymerase, dNTP and reaction buffer premix), PCR enhanced buffer (saline solution, organic small molecule, enhanced high GC region amplification), illumina/BGISEQ 5 'end Index (10 μ M) (sequencing linker), and illumina/BGISEQ3' end Index (10 μ M) (sequencing linker).
In another aspect, the invention provides an application of the primer composition or the kit in detection of drug-resistant genes of mycobacterium tuberculosis.
In another aspect, the present invention provides a method for detecting drug-resistant genes of mycobacterium tuberculosis, wherein the method is a non-disease diagnosis and treatment method, and the method comprises detecting the sites of the drug-resistant genes in the genome of a sample to be detected by using the primer composition or the kit.
Specifically, the method comprises the following steps:
(1) extracting sample DNA;
(2) performing multiple PCR amplification on the DNA extracted in the step (1) by using the primer composition or the kit, and constructing a second-generation sequencing library;
(3) and performing high-throughput sequencing on the amplicon through a second-generation sequencing platform, determining the specific genotype of the drug-resistant gene locus of the sample, and detecting or identifying the drug resistance of the drug according to the genotype.
Compared with the prior art, the invention has the advantages that:
1. the primer composition or the kit is adopted to detect the drug resistance gene of the mycobacterium tuberculosis, a sample does not need to be subjected to isolated culture, the interference of a host and other microorganisms is shielded, more drug types and drug resistance gene sites of the mycobacterium tuberculosis can be detected at one time, the capture efficiency is greatly improved, the omission factor is reduced, the sequence coverage of the drug resistance gene sites is high, the detection accuracy, the sensitivity and the specificity are better, and meanwhile, a high-throughput multi-sample detection system is adopted, so that the primer composition or the kit can be applied to the drug resistance detection of a large sample amount, and the detection cost is reduced.
2. The invention can be widely applied to scientific research, drug efficacy research and clinical medication guidance of drug resistance of mycobacterium tuberculosis.
3. The kit provided by the application has a short sample detection period, and is closer to the real situation of the sample than separation culture. Compared with clinical gold standard phenotype drug sensitive experiments, the primer composition has short detection period, can process data in 2 days from sample acquisition, and can judge the proportion of mixed bacteria; compared with molecular diagnosis (a GeneXpert probe based on a hybridization signal), the primer composition can obtain real data, and in addition, the method based on sequencing has extremely high flux (the maximum of 96 samples can be obtained by one-time construction), the single machine of the GeneXpert is expensive, the maximum of 20 samples can be processed by one machine, and the clinical application only aims at the specific region resistance detection of rifampicin.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 primer composition for drug-resistant gene detection of mycobacterium tuberculosis and kit thereof
1. The primer compositions are shown in table 2 below.
TABLE 2 primer compositions
The mycobacterium tuberculosis-related drugs include rifampicin (rifampicin, RIF), Isoniazid (INH), ethambutol (ethambutol, EMB), streptomycin (streptomycin, Sm), moxifloxacin (moxifloxacin, MFX), Levofloxacin (Levofloxacin, LFX), amikacin (amikacin, AK), kanamycin (kanamycin, Km), capreomycin (capromycin, Cm), pyrazinamide (pyrazinamide, PZA), rifabutin (rifabutin, RFB), ethionamide (ethionamide, Eto), clofazimine (clozimin, Cfz), para-aminosalicylic acid (paraamid, linamide), prothioconazole (PAS, propathromycin), cyclomycin (cyclo-quinoline (cyclomycin, cyclomycin), mannich (cyclomycin, dinoline), mannich (Bdq), mannich (quinoline, nekal), and nekal (nekal, mezzanine, lfoxazidine, mez).
Specifically, the primer composition is used for detecting the following mycobacterium tuberculosis drug-resistant gene detection sites:
(1) rifampicin comprises 105 polymorphic loci of 4 drug-resistant genes, which are respectively:
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, D435A, N36437, N A, S441A, L A, T36444, T444, H A, H13272, S441A, S3678, S A, L36445, S A, L A, S A, L36445, L A, S A, L A, S A, S A, 3678, A, 36445H A, S A, S3678, A, L A, S A, L36445H A, L A, L A, tc A, L A;
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) rpoZ gene 1 site: I55X;
(2) isoniazid, including 207 polymorphic sites of 18 drug-resistant genes, which are respectively:
1) 104 sites of the katG gene: V1A, C20S, Q88E, W90R, W90X, R104L, R104Q, A106V, H108Q, H108G, A109V, A110V, G125D, Q127D, N138D, N138D, A139D, A139D, S140D, L141D, D142D, L148D, L148D, W149D, Y155D, L159D, S175D, T180D, G182D, W191D, W191D, P232D, M257D, D259D, H270D, K274D, T D, G297D, G300, W300, S302, S315, H270D, H270D, K274D, S300, S3673, S315, S D, L D, S315, S300, S D, S315, L D, S D, L300, S D, S315, S D, L D, S315, S D, L300, S D, S315, S D, S315, L D, S315, S D, L D, S315, S D, S315, L D, S D, L D, S3673D D, S315, S D, S3673D D, S D, L D, S3673D D, S D, L D, S3673D D, S D, L D, S1-S3673, S3673D 3673, S3673D D, S3673D 3673, S315, L3673, S1-S D, S3673, S D, S3673, S1-S D, S315, S D, L D, S1-S315, S D, S315, S1-S D, L D, S D, 1668A/-, 1682A/-, 1899-/C, 1955-/G, 2002-/T, 2101-/AACT;
2) 4 sites of furA gene: S5P, -7G/A, -10A/C, -12G/A;
3) 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;
4) ahpC gene 25 sites: -6G/A, -9G/A, -15C/T, -30C/T, -34T/G, -39C/T, -46-/T, -46A/T, -46A/G, -48G/A, -49T/G, -49T/C, -51G/A, -52C/A, -52C/G, -52C/T, -54C/T, -57C/T, -72C/T, -74G/A, -76T/C, -81C/T, -82-/AT, -86T/A, -88G/A;
5) nat gene 3 sites: T175A, D188H, G207R;
6) ndh gene 8 sites: R13C, L104F, L104P, T110A, G225D, R268H, E360L, E360K;
7) the iniA gene has 4 sites: P3A, H481G, H481Q, R537H;
8) the iniB gene has 11 sites: 180C/-, 198G/-, 222T/-, 686G/-, 725-/T, 750C/-, 878T/-, 1019-/C, 1038G/-, 1056A/-, A222T;
9) the iniC gene has 3 sites: 79-/T, 98-/A, W83G;
10) kasA gene 6 sites: D66N, R121K, G269S, G312S, G387D, F413L;
11) 13 sites of fabG1 gene: 609G/A (L203L), -8T/A, -8T/C, -8T/G, -15C/A, -15C/G, -15C/T, -16A/C, -16A/G, -16A/T, -17G/A, -17G/C, -17G/T;
12) rv0340 gene 1 site: V163I;
13) fadE24 gene 1 site: Q85R;
14) rv1592c gene 1 site: P42L;
15) rv1772 gene 1 site: T4A;
16) fabD gene 1 site: A3T;
17) phoR gene 1 site: P186L;
18) 2 sites of the efpA gene: I73T, E520V;
(3) ethambutol, comprising 75 polymorphic sites of 6 drug-resistant genes, which are respectively:
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, M423N, Q445N, Q497N, E504N, Q853N, a 630N, M1000N, H1002N, D361024, D1024N, N1033N;
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, a 278V;
(4) streptomycin, which comprises 77 polymorphic sites of 3 drug-resistant genes, and is respectively:
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 the gidB 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 '-, G202/-202, 202-/351, G' -, G/-347C '-, C/-347, G/-TCG/-20421, G/-TCG/-347, G/-TCG/-S, G/-347, G/-TCG/-20421, G/-347, C' -, C/-347, G/-347, C/-TCG/-347, C/-102, C/-TCG, C/-347, C/-102, C/-TCG/-347, C/-TCG, C/-237, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, C/-347, C/-2, C/-347, C/-102, C/-2, C/-347, C/-102, C/-347, C/-102, C/-2, C/-102;
(5) the moxifloxacin comprises 13 polymorphic sites of 2 drug resistance genes, and the polymorphic sites are respectively as follows:
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;
(6) the ofloxacin comprises 39 polymorphic sites of 2 drug-resistant genes, which are respectively:
1) 16 sites of the gyrA gene: a74S, T80A, N83V, G88C, G88A, D89N, a90V, a90G, S91P, D94A, D94G, D94H, D94N, D94Y, D94V, G668D;
2) the gyrB gene has 23 sites: M291I, R446C, S447Y, D461A, D461H, D461N, G470A, I485C, I486L, D494A, N499D, T500N, E501V, a504T, T511N, Q538D, Q538H, Q538T, Q538K, H539N, I540D, I540V, E592S;
(7) amikacin, which comprises 20 polymorphic sites of 3 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
3) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(8) kanamycin comprises 30 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) 10 loci of the eis gene: -6G/T, -8C/T, -8C/A, -10G/A, -10G/C, -12C/T, -14C/T, -15C/G, -37G/T, -43A/T;
3) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
4) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(9) the capreomycin comprises 32 polymorphic sites of 3 drug-resistant genes, which are respectively:
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;
(10) pyrazinamide, including 190 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) the panD gene has 14 sites: H21R, I49V, I115T, M117T, 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, 410TGT/-, in a preferred embodiment, the composition is formulated as a pharmaceutical composition, for use in a medicament for treating a disease or condition, for example, a disease or a condition, for example, or a disease or a in a or a condition, for the like, or a condition, or a, or a condition, or a, or, 414-/CATT, 418-/, 419-/G, 422A/-, 423-/AG, 455-/C, 457-/, 458-/C, 471-/A, 480A/-, 494-/C, 523-/A, 525-/A, 525CCGCCA/-, 570-/CT, A3, L4, L4, I5, I6, V7, V7, D8, V9, Q10, D12, C14, S18, G23, I31, Y34, Y41, K48, K48, D49, H51, H51, H51, H51, P54, P54, H57, H57, H57, S59, S59, P62, P62, P62, D63, D63, D63, Y64, S66, S67, W68, W68, W68, W68, W69, P62, P96, G97, G94, G85, G97, G96, G85, G97, G85, G95, and G95, g97, Y99, a102, Y103, S104, G108, L116, W119, L120, R123, D129, G132, I133, a134, T135, D136, H137, C138, V139, R140, Q141, T142, a143, a146, L151, T167, T168, V169, L172, M175, V180, L182, X187;
3) rpsA gene 4 sites: T5S, E67D, D123A, 1313 CCG/-;
(11) the rifabutin comprises 15 polymorphic loci of 1 drug-resistant gene, which are respectively:
1) rpoB gene 15 sites: G95F, P206R, Y314C, H323Y, Q429H, Q432A, Q432E, Q432K, Q432P, H445D, H445R, H445Y, S450F, S450L, S450W;
(12) the ethionamide comprises 66 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 56 sites of the ethA gene: -11A/G, -7T/C, M1, V10, G11, H22, Q24, 30G/-, Y32, G43, W45, S57, T61, T88, 107A/-, 110A/-, 157-/T, Q165, M204, L205, Q206, R207, Y211, Q246, W256, S266, P334, 338A/-, 341A/-, a341, D357, S375, P378, 382-/G, S390, V398, L405, Y438, 441-/T, G450, P454, W455, L478, 672-/G, 703/-, 753-/G, 869-/a, 892A/-, G/-, 980T '-, 1175-/CG, 1190-/TGG, 1239T/-, 1242T' -, 1393/-;
2) 1 site of ethR gene: A95T;
3) 3 sites of fabG1 gene: -15C/T, -8T/a, 609G/a;
4) inhA gene 6 sites: -17G/T, -15C/T, -8T/a, I21T, S94A, I194T;
(13) the clofazimine comprises 81 polymorphic sites of 3 drug-resistant genes, and is respectively as follows:
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, V3, N4, D5, E13, V20, 29-/T, T33, A36, G41, W42, L43, C46, Q51, S53, 58G/T, A59, S63, S63, G65, G66, S68, S68, Q76, A84, V85, G87, R89, R90, R90, A102, A102, R105, A110, L114, L117, A118, D119, V120, G121, L122, R123, 125G/A, G126, G126, D127, A128, P130, R134, R135, M139, 141-/GA, D141, M146, N148, V149, L154, R156, G162, D165, 193G/, 193-/G, 198G-/G-202, A-292, G-202, G-C-364, CG/, CG-444;
3) rv1979c gene 1 site: V351A;
(14) the p-aminosalicylic acid comprises 71 polymorphic sites of 4 drug-resistant genes, wherein the polymorphic sites are respectively as follows:
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, a 457X;
3) 1 site of 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, I211R, R222R, P224R, R235R, Y251R, a 259R, V261R, V263R, X263R;
(15) the albuterol hydrochloride comprises 62 polymorphic sites of 2 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 58 sites of the ethA gene: 11-/A, 21-/C, Q24, 30G/-, 65A/-, 107A/-, W109, 129-/C, 136GATT/-, 138T/-, Y147, 157-/T, Q165, S197, 202-/CC, Q206, 229G/-, 243-/A, Q254, Y276, 298T/-, K309, 312-/G, 338A/-, 382-/G, W391, L405, 412-/A, Y438, 440-/AC, 596C/, 672-/G, 703T '-, 752-/G, 752-/GC, 755-/C, 767G/-, GG 7/-, 773G/, 822G, 767G' -, 752-C, 767G/-, and G, 18, 822-/G, 849-/C, 859A/-, 869-/A, 892A/-, 895GG/-, 908G/-, 925A/-, 980T/-, 1121T/-, 1135A/-, 1175-/CG, 1190-/TGG, 1201T/-, 1239T/-, 1304-/C, 1386A/-, 1404-/C;
2) inhA gene 4 sites: -15A/T, -15C/T, S49A, I194T;
(16) the cycloserine comprises 6 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) 3 sites of alr gene: -26G/T, L113R, D344N;
2) 2 sites of the cycA gene: T236A, V301A;
3) ddlA gene 1 site: L372R;
(17) the bedaquiline comprises 62 polymorphic sites of 5 drug-resistant genes, which are respectively:
1) atpE gene 13 sites: -72T/C, -53G/a, D28A, 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-/G193, 193-, -200T/G, 202A/G, 214C/T, 259-/G-, 345-G, 292-G/;
5) rv1979c gene 1 site: M245L;
(18) linezolid, comprising 10 polymorphic sites of 2 drug-resistant genes, which are respectively:
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;
(19) the delamannich comprises 20 polymorphic sites of 5 drug-resistant genes, which are respectively:
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) fgd1 gene 3 sites: G104S, L270M, L296E;
(20) the PrinMannich total comprises 19 polymorphic sites of 4 drug-resistant genes, which are respectively:
1) ddn gene 7 sites: S11X, W20X, Q58X, W88R, Y136X, Q137X, W139X;
2) fbiA gene 1 site: T146A;
3) fbiC gene 9 sites: G310X, W435X, Q436X, W444X, E637X, K644X, W652X, M709I, S715R;
4) fgd1 gene 2 sites: G71D, E230K.
2. Reagent kit
(1) The primer composition described in Table 2 above, each primer was used at a concentration of 0.5. mu.M;
(2) IGT-EM808 polymerase mix: PCR polymerase, dNTP and reaction buffer solution premix;
(3) enhancer buffer NB (1N): primarily a salt solution;
(4) enhancer buffer M: organic small molecules to enhance high GC region amplification;
(5) IGT-I5 Index (10. mu.M): sequencing the adaptor;
(6) IGT-I7 Index (10. mu.M): sequencing the adaptor.
Example 2 detection method of drug-resistant gene of Mycobacterium tuberculosis
1. Reagent
(1) DNA extraction kit: human tissue and cell DNA extraction kits were purchased from QIAGEN, Cat 51304.
(2) IGT-EM808 polymerase mix, Enhancer buffer NB (1N), Enhancer buffer M, IGT-I5 Index (10. mu.M), IGT-I7 Index (10. mu.M): purchased from egetakang biotech.
(3) Agencourt AMPure XP kit: including magnetic beads and YF buffer B, purchased from Beckman.
(4) DynaMag Side magnetic frame: purchased from Life Technologies.
2. The experimental steps are as follows:
2.1, sample DNA extraction:
(1) and collecting the sputum to obtain a sample.
(2) Extracting total DNA from a sample by using a DNA extraction kit, and detecting the concentration of the DNA to obtain a DNA extracting solution with qualified quality (the qualified DNA content is required to be > 50pg, and the band is not degraded).
2.2, multiplex amplification primers:
the synthesis of the multiplex amplification primers was divided into two reaction tubes, T1 and T2, with the primer sequences shown in Table 2 above.
2.3, 1 st round of multiplex PCR reaction:
(1) the 1 st round multiplex PCR reaction system is shown in Table 3 below.
TABLE 31 st round multiplex PCR reaction System
And transferring the prepared PCR reaction solution to a reaction hole for PCR amplification reaction.
(2) 1 st round of multiplex PCR reaction conditions:
heat lid 105 ℃; 30s at 95 ℃ for 3 min; at 98 ℃ for 20s, at 60 ℃ for 4min, 20 cycles; 72 ℃ for 5 min; the reaction was maintained at 4 ℃ until the end of the reaction.
2.4, magnetic bead purification PCR product:
(1) to 30. mu.L of the PCR product, 27. mu.L of the AMPure XP magnetic beads equilibrated at room temperature were added, and gently pipetted and mixed 20 times.
(2) After incubation at room temperature for 5min, the PCR tubes were placed on a DynaMag Side magnetic frame for 3 min.
(3) The supernatant was removed completely, the PCR tube was removed from the magnetic rack, 50. mu.L of YF buffer B was added to the tube, and the mixture was gently pipetted and mixed 20 times.
(4) After incubation at room temperature for 5min, the PCR tubes were placed on a DynaMag Side magnetic frame for 3 min.
(5) The supernatant was removed, the PCR tube was placed on a magnetic stand, 180. mu.L of 80% ethanol solution was added to the tube, and the tube was allowed to stand for 30 seconds.
(6) The supernatant was removed, the PCR tube was further placed on a magnetic stand, 180. mu.L of 80% ethanol solution was added to the tube, and the supernatant was completely removed after standing for 30 seconds. Standing at room temperature for 3min to completely volatilize residual ethanol.
(7) The PCR tube was removed from the magnetic stand, 24. mu.L of nucleic-free water was added, and the resuspension beads were pipetted gently to avoid air bubbles, and allowed to stand at room temperature for 2 min.
(8) The PCR tube was placed on the magnetic stand again and allowed to stand for 3 min. Pipette 13.5. mu.L of the supernatant, and transfer to a new 200. mu.L PCR tube, in which the supernatant is the multiplex PCR product.
2.5, 2 nd round joint sequence PCR reaction:
(1) the 2 nd round adaptor sequence PCR reaction system is shown in Table 4 below.
TABLE 4 round 2 adaptor sequence PCR reaction System
(2) 2 nd round joint sequence PCR reaction conditions:
heat lid 105 ℃; 30s at 95 ℃ for 3 min; 98 ℃ for 20s, 58 ℃ for 1min, 72 ℃ for 30s, 9 cycles; 72 ℃ for 5 min; the reaction was maintained at 4 ℃ until the end of the reaction.
2.6, round 2 magnetic bead purification:
(1) 27 μ L of AMPure XP magnetic beads which are balanced at room temperature are added into a 30 μ L PCR reaction system, and the mixture is gently pipetted and uniformly stirred for 20 times.
(2) After incubation at room temperature for 5min, the PCR tubes were placed on a DynaMag Side magnetic frame for 3 min.
(3) The supernatant was removed completely, the PCR tube was removed from the magnetic rack, 50. mu.L of YF buffer B was added to the tube, and the mixture was gently pipetted and mixed 20 times.
(4) After incubation at room temperature for 5min, the PCR tubes were placed on a DynaMag Side magnetic frame for 3 min.
(5) The supernatant was removed, the PCR tube was placed on a magnetic stand, 180. mu.L of 80% ethanol solution was added to the tube, and the tube was allowed to stand for 30 seconds.
(6) The supernatant was removed, the PCR tube was further placed on a magnetic stand, 180. mu.L of 80% ethanol solution was added to the tube, and the supernatant was completely removed after standing for 30 seconds. Standing at room temperature for 3min to completely volatilize residual ethanol.
(7) The tube was removed from the magnetic holder, 24. mu.L of nucleic-free water or 1 XTE buffer (pH8.0) was added, gently pipetted and mixed by 20 times, the magnetic beads were resuspended to avoid generation of air bubbles, and the mixture was allowed to stand at room temperature for 2 min.
(8) The PCR tube was placed on the magnetic stand again and allowed to stand for 3 min. Pipette 20. mu.L of the supernatant, and transfer to a new PCR tube, where the supernatant is the prepared multiplex PCR library.
2.7, library quantification:
2 μ L of the library was used
3.0 fluorometer (qubit dsDNA HS Assay kit) to determine the concentration of the library, record the library concentration.
2.8, detecting the quality of the library:
a1. mu.L sample of the library was taken for library fragment length and purity measurements using a Qsep400 fully automated nucleic acid protein analysis system.
2.9, sequencing on a machine:
and performing on-machine sequencing on the built sequencing library on an illumina sequencer to generate data. The amount of raw sequencing data per sample was 200Mb bases.
2.10, result analysis:
and performing sequence filtration, sequence comparison and sequence variation identification on the off-line original sequencing data to obtain the genotype of the drug-resistant gene locus of the sample to be detected, and performing drug resistance evaluation by using a mycobacterium tuberculosis drug resistance effect evaluation system.
EXAMPLE 3 construction of drug resistance Effect evaluation System
1. Sample source:
(1) retrospective samples: 19673 whole genome sequencing data and phenotype drug sensitivity information of mycobacterium tuberculosis culture in PATRIC database;
(2) verification sample 1: 1961 whole genome sequencing data of Mycobacterium tuberculosis culture and 9 phenotypic drug susceptibility information in Rapid anti-biotic and resistance predictions from genome sequence data for Mycobacterium tuberculosis and Mycobacterium tuberculosis.
(3) Verification sample 2: evaluation of Whole Genome sequencing data and 13 phenotypic Drug susceptibility information for Mycobacterium tuberculosis culture in Evaluation of hollow-Genome Sequence Method to diagnosis Resistance of 13 Anti-tuberculosis Drugs and characterisation Resistance Genes in Clinical Multi-Drug Resistance Mycobacterium tuberculosis Isolates From China.
2. Calculating by the formula:
sensitivity is true positive/(true positive + false negative);
specificity ═ true negative/(false positive + true negative);
accuracy ═ true positive + true negative)/all samples.
3. Selecting a retrospective sample, evaluating the sensitivity and specificity of drug resistance of the drug-resistant gene locus to identify the drug resistance of the mycobacterium tuberculosis according to the primer composition and the kit described in the embodiment 1 and the detection method described in the embodiment 2, determining the weight of the drug-resistant gene locus, constructing a genome sequence-drug sensitivity/resistance model corresponding to different drugs by analyzing and applying a machine learning method (Random forest), and constructing a final drug resistance effect evaluation system by integrating resistance loci supported by existing literature evidences.
The results of the measurements are shown in Table 5 below.
TABLE 5 retrospective sample results of drug resistance detection of each drug
4. The validity of the evaluation model constructed above is verified by selecting the verification sample 1 and the verification sample 2, and the detection results are shown in tables 6 and 7 below.
Table 6 verification of the results of detection of drug resistance of each drug in sample 1
| Medicine | Total amount of samples | True positive | False negative | False positive | True negative | Sensitivity of the composition | Specificity of | Accuracy of |
| AK | 112 | 5 | 1 | 0 | 106 | 83.33％ | 100.00％ | 99.11％ |
| Cm | 106 | 5 | 2 | 0 | 99 | 71.43％ | 100.00％ | 98.11％ |
| EMB | 1854 | 50 | 2 | 53 | 1749 | 96.15％ | 97.06％ | 97.03％ |
| LFX | 271 | 20 | 0 | 4 | 247 | 100.00％ | 98.41％ | 98.52％ |
| INH | 1877 | 247 | 20 | 13 | 1597 | 92.51％ | 99.19％ | 98.24％ |
| Km | 104 | 9 | 0 | 0 | 95 | 100.00％ | 100.00％ | 100.00％ |
| MFX | 131 | 11 | 4 | 4 | 112 | 73.33％ | 96.55％ | 93.89％ |
| RIF | 1837 | 94 | 5 | 32 | 1706 | 94.95％ | 98.16％ | 97.99％ |
| Sm | 446 | 50 | 10 | 5 | 381 | 83.33％ | 98.70％ | 96.64％ |
Table 7 test results of drug resistance of each drug in verification sample 2
| Medicine | Total amount of samples | True positive | False negative | False positive | True negative | Sensitivity of the composition | Specificity of | Accuracy of |
| AK | 110 | 10 | 1 | 0 | 99 | 90.91％ | 100.00％ | 99.09％ |
| Cm | 110 | 8 | 1 | 2 | 99 | 88.89％ | 98.02％ | 97.27％ |
| EMB | 110 | 56 | 2 | 17 | 35 | 96.55％ | 67.31％ | 82.73％ |
| LFX | 110 | 62 | 3 | 2 | 43 | 95.38％ | 95.56％ | 95.45％ |
| INH | 110 | 96 | 5 | 0 | 9 | 95.05％ | 100.00％ | 95.45％ |
| Km | 110 | 10 | 1 | 1 | 98 | 90.91％ | 98.99％ | 98.18％ |
| MFX | 110 | 37 | 1 | 26 | 46 | 97.37％ | 63.89％ | 75.45％ |
| RIF | 110 | 98 | 2 | 0 | 10 | 98.00％ | 100.00％ | 98.18％ |
| Sm | 110 | 62 | 7 | 1 | 40 | 89.86％ | 97.56％ | 92.73％ |
| PAS | 110 | 6 | 1 | 0 | 103 | 85.71％ | 100.00％ | 99.09％ |
| PTO | 110 | 15 | 8 | 5 | 82 | 65.22％ | 94.25％ | 88.18％ |
| PZA | 110 | 53 | 12 | 7 | 38 | 81.54％ | 84.44％ | 82.73％ |
| Cfz | 80 | 5 | 1 | 4 | 70 | 83.33％ | 94.59％ | 93.75％ |
In the verification sample 2, for four drugs, namely, aminosalicylic acid (PAS), Propylthioisonicotinamine (PTO), Pyrazinamide (PZA) and clofazimine (Cfz), since the existing database has insufficient resistant positive samples and a machine learning-based determination model cannot be constructed, prediction is performed only based on the reported drug resistance gene sites.
According to the result of the verification sample, the primer composition and the kit have good sensitivity, specificity and accuracy on the medicines.
In addition to the 13 drugs in confirmatory samples 1 and 2 (rifampin (rifapexin, RIF), Isoniazid (INH), Ethambutol (EMB), streptomycin (streptomycin, Sm), Moxifloxacin (MFX), Levofloxacin (LFX), amikacin (amikacin, AK), kanamycin (kanamycin, Km), capreomycin (c, Cm), pyrazinamide (pyrazinamide, PZA), clofazimine (clofazimin, Cfz), para-aminosalicylic acid (PAS), prothioisoniamine (PTO )), the present application can also utilize models for predicting resistance to rifampin (rifabutin, RFB) and ethionamide (etonicotinamide ), resistance prediction of five drugs, cyclic serine (Cs), bedaquiline (Bdq), Linezolid (Linezolid), delamannic (Delamanid) and protanib (Pretomanid), is performed according to the resistance gene locus. Since the drug resistance studies of the above 7 drugs are few, no evaluation of the drug resistance test results of the relevant drug resistance gene sites has been performed at present.
Example 4 detection of drug resistance of Mycobacterium tuberculosis
Selecting 100 clinical sputum and 2mL test samples of bronchoalveolar lavage fluid, extracting DNA by adopting a nucleic acid extraction or purification reagent (DA0870), and evaluating the sensitivity and specificity of the drug resistance of the mycobacterium tuberculosis at the drug-resistant gene locus according to the primer composition and the kit described in the embodiment 1 and the detection method described in the embodiment 2. Comparing the clinical Mycobacterium tuberculosis MIC plate (Trek Diagnostic Systems) in vitro Mycobacterium tuberculosis and solid medium drug sensitivity test results. The specific test results are shown in table 8 below.
TABLE 8 test results of drug resistance of each drug in test samples
The above results are all compared with the results of drug sensitivity test. The detection result of the primer composition and the clinical practical sample has lower specificity relative to the drug sensitivity result of the bacteria culture whole genome sequencing sample, because the screening of the drug-resistant bacteria in the bacteria culture process leads to the gradual reduction of the content of the drug-resistant bacteria in the clinical bacteria culture process, thus leading to the sensitivity of the clinical in vitro culture result, and the main flora in the clinical uncultured sample is the drug-resistant flora (Evidence for the clinical bacteria of a secondary site B mutation in the computing and sub-clinical non-cultured sample) (J anticancer bacteria chemistry 2016; 71(2): 324-332). Taking rifampicin (RIF as an example), aiming at clinical uncultured samples, the drug resistance detection result of the application is judged to be a false positive sample (uncultured samples are detected to be drug resistant, and cultured samples are detected to be sensitive in a drug sensitivity test),
MTB/RIF assay also showed drug resistance, and the mutations and ratios are shown in Table 9 below.
TABLE 9 mutation information of samples with false positive drug resistance detection results
Referring to the "catalog of microorganisms in Mycobacterium tuberculosis complex and the infection with drug resistance" 2021 file issued by WHO, based on more than 2 ten thousand Mycobacterium tuberculosis sequencing and drug sensitivity experimental data, the above mutation site has a very high correlation with Mycobacterium tuberculosis drug resistance: "remaining single nucleotide RIF-resistance variables (L430P, D435Y, H445L, H445N, H445S and L452P) yieldd a sensitivity of 92.3% (95% CI, 91.8-92.8%) for predicting pharmaceutical drug selectivity in the ALL database"
The method described in the present application does not require bacterial culture and can be completed within a week from the collection of clinical samples to the analysis of results.
Comparative example 1
This comparative example is different from example 1 in that each primer in the primer composition shown in Table 2 in the kit was used at a concentration of 0.1. mu.M, and the other conditions were the same as in example 1.
The test sample of example 4 was tested according to the test method described in example 2, and the test results are shown in Table 10 below.
TABLE 10 test sample results of drug resistance test for each drug
| Medicine | Total amount of samples | True positive | False negative | False positive | True negative | Sensitivity of the composition | Specificity of | Accuracy of |
| AK | 100 | 3 | 4 | 0 | 93 | 42.86％ | 100.00％ | 96.00％ |
| EMB | 100 | 22 | 11 | 3 | 64 | 66.67％ | 95.52％ | 86.00％ |
| Eto | 100 | 2 | 4 | 3 | 91 | 33.33％ | 96.81％ | 93.00％ |
| LFX | 100 | 22 | 7 | 2 | 69 | 75.86％ | 97.18％ | 91.00％ |
| INH | 100 | 56 | 13 | 0 | 31 | 81.16％ | 100.00％ | 87.00％ |
| Km | 100 | 3 | 7 | 0 | 90 | 30.00％ | 100.00％ | 93.00％ |
| MFX | 100 | 21 | 22 | 1 | 56 | 48.84％ | 98.25％ | 77.00％ |
| PAS | 100 | 6 | 9 | 2 | 83 | 40.00％ | 97.65％ | 89.00％ |
| RFB | 100 | 36 | 20 | 1 | 43 | 64.29％ | 97.73％ | 79.00％ |
| RIF | 100 | 56 | 25 | 5 | 14 | 69.14％ | 73.68％ | 70.00％ |
| Sm | 96 | 38 | 12 | 2 | 48 | 76.00％ | 96.00％ | 86.00％ |
Comparative example 2
This comparative example is different from example 1 in that each primer in the primer composition shown in Table 2 in the kit was used at a concentration of 1.0. mu.M, and the other conditions were the same as in example 1.
The test sample of example 4 was tested according to the test method described in example 2, and the test results are shown in Table 11 below.
TABLE 11 test results of drug resistance of each drug in test samples
| Medicine | Total amount of samples | True positive | False negative | False positive | True negative | Sensitivity of the composition | Specificity of | Accuracy of |
| AK | 100 | 5 | 2 | 1 | 92 | 71.43％ | 98.92％ | 97.00％ |
| EMB | 100 | 26 | 7 | 6 | 61 | 78.79％ | 91.04％ | 87.00％ |
| Eto | 100 | 3 | 3 | 2 | 92 | 50.00％ | 97.87％ | 95.00％ |
| LFX | 100 | 24 | 5 | 6 | 65 | 82.76％ | 91.55％ | 89.00％ |
| INH | 100 | 62 | 7 | 5 | 26 | 89.86％ | 83.87％ | 88.00％ |
| Km | 100 | 6 | 4 | 10 | 80 | 60.00％ | 88.89％ | 86.00％ |
| MFX | 100 | 33 | 10 | 4 | 53 | 76.74％ | 92.98％ | 86.00％ |
| PAS | 100 | 7 | 8 | 1 | 84 | 46.67％ | 98.82％ | 91.00％ |
| RFB | 100 | 42 | 14 | 3 | 41 | 75.00％ | 93.18％ | 83.00％ |
| RIF | 100 | 73 | 8 | 5 | 14 | 90.12％ | 73.68％ | 87.00％ |
| Sm | 100 | 44 | 6 | 7 | 43 | 88.00％ | 86.00％ | 87.00％ |
As is clear from the results in tables 8, 10 and 11, the primer composition of the present invention has high detection sensitivity, specificity and accuracy.
Experimental example 1
One sputum sample from this example 1 was obtained from a clinical patient and informed consent was obtained from this experiment.
The concentration of the sample DNA extract solution of this example 1 was 1.27 ng/. mu.L,
the result of the MTB/RIF assay was rifampicin resistance (rpoB a), and qPCR CT was 26.67.
The drug-resistant genes of the sample of the experimental example 1 were detected according to the detection method described in the example 2 of the present invention, and the patient of the sample of the experimental example 1 was instructed to use the drug-resistant Mycobacterium tuberculosis personalized medicine, and a report was generated. The report is shown in table 12 below.
TABLE 12 sample administration report of this example
| Name of drug | Abbreviations for drugs | Drug classification | Drug resistance assessment |
| Rifampicin | RIF | First line medication | Drug resistance |
| Isoniazid | INH | First line medication | Drug resistance |
| Ethambutol | EMB | First line medication | Sensitivity of |
| Pyrazinamides | PZA | First line medication | Drug resistance |
| Rifabutin | RFB | First line medication | Drug resistance |
| Amikacin | AK | Second line medicine | Drug resistance |
| Capreomycin | Cm | Second line medicine | Sensitivity of |
| Kanamycin | Km | Second line medicine | Drug resistance |
| Streptomycin | Sm | Second line medicine | Sensitivity of |
| Levofloxacin | LFX | Second line medicine | Drug resistance |
| Moxifloxacin hydrate | MFX | Second line medicine | Sensitivity of |
| Ethionamide | Eto | Second line medicine | Drug resistance |
| Clofazimine | Cfz | Second line medicine | Sensitivity of |
| Para-aminosalicylic acid | PAS | Second line medicine | Sensitivity of |
| Propylthioisonicotinamine | PTO | Second line medicine | Sensitivity of |
| Cyclic serine | Cs | Second line medicine | Sensitivity of |
| Bedaquinoline | Bdq | Second line medicine | Sensitivity of |
| Linezolid | Linezolid | Second line medicine | Sensitivity of |
| De Raman Ni | Delamanid | Second line medicine | Sensitivity of |
| Prime Mannich | Pretomanid | Second line medicine | Sensitivity of |
According to the test results of this example, it was possible to determine the drug suitable for the patient.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, although the present patent is described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that the technical solutions described in the foregoing embodiments may be modified or part of the technical features may be equivalently replaced. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> Shenzhen third people hospital of institute of agricultural genome of Chinese academy of agricultural sciences
<120> primer composition for drug resistance gene detection of mycobacterium tuberculosis and application thereof
<130> 20210712
<160> 390
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
gaacgcgatt catagcagca tcg 23
<210> 2
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
gcaagctact gaaggacaag ga 22
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
tcgatgttcc aggcgatact tccgc 25
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
gctaccacaa gatcgtgctg atgg 24
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
ggtgaaatgg acgctaagga gtt 23
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
gccaaggatg ttcggttcct ggat 24
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
gggtgctcta tgcaatgttc gattc 25
<210> 8
<211> 17
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
accccgttgg cgatgga 17
<210> 9
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
ggctggtgaa aaagtccaag ctg 23
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
agggcatgcg gttcaatatc ga 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 11
gccgcaccaa atctcatcgg 20
<210> 12
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
gcaagcgtca tcacgaccga tg 22
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
agccattcgg tgtttgacgt cg 22
<210> 14
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
accgtgatct gaggcgtaaa cga 23
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 15
tggtcatgat tggcatgctg tcgt 24
<210> 16
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 16
tcgacgacat ttcgttcgtc gtca 24
<210> 17
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 17
ccggaaaatc cactctcgtc aa 22
<210> 18
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 18
gctgatcgac ctggaagtcg agt 23
<210> 19
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 19
aagaggtgct ctacgagctg tct 23
<210> 20
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 20
agacggtgtt catgggtgac ttc 23
<210> 21
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 21
atcgaatatc tggtccgctt gcac 24
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 22
accgcagacg ttgatcaaca tc 22
<210> 23
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 23
atcaaggagt tcttcggcac cagcc 25
<210> 24
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 24
gggtcaaccc gttcgggttc atc 23
<210> 25
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 25
ccgactacat cactgtgatg cac 23
<210> 26
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 26
caactacgag gacgcgatca tc 22
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 27
agatgcgcca caatgagctc 20
<210> 28
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 28
tggaggacat ctggagcact ttcac 25
<210> 29
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 29
caaccatgcg cagaacatca agagc 25
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 30
aatgctggtg gaaggcaagg 20
<210> 31
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 31
ggttatccgt tcgtcaacaa gcaga 25
<210> 32
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 32
gcaagaagga gatcctcgac cact 24
<210> 33
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 33
gccttcggaa ccaaagaaag tgc 23
<210> 34
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 34
ccgacatcat ggaattcgtc ga 22
<210> 35
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 35
atgggcggct atttcgagtc cag 23
<210> 36
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 36
aatgcccgga tgctgatcc 19
<210> 37
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 37
ggatcggcgc acctatttcc gg 22
<210> 38
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 38
ctgatgagca acctcgcaat ctga 24
<210> 39
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 39
cgtgaagttg acgagtcagg t 21
<210> 40
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 40
ggtgtcaaga accgcaaaca g 21
<210> 41
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 41
ggtcactggt gctgttgcag 20
<210> 42
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 42
ggctttcgtt ggcgctcaac 20
<210> 43
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 43
tccgttcccg atgtcaatca tg 22
<210> 44
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 44
cagcgattca ccgtttcgac tctct 25
<210> 45
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 45
gcgcaaccca aatacgtaca ggc 23
<210> 46
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 46
ccttcggtga ctgggaatcg atc 23
<210> 47
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 47
tttggcggac ttcttggcat c 21
<210> 48
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 48
gtagtaggcg gtgatcagct gt 22
<210> 49
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 49
aggaacacca ctagtaccgg atgc 24
<210> 50
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 50
ggctgttcac accgttcttc atc 23
<210> 51
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 51
cagtggggaa tattgcacaa tgg 23
<210> 52
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 52
ccggaattac tgggcgtaaa gagct 25
<210> 53
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 53
agctcgtgtc gtgagatgtt gggt 24
<210> 54
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 54
atgtccaggg cttcacacat g 21
<210> 55
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 55
gaagtcggag tcgctagtaa tcg 23
<210> 56
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 56
gctggatcac ctcctttcta agg 23
<210> 57
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 57
ccggaatatc gtgaacaccc ttgc 24
<210> 58
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 58
aaacggcggt ggtaactata acc 23
<210> 59
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 59
taggtgggag actgtgaaac ct 22
<210> 60
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 60
acttacaagt caagcaggga cga 23
<210> 61
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 61
ttgcactact tggtcaccca tgc 23
<210> 62
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 62
cagagaagat gaacggcgaa gc 22
<210> 63
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 63
ttcgtggacg actccgtcat 20
<210> 64
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 64
ctatcaccac cgcaaacgtg a 21
<210> 65
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 65
tcatggtcga agtgtgctga gtca 24
<210> 66
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 66
ccattcgtat cccgttcagt cct 23
<210> 67
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 67
ctgcaattta tcccagcgaa gcg 23
<210> 68
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 68
ctggttagcg gaatcatcac cgact 25
<210> 69
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 69
acgtgcaaaa cgaggagca 19
<210> 70
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 70
atctcggcgt attcgtatgc ttc 23
<210> 71
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 71
ttgccgtaca tgttcgtggt cc 22
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 72
cactttgcgc tgcaattcct 20
<210> 73
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 73
gctcaccaag gaggacaaag agggc 25
<210> 74
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 74
gagatcgagg ccaagatcat cg 22
<210> 75
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 75
tgtggtcgct attgctgatg g 21
<210> 76
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 76
gctccggacc gttgaaatgg at 22
<210> 77
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 77
tggcgcgatc acgtcaac 18
<210> 78
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 78
ggtgggttca ccgaagtact g 21
<210> 79
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 79
cgcagtttga ggtggggaa 19
<210> 80
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 80
gggattggag gatgcggtgc a 21
<210> 81
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 81
caacgttcac gccgaatatg c 21
<210> 82
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 82
cgagatgtcc aagcacatcg aca 23
<210> 83
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 83
caaatcagcc aggcgataaa gccg 24
<210> 84
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 84
gccatatcgc ccaccacgaa cac 23
<210> 85
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 85
ggtcgttgcc gaaataagac tgg 23
<210> 86
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 86
gcgatttctc cctcggagat aatcc 25
<210> 87
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 87
tcgtaccgat ctatctgggg tacgt 25
<210> 88
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 88
ttcctgctga tgtcgcagat g 21
<210> 89
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 89
tcacgaagaa gtcgttggtc agtg 24
<210> 90
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 90
cctttccgag gtagtttcgg aagc 24
<210> 91
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 91
ggcgaaggac actttgatgt tccc 24
<210> 92
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 92
atgcggtcga aactagctgt 20
<210> 93
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 93
ctggctctta aggctggcaa tc 22
<210> 94
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 94
cgaactcgtc ggccaattcc t 21
<210> 95
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 95
atccgctcat agatcggatc cacc 24
<210> 96
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 96
gtgttcgtcc atacgacctc gat 23
<210> 97
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 97
gtgccatacg agctcttcca gcc 23
<210> 98
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 98
ccgaaacgtc tcgcgaatgt c 21
<210> 99
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 99
ttaccgctgt aacgctcatc g 21
<210> 100
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 100
atgagagctt cttgccgtac ttc 23
<210> 101
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 101
ataaacagcg gcccgtagtg gcc 23
<210> 102
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 102
agtaccttca gattgagccg gtt 23
<210> 103
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 103
ttcggcgtag tccggtatag agga 24
<210> 104
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 104
gcacggcagg aaggtggtag ag 22
<210> 105
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 105
attccctggc attaccgtgg tgc 23
<210> 106
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 106
gagccctcgt atgtaaattc gcttc 25
<210> 107
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 107
gccaacagtt catcccggtt cg 22
<210> 108
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 108
gtctggcgca cacaatgatc g 21
<210> 109
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 109
tagaacaccg cctcgattgc cg 22
<210> 110
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 110
ccctcgcaga agtcgttctg ca 22
<210> 111
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 111
gcttacaggc ccgttttgtt gg 22
<210> 112
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 112
gaaagccgct gatctagatc ttgc 24
<210> 113
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 113
aagacgagtt cgtcaccaag tgg 23
<210> 114
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 114
agagctacga cctgatgaat gc 22
<210> 115
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 115
ggacatcgac cacgtcaacg cg 22
<210> 116
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 116
cacccgatcc cgagatcgac ctt 23
<210> 117
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 117
atcccataga cggcgaagaa cacc 24
<210> 118
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 118
gccttcgatg accgcataca cca 23
<210> 119
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 119
gccgatgaaa tcggtgaaac tggcc 25
<210> 120
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 120
cgacagtgtc tggcaaagga tcac 24
<210> 121
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 121
tgaatccctt ccacgggtca gc 22
<210> 122
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 122
ggtggcaacg tcgatagcat 20
<210> 123
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 123
atgccgtcca cgtccttgtc g 21
<210> 124
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 124
aaaggcctcg acggaagc 18
<210> 125
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 125
cctggtgttc accgtgca 18
<210> 126
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 126
cgagtgatga tgcccgcctt 20
<210> 127
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 127
cggtaggtcg ccacatactg cgc 23
<210> 128
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 128
gatcgacgga tagctgcgtt 20
<210> 129
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 129
tggctgtttg ttcgacacga 20
<210> 130
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 130
tccaaaatgt gcgcgaccta 20
<210> 131
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 131
tatcaggtcg ctgatcagca tcgca 25
<210> 132
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 132
ggcattgatc aacgcgtggc aga 23
<210> 133
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 133
ccgatgggaa tcagtgaaga cacc 24
<210> 134
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 134
cgatgatgct ggtcaacaac ag 22
<210> 135
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 135
ttcaggtcca cgatgatgtc cttga 25
<210> 136
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 136
cgaagaagcg caggttctca tacc 24
<210> 137
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 137
atagacttgt ccgcctccga t 21
<210> 138
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 138
cccatggtga tctcccggaa atgc 24
<210> 139
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 139
actccttgat tccggcttga aggct 25
<210> 140
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 140
cttgctcgac gtgattgtcg ta 22
<210> 141
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 141
gcatagctgg cgatgttgaa c 21
<210> 142
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 142
tgatctggtc gatgtgctca c 21
<210> 143
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 143
gatggagccg gccacgatc 19
<210> 144
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 144
aacaggctgc gggttccg 18
<210> 145
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 145
tacccgtttc gagcacgaag c 21
<210> 146
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 146
tcgacgggaa tacacccaga tcc 23
<210> 147
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 147
aatgcgccga cgaatagaca tc 22
<210> 148
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 148
cacggcataa tcatcgccga a 21
<210> 149
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 149
ctctacagca ttgttccgct gatc 24
<210> 150
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 150
ggtggtattg ggtttccggt ttcga 25
<210> 151
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 151
gcagttgatc ccaactcgac acc 23
<210> 152
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 152
ttggtgaact ccttgtccat gc 22
<210> 153
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 153
cgaaaatccc gcacgaaagc c 21
<210> 154
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 154
ttggtgacca tatcgcagct gct 23
<210> 155
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 155
gtgaagacga tgtttgccac tgc 23
<210> 156
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 156
tcgacgaatt cgccgaagag gtac 24
<210> 157
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 157
gcgtcaataa gtagcccaag ctc 23
<210> 158
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 158
ccggcaaata gcggcagatt cc 22
<210> 159
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 159
cgacttggtc aaaggcaaac tgacc 25
<210> 160
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 160
cgccagcaag cccaactgtt gc 22
<210> 161
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 161
tcctgtctgg attagaggca gtc 23
<210> 162
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 162
gcttgatctt cacctcgtcg atgg 24
<210> 163
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 163
gtcacggatt aatacctgga tgtacc 26
<210> 164
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 164
caaccccaag gttcaggtac aga 23
<210> 165
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 165
caaggcggat cttctcggta cc 22
<210> 166
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 166
gaccatcgcc tgcaagacaa agc 23
<210> 167
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 167
gtaagcgtcg acaaacacga tccgc 25
<210> 168
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 168
cgatggttac ctgttcgcct tcc 23
<210> 169
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 169
cggtctatga tgcaatcgtc gt 22
<210> 170
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 170
cgccatccat ttggtcactc tgt 23
<210> 171
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 171
tgaagatggc cgccatgatc 20
<210> 172
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 172
gcatgcgggc tatatggcca a 21
<210> 173
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 173
tgggtaatca gccgtgaggt 20
<210> 174
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 174
tgcatcatcg gtgccttgac 20
<210> 175
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 175
tcaattgccc agctcctcct cag 23
<210> 176
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 176
tgtccaacta tttccgctgg ttcg 24
<210> 177
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 177
gcgctgatga cccatgtcag 20
<210> 178
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 178
gctggtcacc tatgtgctga tcg 23
<210> 179
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 179
cttcggcacc atgttcttcc tgat 24
<210> 180
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 180
tggtatgtct ccagctacgg tgtg 24
<210> 181
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 181
atcgccgaaa tcccgaagtt cc 22
<210> 182
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 182
cgcaagttcg acaccctggt cgat 24
<210> 183
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 183
gcgatggtga acggaatcat c 21
<210> 184
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 184
cgaaccgaat gccatgatca g 21
<210> 185
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 185
gaaccttttg gtggggtgct cc 22
<210> 186
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 186
ggtttgccgg aggttgagtc a 21
<210> 187
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 187
ttcttagcgt cgtagtcgag gtcct 25
<210> 188
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 188
cactgatcac cttgtggtgg aa 22
<210> 189
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 189
ggataacgga acaaatccca ggtgc 25
<210> 190
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 190
agatgtcgac actatcgaca cgtag 25
<210> 191
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 191
tcggtgtcga tgctgaacac gc 22
<210> 192
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 192
atcgaacttc gattctggcc ggat 24
<210> 193
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 193
ggcatgctcc atttcgtcaa cttg 24
<210> 194
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 194
cggttctagg agaactacct gga 23
<210> 195
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 195
gttcgaggag ctcaccgatc a 21
<210> 196
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 196
gaaacattct ccgctctcga ga 22
<210> 197
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 197
gcgaaagtcg ttgtgaacaa ggc 23
<210> 198
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 198
tcgatgttcc aggcgatact tc 22
<210> 199
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 199
taccacaaga tcgtgctgat ggcc 24
<210> 200
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 200
gatcaacaag gaagacggca ttcag 25
<210> 201
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 201
ttgacatcga gcaggagatg cagc 24
<210> 202
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 202
tgccgagacc atgggcaact ac 22
<210> 203
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 203
gttggcgatg gagatgctga g 21
<210> 204
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 204
cggtcggcat ggcaaccaat atcc 24
<210> 205
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 205
ctgaaatacc cgacacgacc agcg 24
<210> 206
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 206
cccatttccc ctaatcccct aacg 24
<210> 207
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 207
aatggccaga tcggagttgc c 21
<210> 208
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 208
gcagtgttgg cttccaacgc aag 23
<210> 209
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 209
acaacttcgt gtgggcatac c 21
<210> 210
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 210
gtggtcatga ttggcatgct gt 22
<210> 211
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 211
tatcctgagc caggtcaacg aca 23
<210> 212
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 212
actcaaggcc ggaaaatcca ctct 24
<210> 213
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 213
tgaatcggtg gggctaatgc ggc 23
<210> 214
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 214
ggcttcatct gtacgtccgg caa 23
<210> 215
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 215
catcaacaac aacaccggtg ag 22
<210> 216
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 216
cgacgagacc attgacaagt cca 23
<210> 217
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 217
atcacaccgc agacgttgat caa 23
<210> 218
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 218
ccggatgtgc ccgatcgaaa cc 22
<210> 219
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 219
tcaacccgtt cgggttcatc gaa 23
<210> 220
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 220
ctgaggtgga ctacatggac gtct 24
<210> 221
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 221
agccgaacgc cattgtgtcg a 21
<210> 222
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 222
catcatgaag ctgcaccacc t 21
<210> 223
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 223
tcgatccaga agctgatcga gaac 24
<210> 224
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 224
cgtccgactt gaacgacctg tac 23
<210> 225
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 225
tgtcctccaa caacatcctg tcgcc 25
<210> 226
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 226
ccaagatcaa ggtgcggctg a 21
<210> 227
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 227
tggtggagat ttggaaggaa gcc 23
<210> 228
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 228
gaccgtgctg gagtacttca tc 22
<210> 229
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 229
cgcttgagag ttccaatcat cg 22
<210> 230
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 230
tatttcgagt ccaggagttt gactc 25
<210> 231
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 231
ttggcgggtc gattgttgg 19
<210> 232
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 232
caatggccga actgcagga 19
<210> 233
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 233
atggccgaca aacagaacgt 20
<210> 234
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 234
ttgacgagtc aggtcgaggt 20
<210> 235
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 235
taccaggttg ggcaagagtt ga 22
<210> 236
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 236
cgggtgaccg ttcttaacct t 21
<210> 237
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 237
gttctggaac aagcgcatgt cta 23
<210> 238
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 238
gcgatctctt gggaacacga t 21
<210> 239
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 239
agatagaccg cgaacgggat c 21
<210> 240
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 240
cgaagtacac gccggaccac t 21
<210> 241
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 241
tggtggtcgg tgaagtattt cggga 25
<210> 242
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 242
tcgcggtaat ccacattgtg gtga 24
<210> 243
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 243
gagcttacgc agattagaca cgta 24
<210> 244
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 244
tcgatagtca tctgcaatgg tcca 24
<210> 245
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 245
taccagagcc atcaaggagg ataa 24
<210> 246
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 246
acttcatcaa cctggcgttt atgg 24
<210> 247
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 247
gggagcgaac aggattagat acc 23
<210> 248
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 248
ggcggagcat gtggattaat tcg 23
<210> 249
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 249
cttaaaagcc ggtctcagtt cg 22
<210> 250
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 250
aagtcgtaac aaggtagccg tac 23
<210> 251
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 251
cggctggatc acctcctttc taagg 25
<210> 252
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 252
ggtggggtgt ggtgtttgag aactg 25
<210> 253
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 253
aacggcggtg gtaactataa cca 23
<210> 254
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 254
ggataggtgg gagactgtga aacct 25
<210> 255
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 255
cttacaagtc aagcagggac gaa 23
<210> 256
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 256
gctgggttta gaacgtcgtg agaca 25
<210> 257
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 257
ttagctttcc aagccgctct acg 23
<210> 258
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 258
tcaacgacta ctacaaccag cttgg 25
<210> 259
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 259
gtgtctcgat gtcgaacttc ct 22
<210> 260
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 260
agaactgctc aagcccaaga tc 22
<210> 261
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 261
ggctacatcg acaccgatat gacc 24
<210> 262
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 262
cacaacacaa ggacgcacat g 21
<210> 263
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 263
ttagcggaat catcaccgac tcgtc 25
<210> 264
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 264
ggtggtgcat tcgattgggt tcatg 25
<210> 265
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 265
aagtacggtg tgcgttcgaa t 21
<210> 266
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 266
gatcggctgg aacatgaagg at 22
<210> 267
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 267
gatttttccg ccctgagttc ac 22
<210> 268
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 268
aagtagccgt caacgacata gg 22
<210> 269
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 269
cgaatggctt gagggattcg aaa 23
<210> 270
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 270
cagcgcttga tcttgcagct gatc 24
<210> 271
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 271
agtcggcgga gaagggttga g 21
<210> 272
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 272
actagtcggt gcgctgga 18
<210> 273
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 273
ctgtcgttca tctcgttggc taccg 25
<210> 274
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 274
catcgggcaa tgtcgagtac ttc 23
<210> 275
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 275
cggacgctga acaaattgac agc 23
<210> 276
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 276
cgacttgtag ttcaggttcg aca 23
<210> 277
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 277
tgagttggcg gatatcggtt gggt 24
<210> 278
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 278
gattgccggt cttcgaaata gc 22
<210> 279
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 279
gagctcaacc cgtgattgct cg 22
<210> 280
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 280
atttccacgc ccagcttctg 20
<210> 281
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 281
cgatttctcc ctcggagata atcccg 26
<210> 282
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 282
cctcgatgag cgagttcagt tagct 25
<210> 283
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 283
aacctgtcga ggttcatcac cttgt 25
<210> 284
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 284
aggttcacga agaagtcgtt ggt 23
<210> 285
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 285
tttccgaggt agtttcggaa gcca 24
<210> 286
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 286
tcggcgaagg acactttgat gttcc 25
<210> 287
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 287
ttccagggtg cgaatgacct tgcg 24
<210> 288
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 288
cttaaggctg gcaatctcgg ctt 23
<210> 289
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 289
gtctcggtgg atcagcttgt acc 23
<210> 290
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 290
actcgtagcc gtacaggatc tc 22
<210> 291
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 291
gaggaaactg ttgtcccatt tcg 23
<210> 292
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 292
ccgctgtttc gacgtcgttc atg 23
<210> 293
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 293
aaacgtctcg cgaatgtcga ccg 23
<210> 294
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 294
aacgtcttga agcccatcga ttcca 25
<210> 295
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 295
ttgccggcga aaacaatcag g 21
<210> 296
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 296
ataaacagcg gcccgtagtg 20
<210> 297
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 297
aggtggtcat cacttcctcg atg 23
<210> 298
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 298
ggtttctgta atgggtgggt gt 22
<210> 299
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 299
caacaagcgg taacaccttg tc 22
<210> 300
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 300
acgtcgtatt ggttcttgcg tgac 24
<210> 301
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 301
atcgcgatgg aacgtgatat ccc 23
<210> 302
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 302
ccatcaggag ctgcaaacca actc 24
<210> 303
<211> 19
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 303
tggccaagcc attgcgtac 19
<210> 304
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 304
gtgtccagac tgggatggaa g 21
<210> 305
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 305
gttgccacga cgtgatggta g 21
<210> 306
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 306
gacgatcgcg aacatgacca 20
<210> 307
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 307
aagacgagtt cgtcaccaag tg 22
<210> 308
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 308
agaggattgt cgagagctac ga 22
<210> 309
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 309
ttctccatga tgcgggccat gtc 23
<210> 310
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 310
acctcggacg cctttcatat ggt 23
<210> 311
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 311
ctgaactacg agacacccga tccc 24
<210> 312
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 312
ggaccgatat ggaaggaacg ttcg 24
<210> 313
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 313
tcaaccgcag atccatgtac agc 23
<210> 314
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 314
cggccacgtg cacgtgaata ttacg 25
<210> 315
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 315
ggctcatatc gagaatgctt gcg 23
<210> 316
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 316
tgtccgcttt gatgatgagg agagt 25
<210> 317
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 317
tggcaacgtc gatagcatcg c 21
<210> 318
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 318
accccgacca gaaatcggaa 20
<210> 319
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 319
cgaaaaaggc ctcgacggaa gcg 23
<210> 320
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 320
caccgcgaat tcggaatcct 20
<210> 321
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 321
cggtgcgttg atcacgttgg tg 22
<210> 322
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 322
ggcttgccgt cgatcgaaat gc 22
<210> 323
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 323
gcgatatgga tcgacggata gc 22
<210> 324
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 324
gggccggaat tcgtcgaatt cattgc 26
<210> 325
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 325
gtaaatagac accgggctcc a 21
<210> 326
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 326
gccaccagct gatagatctc tag 23
<210> 327
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 327
tgcatggtgg cactgtagcg a 21
<210> 328
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 328
gaccgattcg tcttcaacct gt 22
<210> 329
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 329
aacggcaacg aggaactgaa gcc 23
<210> 330
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 330
acagttagtt tggcagccag gta 23
<210> 331
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 331
gtggtagctg tacaaccggt ac 22
<210> 332
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 332
gcatagactt gtccgcctcc gat 23
<210> 333
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 333
gctggccata aagtcagctt g 21
<210> 334
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 334
cactccttga ttccggcttg aagg 24
<210> 335
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 335
cggtcggcta gaagtagttt cgg 23
<210> 336
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 336
gcgttggtag agctgacagc tcag 24
<210> 337
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 337
cgtagaactg gaagaacgca tg 22
<210> 338
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 338
gatcgccatt gtacaccgta gatc 24
<210> 339
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 339
aaaaccacag cagctcgtag gct 23
<210> 340
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 340
tcgtatggcg tcacgattga c 21
<210> 341
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 341
cacgatgaca aacgcgacag cg 22
<210> 342
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 342
ggtagatcat caccagcccg atc 23
<210> 343
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 343
gtcaatcgtc ggtttcagtc catgc 25
<210> 344
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 344
tttgcctact ggttatcgcg atc 23
<210> 345
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 345
cgatgtggtg gtgatcaaat ccg 23
<210> 346
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 346
cggtgactgc gatcaaatcg atg 23
<210> 347
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 347
cgaaagcgtg caggtatatc gc 22
<210> 348
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 348
cgtattgcca aaggcggtgt tc 22
<210> 349
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 349
atacctcttg tgcgcgatct tcg 23
<210> 350
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 350
ctcgccgata tctgtgatga agg 23
<210> 351
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 351
gccttggtcg aatatctcac caccc 25
<210> 352
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 352
gcaatctttg tctgggggtc tgt 23
<210> 353
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 353
cgttgcgaaa cccatgtagt ga 22
<210> 354
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 354
cgtctaatcc ggtggtcagc atc 23
<210> 355
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 355
ttctcccaga accgtttcac tga 23
<210> 356
<211> 20
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 356
tggcaggaaa cagttgcagt 20
<210> 357
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 357
cgcgggctga ttgaattctt ggtc 24
<210> 358
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 358
ttccagcttg cccttatcgt tc 22
<210> 359
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 359
tatctgccac gttgatcccg aa 22
<210> 360
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 360
tgggtggtga attcgtacag tgt 23
<210> 361
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 361
cctttgcgtg ctctttcatt ccgga 25
<210> 362
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 362
tgtaagcgtc gacaaacacg atc 23
<210> 363
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 363
accgttgtcg atatcgacga tgg 23
<210> 364
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 364
ccggcgaaag ttccgatttc aatcg 25
<210> 365
<211> 18
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 365
ctgtcgctgg gtcgcatc 18
<210> 366
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 366
tgcaagcagg ctcgtatttg ttc 23
<210> 367
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 367
cgggctatat ggccaactac tac 23
<210> 368
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 368
ggtaatcagc cgtgaggtca ttcc 24
<210> 369
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 369
aagacgactg gtttagggac tg 22
<210> 370
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 370
ggtaatgagc gatctcaccg gatc 24
<210> 371
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 371
tgatattcgg cttcctgctc tgg 23
<210> 372
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 372
ggtattacaa cctgctggcg ctga 24
<210> 373
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 373
gtcatcctga ccgtggtgtt cg 22
<210> 374
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 374
cggctttttg atcaccgcgc tatgc 25
<210> 375
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 375
cgctcactgc aggaatatgt ggg 23
<210> 376
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 376
gctaagaagc tggacaccga cac 23
<210> 377
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 377
catgggctca tacggaaaca cc 22
<210> 378
<211> 21
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 378
ggtgaacgga atcatcgaca c 21
<210> 379
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 379
ataggtgctg gtgtagcttt cca 23
<210> 380
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 380
gagaccaaag caatacgcca ac 22
<210> 381
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 381
agcacgttct tccaccgtac cg 22
<210> 382
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 382
tgttcttagc gtcgtagtcg aggt 24
<210> 383
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 383
tcggaaacct agcgtgtaca tg 22
<210> 384
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 384
tcatggatcc acgctatcaa cg 22
<210> 385
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 385
tggtcaatca agccgacatg gccc 24
<210> 386
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 386
aactgtggtc gacgtttatg caga 24
<210> 387
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 387
tcatttcccg ctggaatggt tcga 24
<210> 388
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 388
cacatgtcac caccctgaca tcaac 25
<210> 389
<211> 26
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 389
cagatctgtc accatctctc gaagaa 26
<210> 390
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 390
agttcagtag atgccggtcc cat 23
Claims (10)
1. A primer composition for drug-resistant gene detection of mycobacterium tuberculosis is characterized in that: the primer composition comprises:
2. the primer composition of claim 1, wherein: the mycobacterium tuberculosis drug comprises rifampicin, isoniazid, ethambutol, streptomycin, moxifloxacin, ofloxacin, amikacin, kanamycin, capreomycin, pyrazinamide, rifabutin, ethionamide, clofazimine and para-aminosalicylic acid.
3. The primer composition of claim 2, wherein: the primer composition is used for detecting the following drug-resistant gene detection sites of the mycobacterium tuberculosis:
(1) rifampicin comprises 105 polymorphic loci of 4 drug-resistant genes, which are respectively:
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, D435A, N36437, N A, S441A, L A, T36444, T444, H A, H13272, S441A, S3678, S A, L36445, S A, L A, S A, L36445, L A, S A, L A, S A, S A, 3678, A, 36445H A, S A, S3678, A, L A, S A, L36445H A, L A, L A, tc A, L A;
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) rpoZ gene 1 site: I55X;
(2) isoniazid, including 207 polymorphic sites of 18 drug-resistant genes, which are respectively:
1) 104 sites of the katG gene: V1A, C20S, Q88E, W90R, W90X, R104L, R104Q, A106V, H108Q, H108G, A109V, A110V, G125D, Q127D, N138D, N138D, A139D, A139D, S140D, L141D, D142D, L148D, L148D, W149D, Y155D, L159D, S175D, T180D, G182D, W191D, W191D, P232D, M257D, D259D, H270D, K274D, T D, G297D, G300, W300, S302, S315, H270D, H270D, K274D, S300, S3673, S315, S D, L D, S315, S300, S D, S315, L D, S D, L300, S D, S315, S D, L D, S315, S D, L300, S D, S315, S D, S315, L D, S315, S D, L D, S315, S D, S315, L D, S D, L D, S3673D D, S315, S D, S3673D D, S D, L D, S3673D D, S D, L D, S3673D D, S D, L D, S1-S3673, S3673D 3673, S3673D D, S3673D 3673, S315, L3673, S1-S D, S3673, S D, S3673, S1-S D, S315, S D, L D, S1-S315, S D, S315, S1-S D, L D, S D, 1668A/-, 1682A/-, 1899-/C, 1955-/G, 2002-/T, 2101-/AACT;
2) 4 sites of furA gene: S5P, -7G/A, -10A/C, -12G/A;
3) 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;
4) ahpC gene 25 sites: -6G/A, -9G/A, -15C/T, -30C/T, -34T/G, -39C/T, -46-/T, -46A/T, -46A/G, -48G/A, -49T/G, -49T/C, -51G/A, -52C/A, -52C/G, -52C/T, -54C/T, -57C/T, -72C/T, -74G/A, -76T/C, -81C/T, -82-/AT, -86T/A, -88G/A;
5) nat gene 3 sites: T175A, D188H, G207R;
6) ndh gene 8 sites: R13C, L104F, L104P, T110A, G225D, R268H, E360L, E360K;
7) the iniA gene has 4 sites: P3A, H481G, H481Q, R537H;
8) the iniB gene has 11 sites: 180C/-, 198G/-, 222T/-, 686G/-, 725-/T, 750C/-, 878T/-, 1019-/C, 1038G/-, 1056A/-, A222T;
9) the iniC gene has 3 sites: 79-/T, 98-/A, W83G;
10) kasA gene 6 sites: D66N, R121K, G269S, G312S, G387D, F413L;
11) 13 sites of fabG1 gene: 609G/A (L203L), -8T/A, -8T/C, -8T/G, -15C/A, -15C/G, -15C/T, -16A/C, -16A/G, -16A/T, -17G/A, -17G/C, -17G/T;
12) rv0340 gene 1 site: V163I;
13) fadE24 gene 1 site: Q85R;
14) rv1592c gene 1 site: P42L;
15) rv1772 gene 1 site: T4A;
16) fabD gene 1 site: A3T;
17) phoR gene 1 site: P186L;
18) 2 sites of the efpA gene: I73T, E520V;
(3) ethambutol, comprising 75 polymorphic sites of 6 drug-resistant genes, which are respectively:
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, M423N, Q445N, Q497N, E504N, Q853N, a 630N, M1000N, H1002N, D361024, D1024N, N1033N;
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, a 278V;
(4) streptomycin, which comprises 77 polymorphic sites of 3 drug-resistant genes, and is respectively:
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 the gidB 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 '-, G202/-202, 202-/351, G' -, G/-347C '-, C/-347, G/-TCG/-20421, G/-TCG/-347, G/-TCG/-S, G/-347, G/-TCG/-20421, G/-347, C' -, C/-347, G/-347, C/-TCG/-347, C/-102, C/-TCG, C/-347, C/-102, C/-TCG/-347, C/-TCG, C/-237, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, 2, C/-102, C/-TCG, C/-347, C/-102, C/-TCG, C/-347, C/-102, C/-347, C/-102, C/-347, C/-102, C/-347, C/-2, C/-347, C/-102, C/-2, C/-347, C/-102, C/-347, C/-102, C/-2, C/-102;
(5) the moxifloxacin comprises 13 polymorphic sites of 2 drug resistance genes, and the polymorphic sites are respectively as follows:
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;
(6) the ofloxacin comprises 39 polymorphic sites of 2 drug-resistant genes, which are respectively:
1) 16 sites of the gyrA gene: a74S, T80A, N83V, G88C, G88A, D89N, a90V, a90G, S91P, D94A, D94G, D94H, D94N, D94Y, D94V, G668D;
2) the gyrB gene has 23 sites: M291I, R446C, S447Y, D461A, D461H, D461N, G470A, I485C, I486L, D494A, N499D, T500N, E501V, a504T, T511N, Q538D, Q538H, Q538T, Q538K, H539N, I540D, I540V, E592S;
(7) amikacin, which comprises 20 polymorphic sites of 3 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
3) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(8) kanamycin comprises 30 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively:
1) rrs gene 5 sites: 513C/T, 514A/C, 1401A/G, 1402C/T, 1484G/T;
2) 10 loci of the eis gene: -6G/T, -8C/T, -8C/A, -10G/A, -10G/C, -12C/T, -14C/T, -15C/G, -37G/T, -43A/T;
3) the whiB7 gene has 6 sites: 86C/-, 124C/-, 128G/-, 133C/-, 133-/C, 179G/-;
4) 9 sites of the gidB gene: 102G/-, 104T/G, 230T/C, 254A/G, 286C/T, L35R, V77A, D85G, R96C;
(9) the capreomycin comprises 32 polymorphic sites of 3 drug-resistant genes, which are respectively:
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;
(10) pyrazinamide, including 190 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) the panD gene has 14 sites: H21R, I49V, I115T, M117T, 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, 410TGT/-, in a preferred embodiment, the composition is formulated as a pharmaceutical composition, for use in a medicament for treating a disease or condition, for example, a disease or a condition, for example, or a disease or a in a or a condition, for the like, or a condition, or a, or a condition, or a, or, 414-/CATT, 418-/, 419-/G, 422A/-, 423-/AG, 455-/C, 457-/, 458-/C, 471-/A, 480A/-, 494-/C, 523-/A, 525-/A, 525CCGCCA/-, 570-/CT, A3, L4, L4, I5, I6, V7, V7, D8, V9, Q10, D12, C14, S18, G23, I31, Y34, Y41, K48, K48, D49, H51, H51, H51, H51, P54, P54, H57, H57, H57, S59, S59, P62, P62, P62, D63, D63, D63, Y64, S66, S67, W68, W68, W68, W68, W69, P62, P96, G97, G94, G85, G97, G96, G85, G97, G85, G95, and G95, g97, Y99, a102, Y103, S104, G108, L116, W119, L120, R123, D129, G132, I133, a134, T135, D136, H137, C138, V139, R140, Q141, T142, a143, a146, L151, T167, T168, V169, L172, M175, V180, L182, X187;
3) rpsA gene 4 sites: T5S, E67D, D123A, 1313 CCG/-;
(11) the rifabutin comprises 15 polymorphic loci of 1 drug-resistant gene, which are respectively:
1) rpoB gene 15 sites: G95F, P206R, Y314C, H323Y, Q429H, Q432A, Q432E, Q432K, Q432P, H445D, H445R, H445Y, S450F, S450L, S450W;
(12) the ethionamide comprises 66 polymorphic sites of 4 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 56 sites of the ethA gene: -11A/G, -7T/C, M1, V10, G11, H22, Q24, 30G/-, Y32, G43, W45, S57, T61, T88, 107A/-, 110A/-, 157-/T, Q165, M204, L205, Q206, R207, Y211, Q246, W256, S266, P334, 338A/-, 341A/-, a341, D357, S375, P378, 382-/G, S390, V398, L405, Y438, 441-/T, G450, P454, W455, L478, 672-/G, 703/-, 753-/G, 869-/a, 892A/-, G/-, 980T '-, 1175-/CG, 1190-/TGG, 1239T/-, 1242T' -, 1393/-;
2) 1 site of ethR gene: A95T;
3) 3 sites of fabG1 gene: -15C/T, -8T/a, 609G/a;
4) inhA gene 6 sites: -17G/T, -15C/T, -8T/a, I21T, S94A, I194T;
(13) the clofazimine comprises 81 polymorphic sites of 3 drug-resistant genes, and is respectively as follows:
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, V3, N4, D5, E13, V20, 29-/T, T33, A36, G41, W42, L43, C46, Q51, S53, 58G/T, A59, S63, S63, G65, G66, S68, S68, Q76, A84, V85, G87, R89, R90, R90, A102, A102, R105, A110, L114, L117, A118, D119, V120, G121, L122, R123, 125G/A, G126, G126, D127, A128, P130, R134, R135, M139, 141-/GA, D141, M146, N148, V149, L154, R156, G162, D165, 193G/, 193-/G, 198G-/G-202, A-292, G-202, G-C-364, CG/, CG-444;
3) rv1979c gene 1 site: V351A;
(14) the p-aminosalicylic acid comprises 71 polymorphic sites of 4 drug-resistant genes, wherein the polymorphic sites are respectively as follows:
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, a 457X;
3) 1 site of 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, I211R, R222R, P224R, R235R, Y251R, a 259R, V261R, V263R, X263R;
(15) the albuterol hydrochloride comprises 62 polymorphic sites of 2 drug-resistant genes, and the polymorphic sites are respectively as follows:
1) 58 sites of the ethA gene: 11-/A, 21-/C, Q24, 30G/-, 65A/-, 107A/-, W109, 129-/C, 136GATT/-, 138T/-, Y147, 157-/T, Q165, S197, 202-/CC, Q206, 229G/-, 243-/A, Q254, Y276, 298T/-, K309, 312-/G, 338A/-, 382-/G, W391, L405, 412-/A, Y438, 440-/AC, 596C/, 672-/G, 703T '-, 752-/G, 752-/GC, 755-/C, 767G/-, GG 7/-, 773G/, 822G, 767G' -, 752-C, 767G/-, and G, 18, 822-/G, 849-/C, 859A/-, 869-/A, 892A/-, 895GG/-, 908G/-, 925A/-, 980T/-, 1121T/-, 1135A/-, 1175-/CG, 1190-/TGG, 1201T/-, 1239T/-, 1304-/C, 1386A/-, 1404-/C;
2) inhA gene 4 sites: -15A/T, -15C/T, S49A, I194T;
(16) the cycloserine comprises 6 polymorphic sites of 3 drug-resistant genes, which are respectively:
1) 3 sites of alr gene: -26G/T, L113R, D344N;
2) 2 sites of the cycA gene: T236A, V301A;
3) ddlA gene 1 site: L372R;
(17) the bedaquiline comprises 62 polymorphic sites of 5 drug-resistant genes, which are respectively:
1) atpE gene 13 sites: -72T/C, -53G/a, D28A, 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-/G193, 193-, -200T/G, 202A/G, 214C/T, 259-/G-, 345-G, 292-G/;
5) rv1979c gene 1 site: M245L;
(18) linezolid, comprising 10 polymorphic sites of 2 drug-resistant genes, which are respectively:
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;
(19) the delamannich comprises 20 polymorphic sites of 5 drug-resistant genes, which are respectively:
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) fgd1 gene 3 sites: G104S, L270M, L296E;
(20) the PrinMannich total comprises 19 polymorphic sites of 4 drug-resistant genes, which are respectively:
1) ddn gene 7 sites: S11X, W20X, Q58X, W88R, Y136X, Q137X, W139X;
2) fbiA gene 1 site: T146A;
3) fbiC gene 9 sites: G310X, W435X, Q436X, W444X, E637X, K644X, W652X, M709I, S715R;
4) fgd1 gene 2 sites: G71D, E230K.
4. Use of the primer composition of any one of claims 1 to 3 for preparing a kit for detecting drug resistance of Mycobacterium tuberculosis and/or a kit for guiding drug administration of Mycobacterium tuberculosis.
5. A mycobacterium tuberculosis drug resistance detection kit and/or a kit for guiding the drug administration of mycobacterium tuberculosis is characterized in that: the kit comprises the primer composition of any one of claims 1 to 3.
6. The kit of claim 5, wherein: the concentration of each primer in the primer composition is 0.2-0.8. mu.M, preferably 0.5. mu.M.
7. The kit of claim 6, wherein: the kit also comprises a Polymerase mix, a PCR enhanced buffer, an illumina/BGISEQ 5 'end Index sequencing joint and an illumina/BGISEQ3' end Index sequencing joint.
8. Use of the primer composition according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 7 for the detection of a drug resistance gene of mycobacterium tuberculosis.
9. A method for detecting drug-resistant genes of mycobacterium tuberculosis, which is a non-disease diagnosis and treatment method and is characterized in that: the method comprises detecting the site of a drug-resistant gene in the genome of a test sample using the primer composition of any one of claims 1 to 3 or the kit of any one of claims 5 to 7.
10. The method of claim 9, wherein: the method comprises the following steps:
(1) extracting sample DNA;
(2) performing multiplex PCR amplification on the DNA extracted in the step (1) by using the primer composition according to any one of claims 1 to 3 or the kit according to any one of claims 5 to 7, and constructing a second-generation sequencing library;
(3) and performing high-throughput sequencing on the amplicon through a second-generation sequencing platform, determining the specific genotype of the drug-resistant gene locus of the sample, and detecting or identifying the drug resistance of the drug according to the genotype.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111044169.3A CN113817850A (en) | 2021-09-07 | 2021-09-07 | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111044169.3A CN113817850A (en) | 2021-09-07 | 2021-09-07 | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113817850A true CN113817850A (en) | 2021-12-21 |
Family
ID=78922116
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111044169.3A Pending CN113817850A (en) | 2021-09-07 | 2021-09-07 | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113817850A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292935A (en) * | 2022-01-05 | 2022-04-08 | 深圳汉亚生物科技合伙企业(有限合伙) | Nucleic acid composition and kit for detecting drug resistance gene of mycobacterium tuberculosis and method for detecting drug resistance of mycobacterium tuberculosis |
| CN114574606A (en) * | 2022-04-02 | 2022-06-03 | 予果生物科技（北京）有限公司 | Primer group for detecting mycobacterium tuberculosis in metagenome and high-throughput sequencing method |
| CN116356056A (en) * | 2023-04-07 | 2023-06-30 | 江苏先声医学诊断有限公司 | Primer group, product and application for detecting drug-resistant genes of mycobacterium tuberculosis |
| WO2023170395A1 (en) * | 2022-03-08 | 2023-09-14 | Quadram Institute Bioscience | Methods and compositions for drug resistance screening |
| CN117230222A (en) * | 2023-10-18 | 2023-12-15 | 鲲鹏基因（北京）科技有限责任公司 | Composition, kit and method for detecting quinolone resistance of mycobacterium tuberculosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342307A (en) * | 2020-11-09 | 2021-02-09 | 盛世中方(北京)生物科技有限公司 | Mycobacterium tuberculosis drug resistance detection kit and method based on NGS technology |
| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
| CN113330123A (en) * | 2018-11-29 | 2021-08-31 | 苏黎世大学 | Tuberculosis drug resistance prediction method |
-
2021
- 2021-09-07 CN CN202111044169.3A patent/CN113817850A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113330123A (en) * | 2018-11-29 | 2021-08-31 | 苏黎世大学 | Tuberculosis drug resistance prediction method |
| CN112342307A (en) * | 2020-11-09 | 2021-02-09 | 盛世中方(北京)生物科技有限公司 | Mycobacterium tuberculosis drug resistance detection kit and method based on NGS technology |
| CN113249502A (en) * | 2021-05-08 | 2021-08-13 | 上海康黎诊断技术有限公司 | Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection |
Non-Patent Citations (5)
| Title |
|---|
| ABHIRUPA GHOSH ET AL.: "Survey of drug resistance associated gene mutations in Mycobacterium tuberculosis, ESKAPE and other bacterial species", 《SCIENTIFIC REPORTS 》 * |
| LICENCE: CC BY-NC-SA 3.0 IGO: "Catalogue of mutations in Mycobacterium tuberculosis complex and their association with drug resistance", 《GENEVA: WORLD HEALTH ORGANIZATION》 * |
| SUHA KADURA ET AL.: "Systematic review of mutations associated with resistance to the new and repurposed Mycobacterium tuberculosis drugs bedaquiline, clofazimine, linezolid, delamanid and pretomanid", 《J ANTIMICROB CHEMOTHER》 * |
| Y. ZHANG & W-W. YEW: "Mechanisms of drug resistance in Mycobacterium tuberculosis:update 2015", 《INT J TUBERC LUNG DIS》 * |
| 王浩 等: "Sensititre结核药敏板检测结核分枝杆菌药敏的方法学研究", 《河北医科大学学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114292935A (en) * | 2022-01-05 | 2022-04-08 | 深圳汉亚生物科技合伙企业(有限合伙) | Nucleic acid composition and kit for detecting drug resistance gene of mycobacterium tuberculosis and method for detecting drug resistance of mycobacterium tuberculosis |
| WO2023170395A1 (en) * | 2022-03-08 | 2023-09-14 | Quadram Institute Bioscience | Methods and compositions for drug resistance screening |
| CN114574606A (en) * | 2022-04-02 | 2022-06-03 | 予果生物科技（北京）有限公司 | Primer group for detecting mycobacterium tuberculosis in metagenome and high-throughput sequencing method |
| CN116356056A (en) * | 2023-04-07 | 2023-06-30 | 江苏先声医学诊断有限公司 | Primer group, product and application for detecting drug-resistant genes of mycobacterium tuberculosis |
| CN116356056B (en) * | 2023-04-07 | 2023-12-15 | 江苏先声医学诊断有限公司 | Primer group, product and application for detecting drug-resistant genes of mycobacterium tuberculosis |
| CN117230222A (en) * | 2023-10-18 | 2023-12-15 | 鲲鹏基因（北京）科技有限责任公司 | Composition, kit and method for detecting quinolone resistance of mycobacterium tuberculosis |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113817850A (en) | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof | |
| Gao et al. | RT-qPCR based quantitative analysis of gene expression in single bacterial cells | |
| CN110878343B (en) | Cpf1 kit for rapidly detecting genetic deafness pathogenic gene SLC26A4 mutation and detection method thereof | |
| JP6835712B2 (en) | Polymerase Chain Reaction Primers and Probes for Mycobacterium Tuberculosis | |
| JP2013081450A (en) | Probe for detecting polymorphism, method of detecting polymorphism, method of evaluating efficacy of drug, and reagent kit for detecting polymorphism | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| JP6007652B2 (en) | Primers and probes for detecting methicillin-resistant Staphylococcus aureus, and methods for detecting methicillin-resistant Staphylococcus aureus using them | |
| JP2009039106A (en) | Oligonucleotide for species identification of acid-fast bacterium and use thereof | |
| CN115852002A (en) | Primer composition for detecting mycobacterium tuberculosis drug-resistant gene and application thereof | |
| EP1992703B1 (en) | Method for detection of mutant gene | |
| US20080176220A1 (en) | Method, Kit and System for Enhanced Nested Pcr | |
| CN106957904A (en) | A kind of PCR fluorescent molecular bacon probes for detecting drug-induced deafness | |
| Ciervo et al. | Rapid detection and differentiation of Bartonella spp. by a single-run real-time PCR | |
| US20220136046A1 (en) | Detection and antibiotic resistance profiling of microorganisms | |
| Grillova et al. | Core genome sequencing and genotyping of Leptospira interrogans in clinical samples by target capture sequencing | |
| Kooti et al. | Modified gold nanoparticle colorimetric probe-based biosensor coupled with allele-specific PCR for rapid detection of G944C mutation associated with isoniazid resistance | |
| CN113930500A (en) | Digital PCR (polymerase chain reaction) detection method for human PIK3CA gene mutation and application | |
| CN112662748B (en) | Primer pair combination, method and kit for single cell DNA sequencing library quality identification | |
| KR20230057778A (en) | Method and Kit for Diagnosing Bovine tuberculosis using PNA | |
| JP6205216B2 (en) | Mutation detection probe, mutation detection method, efficacy determination method, and mutation detection kit | |
| Glennon et al. | Detection and diagnosis of mycobacterial pathogens using PCR | |
| CN113604589A (en) | Kit for simultaneously detecting drug-resistant sites, virulence genotyping and proton pump inhibitor metabolic genotyping of helicobacter pylori | |
| CN112080556A (en) | Method for performing multiple rapid sequencing on gonococcus drug-resistant gene | |
| US20030077631A1 (en) | Materials and methods for detection and characterization of nucleic acid sequence variability | |
| CN105316350B (en) | Mycobacterium tuberculosis EmbB mutators and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |