CN113249502A

CN113249502A - Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection

Info

Publication number: CN113249502A
Application number: CN202110500898.9A
Authority: CN
Inventors: 何熲; 石忆湘; 黄成琛; 谢海波; 杨军; 魏宁; 王保曼; 张辉
Original assignee: Shanghai Kangli Diagnostic Technology Co ltd
Current assignee: Shanghai Kangli Diagnostic Technology Co ltd
Priority date: 2021-05-08
Filing date: 2021-05-08
Publication date: 2021-08-13

Abstract

The invention provides a related gene, a method, a primer group and a kit for identification and drug resistance detection of mycobacterium tuberculosis complex flora. According to the invention, the mycobacterium tuberculosis complex flora can be identified, and the high-resolution differential detection can be carried out on 16 common primary and secondary drug resistance gene mutations of the mycobacterium tuberculosis complex flora, so as to realize rapid, simultaneous and accurate drug resistance detection.

Description

Related gene, method, primer group and kit for mycobacterium tuberculosis complex flora identification and drug resistance detection

Technical Field

The invention relates to pathogenic microorganism detection, in particular to a related gene, a method, a primer group and a kit for mycobacterium tuberculosis complex flora identification and drug resistance detection.

Background

Mycobacterium tuberculosis is the most important factor causing various tuberculosis (mainly pulmonary tuberculosis). Although epidemiological data indicate that tuberculosis is prevalent in large areas, the effective rate of treatment for tuberculosis is only 55%, and drug resistance is an important cause for this. In 2017, 5.6% of tuberculosis patients in China are multi-drug resistant tuberculosis (MDR-TB) patients, and 8.5% of the patients are estimated to be wide drug resistant tuberculosis (XDR-TB). Therefore, the identification of the mycobacterium tuberculosis and the drug sensitivity detection thereof have important public health significance.

Since there are very small genomic differences from each other, for example, m.bovis (m.bovis) and m.tuberculosis (m.tuberculosis) are distinguished by ANI (mean nucleotide identity) with less than 1%, m.bovis and m.tuberculosis, together with 6 other mycobacteria (m.africanum, m.bovis BCG, m.canetti, m.caprae, m.microti, m.pinnipedii) are called mycobacterium tuberculosis complex (MTBC). Wherein, M.bovis and M.bovis BCG are resistant to pyrazinamide naturally, and have important clinical significance for identifying and distinguishing the pyrazinamide.

The detection of the drug resistance of the mycobacterium tuberculosis also has important clinical significance. The current detection method cannot completely meet clinical requirements, for example, the smear method has low sensitivity, the culture method consumes long time, the commercialized product has high cost, the operation requirement is complex and other adverse factors limit the detection efficiency of germs in clinical practice, the diagnosis confirming time is prolonged, and the effective treatment of patients is not facilitated.

Disclosure of Invention

The invention aims to provide a primer group and a kit for identifying mycobacterium tuberculosis complex flora and detecting drug resistance by using flight time mass spectrum, thereby solving the problems that the detection method for mycobacterium tuberculosis in the prior art has the defects of low sensitivity, long period, high cost and narrow detection range, and cannot simultaneously identify the mycobacterium tuberculosis complex flora and detect the drug resistance.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to the first aspect of the invention, the related gene for identifying the mycobacterium tuberculosis complex flora is provided, and comprises the following gene sites and characteristic bases:

gene locus	Characteristic base
		TB81	T
mce3B	C
		TB10	G
TB78	G
		TB61	G

；

According to a second aspect of the present invention, there is provided a gene related to drug resistance detection of mycobacterium tuberculosis, the gene related to drug resistance detection of mycobacterium tuberculosis comprises the following drugs, detection genes and detection mutations:

1) rifampicin

2) Isoniazid

Serial number	Detection of genes	Detecting sites or mutations
			1	katG	S315G
	katG	S315R
				katG	S315T
	katG	S315N
				katG	S315I
2	katG	G316S
			3	inhA	-15
	inhA	-102
				inhA	-17
	inhA	S94A
				inhA	-8
4	mshA	A187V

3) Pyrazinamides

Serial number	Detection of genes	Detecting sites or mutations
			1	pncA	H57D

4) Ethambutol

Serial number	Detection of genes	Detecting sites or mutations
			1	embB	M306V
	embB	M306L
				embB	M306I
2	embB	G406D

5) Fluoroquinolones

6) Streptomycin

7) Ethionamide

8) Propylthioisonicotinamine

9) Para-aminosalicylic acid sodium salt

Serial number	Detection of genes	Detecting sites or mutations
			1	folC	I43T
2	thyA	T202A
			3	thyA	H75N

10) Amikacin

Serial number	Detection of genes	Detecting sites or mutations
			1	rrs	A1401G
2	rrs	G1484T

11) Kanamycin

12) Capreomycin

13) Cyclic serine

Serial number	Detection of genes	Detecting sites or mutations
			1	alr	S261N

14) Clofazimine

Serial number	Detection of genes	Detecting sites or mutations
			1	rv0678	G193DEL
2	rv0678	C466T

15) Bedaquinoline

16) Benefit toNazolamide

Serial number	Detection of genes	Detecting sites or mutations
			1	rplC	T460C

According to a third aspect of the present invention there is provided a method of complex bacterial flora identification of mycobacterium tuberculosis for non-disease diagnostic purposes, the method comprising detecting the identity of characteristic bases in relevant genetic loci identified by complex bacterial flora of mycobacterium tuberculosis, and if so determining the presence of a corresponding complex bacterial flora of mycobacterium tuberculosis:

according to a fourth aspect of the present invention, there is provided a method for the detection of drug resistance in mycobacterium tuberculosis for non-disease diagnostic purposes, the method further comprising detecting the presence or absence of the following sensitive, drug-resistant nucleic acid mutations in the mycobacterium tuberculosis drug resistance-associated genetic loci as follows:

1) rifampicin

2) Isoniazid

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	katG	S315G	AGC>GGC
	katG	S315R	AGC>AGG
					katG	S315T	AGC>ACC
	katG	S315N	AGC>AAC
					katG	S315I	AGC>ATC
2	katG	G316S	GGC>AGC
				3	inhA	-15	C>T
	inhA	-102	G>T
					inhA	-17	C>T
	inhA	S94A	TCG>GCG
					inhA	-8	T>A
4	mshA	A187V	GCA>GTA

3) Pyrazinamides

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	pncA	H57D	CAC>GAC

4) Ethambutol

5) Fluoroquinolones

6) Streptomycin

7) Ethionamide

8) Propylthioisonicotinamine

9) Para-aminosalicylic acid sodium salt

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	folC	I43T	ATC>ACC
2	thyA	T202A	ACC>GCC
				3	thyA	H75N	CAC>AAC

10) Amikacin

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	rrs	A1401G	A>G
2	rrs	G1484T	G>T

11) Kanamycin

12) Capreomycin

13) Cyclic serine

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	alr	S261N	AGC>AAC

14) Clofazimine

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	rv0678	G193DEL	G>-
2	rv0678	C466T	C>T

15) Bedaquinoline

16) Linezolid

Serial number	Detection of genes	Detecting sites or mutations	Sensitive drug-resistant nucleic acid mutation
				1	rplC	T460C	T>C

Wherein, when any one of the detection sites is mutated, the drug resistance of the drug corresponding to the detection gene can be determined.

According to a fifth aspect of the present invention, a method for simultaneously performing identification of Mycobacterium tuberculosis complex flora and detection of drug resistance is provided.

The method enables simultaneous detection of resistance to 16 drugs, including: rifampin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, ethionamide, prothiocyanamide, sodium para-aminosalicylate, amikacin, kanamycin, capreomycin, cycloserine, clofazimine, bedaquiline, linezolid.

According to the method provided by the invention, the identification of the Mycobacterium tuberculosis complex flora and the detection of drug resistance are simultaneously realized by using the flight time mass spectrum.

According to the sixth aspect and the seventh aspect of the present invention, a primer set for mycobacterium tuberculosis complex flora identification and drug resistance detection and a kit comprising the same are provided, the primer set comprises a drug resistance detection primer set and a mycobacterium tuberculosis complex flora identification primer set, and the drug resistance detection primer set comprises the following gene variation sites and primers:

1.rpoB_S522_2F:

amplification primer pair:

F(SEQ ID NO：1)：ACGTTGGATG TCACACCGCCTACCATCAT

R(SEQ ID NO：2)：ACGTTGGATGATTCTCAGCTGACAGGTCG

the concentration range of the primers (concentration of the PCR primers in the reaction system, the same below) is 0.1-0.3. mu.M each

Extension primer (SEQ ID NO: 3): AATACCTCAACAAGCCGCAGT (final concentration of primer 8.0-10. mu.M)

2.rpoB_H526-1F：

F(SEQ ID NO：4)：ACGTTGGATGTCACACCGCCTACCATCAT

R (SEQ ID NO: 5): ACGTTGGATGATTCTCAGCTGACAGGTCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 6): AACTGTCCGGCTTGAGG (7.0 to 9.0 μ M)

3.Rpob_S531_2F

F(SEQ ID NO：7)：ACGTTGGATGTCACACCGCCTACCATCAT

R (SEQ ID NO: 8): ACGTTGGATGATTCTCAGCTGACAGGTCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 9): CAGAAGCGGCCACTCT (4.0 to 6.0 μ M)

4.rpob511R

F(SEQ ID NO：10)：ACGTTGGATGTCACACCGCCTACCATCAT

R (SEQ ID NO: 11): ACGTTGGATGATTCTCAGCTGACAGGTCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 12): GGGTCCATGAATTGCCTG (6.0 to 8.0 μ M)

5.rpoB_F505_1F

F(SEQ ID NO：13)：ACGTTGGATGTCACACCGCCTACCATCAT

R (SEQ ID NO: 14): ACGTTGGATGATTCTCAGCTGACAGGTCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 15): TTATTG GTCGCGGCGATCAAGCAG (6.5 to 8.0 μ M)

6.Rpob_Q513_2F

F(SEQ ID NO：16)：ACGTTGGATGTCACACCGCCTACCATCAT

R (SEQ ID NO: 17): ACGTTGGATGATTCTCAGCTGACAGGTCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 18): TTTTTACACCAGCCAGCAGACGC (5.0 to 7.0 μ M)

7.rpoB_Q513_1F

F(SEQ ID NO：19)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 20): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 21): ACCAGCCAGCAGTGC (6.0 to 8.0 μ M)

8.rpoB_Q513_5R

F(SEQ ID NO：22)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 23): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 24): TTGTTCTGGTCCGATAA (7.0 to 9.0 μ M)

9.rpoB_D516_4R

F(SEQ ID NO：25)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 26): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 27): AGCGGGTTGTGTCTG (10.0 to 12.0 μ M)

10.rpoB_S522_5R_1126E

F(SEQ ID NO：28)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 29): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 30): ACTTGTGGGTCCCAAC (8.0 to 10.0 μ M)

11.rpoB_D516_1F

F(SEQ ID NO：31)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 32): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 33): GCTGAGCCAATATTCG (6.0 to 7.0 μ M)

12.rpoB_S531_1F_1126E

F(SEQ ID NO：34)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 35): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 36): GTTGACCCACAAGCGCTCAGCG (4.0 to 6.0 μ M)

13.rpoB_L533_4R

F(SEQ ID NO：37)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 38): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 39): CAGACCGCCGCCCGGC (6.0 to 7.0 μ M)

14.rpoB_S522_1F

F(SEQ ID NO：40)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 41): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 42): ACCAGAACAACCTGCCG (8.0 to 9.0 μ M)

15.rpoB_H526_4R

F(SEQ ID NO：43)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 44): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 45): AACTACAGTCGGCTTCGG (6.0 to 8.0 μ M)

16.rpoB_D516_5R_1126E

F(SEQ ID NO：46)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 47): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 48): AACAGCGGGTTCGTTTG (9.0 to 11.0 μ M)

17.rpoB_S512_2F

F(SEQ ID NO：49)：ACGTTGGATGGATCAAGGAGTTCTCTGCGA

R (SEQ ID NO: 50): ACGTTGGATGACGCTCACGTGCAAGCACG (0.8 to 1.2 μ M each)

Extension primer (SEQ ID NO: 51): TCTTCGGCACCAGCCATGGCA (5.0 to 7.0 μ M)

18.rpoB_D626E

F(SEQ ID NO：52)：ACGTTGGATGTAGCGACGAGATCGTCATGC

R (SEQ ID NO: 53): ACGTTGGATGCATGTAGTCCACCTCAGAGACG (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 54): AATTCGCCGATCGCGTAGGA (7.0 to 8.0 μ M)

19.rpoB_D516_2F

F(SEQ ID NO：55)：ACGTTGGATGGATCAAGGAGTTCTTGCCGA

R (SEQ ID NO: 56): ACGTTGGATGACGCTCACGTGACACCAGG (0.6 to 1.0 μ M each)

Extension primer (SEQ ID NO: 57): TGAGCCAATTTGCAG (6.0 to 7.0 μ M)

20.katG316-1

F(SEQ ID NO：58)：ACGTTGGATGCTGTTGTCCCATTCTGTCGG

R (SEQ ID NO: 59): ACGTTGGATGTGGAAGAGCTCGTGTGACAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 60): TCCATACGACCTAGTCGC (7.0 to 8.0 μ M)

21.katG_S315_5R_1126E

F(SEQ ID NO：61)：ACGTTGGATGCTGTTGTCCCATTCTGTCGG

R (SEQ ID NO: 62): ACGTTGGATGTGGAAGAGCTCGTGTGACAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 63): TGTTCGTCCATACGACCTTAGGCCC (8.0 to 10.0 μ M)

22.katG_S315_1F

F(SEQ ID NO：64)：ACGTTGGATGCTGTTGTCCCATTCTGTCGG

R (SEQ ID NO: 65): ACGTTGGATGTGGAAGAGCTCGTGTGACAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 66): AAGGACGCGTAACCC (9.0 to 10.0 μ M)

23.inhA_t-8a

F(SEQ ID NO：67)：ACGTTGGATGACTGAACGGGATAAGCTAGG

R (SEQ ID NO: 68): ACGTTGGATGCTCGTGGACATACTGCTGTC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 69): TTGGCAGTCACCACGCCA (4.0 to 6.0 μ M)

24.inhA g-102t

F(SEQ ID NO：70)：ACGTTGGATGCCTCGCTGCCCAGAGAGAGA

R (SEQ ID NO: 71): ACGTTGGATGGAAATCGGTATGTCGACCAG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 72): TCACACCGACAAACGCTACGA (4.0 to 6.0 μ M)

25.inhA_c-17t

F(SEQ ID NO：73)：ACGTTGGATGACTGAACGGGATACTAGAGG

R (SEQ ID NO: 74): ACGTTGGATGCTCGTGGACATACTCATGTC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 75): CCCCGACAACCTGTACT (4.0 to 6.0 μ M)

26.pncA-57

F(SEQ ID NO：76)：ACGTTGGATGACGAGGAATAGTGTCGCGTG

R (SEQ ID NO: 77): ACGTTGGATGCAACCAAGGACTAATCCCTC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 78): AGGTGTGCCGAAGGAGT (7.0 to 8.0 μ M)

27.embB306-3

F(SEQ ID NO：79)：ACGTTGGATGACCAGCGGAAATAGGTGATC

R (SEQ ID NO: 80): ACGTTGGATGAATTCGTCGGACGAGCGTCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 81): GGTGGGTCGGCGACGTGCGC (8.0 to 9.0 μ M)

28.embB-497

F(SEQ ID NO：82)：ACGTTGGATGTTCTCGGTATACCATCCGCG

R (SEQ ID NO: 83): ACGTTGGATGTCATCCTGACCGTGTGTGTC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 84): CCAACACCGTTGACGAGTGC (8.0 to 9.0 μ M)

29.embB-406

F(SEQ ID NO：85)：ACGTTGGATGTCGATCAGCACATTGAGGC

R (SEQ ID NO: 86): ACGTTGGATGCGTGGATGCCGTACCATAAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 87): GCTAGCCGAGCGCGTGTAAG (8.0 to 9.0 μ M)

30.embB306-1

F(SEQ ID NO：88)：ACGTTGGATG TGATATTCGGCTTCCGCTTC

R (SEQ ID NO: 89): ACGTTGGATG AGCGCCAGCAGGTTATATGA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 90): GGACGAGGCTACATCGTGCGC (9.0 to 10.0 μ M)

31.gyrA90_CT__1126E

F(SEQ ID NO：91)：ACGTTGGATGAGACCATGGGCAACCACATC

R (SEQ ID NO: 92): ACGTTGGATGTCCACCAGCGGGTCGCGAA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 93): TAACCACCCGCAGCGACGCG (6.0 to 7.0 μ M)

32.gyrA94_AT

F(SEQ ID NO：94)：ACGTTGGATGAGACCATGGGCAACTCAACC

R (SEQ ID NO: 95): ACGTTGGATGTCCACCAGCGGGTGAGCCA (0.1 to 0.3 μ M each)

Extension primers (SEQ ID NO: 96 or 229): ATGCGCACCGAGTGG or ATGGGCACCGAGCGG (5.0 to 7.0 μ M)

33.gyrA91

F(SEQ ID NO：97)：ACGTTGGATGGCGGCAAGATCATCTATAGG

R (SEQ ID NO: 98): ACGTTGGATGCGTGATGATCGCCTAGTCAT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 99): CGCACCAGGGTGTCGTACGTAG (5.0 to 7.0 μ M)

34.gyrA89

F(SEQ ID NO：100)：ACGTTGGATGAGACCATGGGCAACCAATCC

R (SEQ ID NO: 101): ACGTTGGATGTCCACCAGCGGGTCGCGAA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 102): CAGGCTGTCGTAGATCGCACGGT (5.0 to 7.0 μ M)

35.gyrA_G88_2F

F(SEQ ID NO：103)：ACGTTGGATGGCGGCAAGATCATCATATGG

R (SEQ ID NO: 104): ACGTTGGATGCGTGATGATCGCCTGTACAT (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 105): AAGCAACTACCACCCCACGG (5.0 to 7.0 μ M)

36.gyrA_G88C_1126E

F(SEQ ID NO：106)：ACGTTGGATGGCGGCAAGATCATCATGTAG

R (SEQ ID NO: 107): ACGTTGGATGCGTGATGATCGCCTGTAACT (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 108): ACAACTACCACCACCGC (5.0 to 7.0 μ M)

37.gyrA_D94_3R-1

F(SEQ ID NO：109)：ACGTTGGATGAGACCATGGGCAACTCAACC

R (SEQ ID NO: 110): ACGTTGGATGTCCACCAGCGGGTGAGCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 111): AAG GCCATGCGCACCGAAGGTT (6.0 to 8.0 μ M)

38.gyrA_D94_3R-2

F(SEQ ID NO：112)：ACGTTGGATGAGACCATGGGCAACTCAACC

R (SEQ ID NO: 113): ACGTTGGATGTCCACCAGCGGGTGAGCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 114): AAGGGCATGCGCACCGTCGAGT (6.0 to 8.0 μ M)

39.gyrB-180

F(SEQ ID NO：115)：ACGTTGGATG GCGGCAAGATCATCTATAGG

R (SEQ ID NO: 116): ACGTTGGATGCGTGATGATCGCCTAGTCAT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 117): GACCGGGTGAATACG (5.0 to 7.0 μ M)

40.gyrB539

F(SEQ ID NO：118)：ACGTTGGATGGCGGCAAGATCATCTATAGG

R (SEQ ID NO: 119): ACGTTGGATGCGTGATGATCGCCTAGTCAT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 120): TGATCGCCTGACATCTG (5.0 to 7.0 μ M)

41.gyrB_N538R_4R

F(SEQ ID NO：121)：ACGTTGGATGGGCAAGATCATCAGATGTGA

R (SEQ ID NO: 122): ACGTTGGATGATCTTGTGGTAGCGACTCGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 123): GTGATGATCGCCTGAACTCTGTGG (5.0 to 7.0 μ M)

42.gyrB_A543T

F(SEQ ID NO：124)：ACGTTGGATGGGCAAGATCATCAGATGTGA

R (SEQ ID NO: 125): ACGTTGGATGATCTTGTGGTAGCGACTCGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 126): GTGCTAAAGAACACCGAATGACTG (5.0 to 7.0 μ M)

43.gyrB_A543V

F(SEQ ID NO：127)：ACGTTGGATGGGCAAGATCATCAGATGTGA

R (SEQ ID NO: 128): ACGTTGGATGATCTTGTGGTAGCGACTCGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 129): GCCCAGCGCCGTGAGTTAC (5.0 to 7.0 μ M)

44.gyrB_G551R

F(SEQ ID NO：130)：ACGTTGGATG GGCAAGATCATCAGATGTGA

R (SEQ ID NO: 131): ACGTTGGATG ATCTTGTGGTAGCGACTCGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 132): CTATCGAACTCGTCGTGAGCTC (6.0 to 8.0 μ M)

45.gyrB498

F(SEQ ID NO：133)：ACGTTGGATGCGCAAGTCCGAACTTGGATT

R (SEQ ID NO: 134): ACGTTGGATGCGCTTTCTCCACATGTGATA (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 135): CAAGTCCGAACTGTATGCTTGA (5.0 to 7.0 μ M)

46.gyrB509

F(SEQ ID NO：136)：ACGTTGGATG CGCAAGTCCGAACTTGGATT

R (SEQ ID NO: 137): ACGTTGGATG CGCTTTCTCCACATGTGATA (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 138): GCCATGGAACATCGAATCCGAGC (7.0 to 9.0 μ M)

47.gyrB500-3

F(SEQ ID NO：139)：ACGTTGGATG CGCAAGTCCGAACTTGGATT

R (SEQ ID NO: 140): ACGTTGGATG CGCTTTCTCCACATGTGATA (0.3 to 0.5 μ M each)

Extension primer (SEQ ID NO: 141): TTAGAACCGCCGGCGCGAT (6.0 to 8.0 μ M)

48.rpsL-88

F(SEQ ID NO：142)：ACGTTGGATGGATGATCTTGTAGCCAGACC

R (SEQ ID NO: 143): ACGTTGGATGAACCTGCAGGAGCATCACGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 144): ATCGCACACCAGGCATGCGC (7.0 to 9.0 μ M)

49.rpsl_T40_2F

F(SEQ ID NO：145)：ACGTTGGATGGATGATCTTGTAGCCAGACC

R (SEQ ID NO: 146): ACGTTGGATGAACCTGCAGGAGCATCACGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 147): GTATGCACCCGCGTGTCACCAA (6.0 to 8.0 μ M)

50.rpsL_K43_4R

F(SEQ ID NO：148)：ACGTTGGATGGATGATCTTGTAGCCAGACC

R (SEQ ID NO: 149): ACGTTGGATGAACCTGCAGGAGCATCACGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 150): TTTCCGAAGCGCCGAGTTCTTCGGC (5.0 to 7.0 μ M)

51.folC-43

F(SEQ ID NO：151)：ACGTTGGATGTGAGCATCTACTCCAAGACC

R (SEQ ID NO: 152): ACGTTGGATGTCCATCAACGCGATGCTCC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 153): GGGTCAGGCTCATCGGG (6.0 to 8.0 μ M)

52.thyA-202

F(SEQ ID NO：154)：ACGTTGGATGTTGCTCACCCACAGGTAGTC

R (SEQ ID NO: 155): ACGTTGGATGTTGTCGTAGATGTAGTGGCC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 156): CGGTCGGCGAGTTCTGCATG (7.0 to 9.0 μ M)

53.tlyA_N236K

F(SEQ ID NO：157)：ACGTTGGATGAGTTTGAGGTGGGGGAGAAT

R (SEQ ID NO: 158): ACGTTGGATGAATCCCTTGGCAAGACCT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 159): ACAGGAAGTCGAACTC (10.0 to 11.0 μ M)

54.rrs-G1484T_2

F(SEQ ID NO：160)：ACGTTGGATGCCGGTACGGCTACTTTTGCA

R (SEQ ID NO: 161): ACGTTGGATGAAAGTCGGTAACACACAGCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 162): AAGGTGGGATCGGCGATGTGAGC (7.0 to 9.0 μ M)

55.rrs_a514ct

F(SEQ ID NO：163)：ACGTTGGATG TCGGGTTGTAAACCTTCTCTA

R (SEQ ID NO: 164): ACGTTGGATGCGAGCTCTTTACGAGTCCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 165): AAGCACCGGCCAACTACGCAGTGCC (5.0 to 7.0 μ M)

56.rrs_c517t

F(SEQ ID NO：166)：ACGTTGGATGTCGGGTTGTAAACCTTCTCTA

R (SEQ ID NO: 167): ACGTTGGATGCGAGCTCTTTACGAGTCCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 168): CTCGCACCCTACGTACCGTTAC (8.0 to 10.0 μ M)

57.rrs-A1401G

F(SEQ ID NO：169)：ACGTTGGATGTTCGGGTGTTACCTTCGATC

R (SEQ ID NO: 170): ACGTTGGATGTAATCGCAGATCAACGGCAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 171): TGTTACCGACTTTCAACTGG (7.0 to 9.0 μ M)

58.rrs_c513t_1126E

F(SEQ ID NO：172)：ACGTTGGATG TCGGGTTGTAAACTTTCCTCA

R (SEQ ID NO: 173): ACGTTGGATGCGAGCTCTTTACGAGTCCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 174): AAGCACCGGCCAACTACGTCAGCG (5.0 to 7.0 μ M)

59.rrs_c462t

F(SEQ ID NO：175)：ACGTTGGATG TCGGGTTGTAAACTTTCCTCA

R (SEQ ID NO: 176): ACGTTGGATGCGAGCTCTTTACGAGTCCCA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 177): AACGACGAAGGTGGTCCGT (8.0 to 10.0 μ M)

60.rrl-A2059G

F(SEQ ID NO：178)：ACGTTGGATGAAGCTATAGTGAACCCGGTG

R (SEQ ID NO: 179): ACGTTGGATGACTCGGCGAAATTCTAGCAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 180): TATATTGAAGGTCCCGTCGGGT (8.0 to 10.0 μ M)

61.rrl-A2058G

F(SEQ ID NO：181)：ACGTTGGATGAAGCTATAGTGACCCTGGAG

R (SEQ ID NO: 182): ACGTTGGATGACTCGGCGAAATTCTAGCAC (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 183): GTGAAGGTCCCGTCTGGGT (7.0 to 9.0 μ M)

62.alr-261_E3

F(SEQ ID NO：184)：ACGTTGGATGACCTGACGTTCGAGGTTCCG

R (SEQ ID NO: 185): ACGTTGGATGAGCACATTTCACGCTACTGG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 186): ATCGCGGTGTATGTGAGCTA (5.0 to 7.0 μ M)

63.gidB_E92D

F(SEQ ID NO：187)：ACGTTGGATGACCGGCATCTACTCTGGAAC

R (SEQ ID NO: 188): ACGTTGGATGACGCCCAGATCCACTGTC (0.5 to 0.7 μ M each)

Extension primer (SEQ ID NO: 189): TGC CGA CCA GGT AGT TCTAGTCCA (6.0 to 8.0 μ M)

64.rv0678-C466T

F(SEQ ID NO：190)：ACGTTGGATGTCAGTCGTCCTCTGTTCCGC

R (SEQ ID NO: 191): ACGTTGGATGGGATCTGTTGGCAATATGTG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 192): CCCCTTCGCTGGTATCTGC (7.0 to 9.0 μ M)

65.rv0678-G193D

F(SEQ ID NO：193)：ACGTTGGATGCCAAATTGGATCATCCGCAG

R (SEQ ID NO: 194): ACGTTGGATGTGCTGGTGTGTGACGATCCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 195): TGGTGCTCCCGATCC (5.0 to 7.0 μ M)

66.Erm-C28T

F(SEQ ID NO：196)：ACGTTGGATGGTCATCAGTGAGCTACGGAC

R (SEQ ID NO: 197): ACGTTGGATGCGATATCTTTGGATGGGCAG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 198): AGCTGCGGATACCCCCAGC (7.0 to 9.0 μ M)

67.rplC-T460C_E2

F(SEQ ID NO：199)：ACGTTGGATGTTTCGCCGGCACGAACATG

R (SEQ ID NO: 200): ACGTTGGATGATCCGGGTGCCCAACTTGA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 201): TTATTTGGGCTCCATGCGGCGA (7.0 to 9.0 μ M)

68.eis_g-10a_1126E

F(SEQ ID NO：202)：ACGTTGGATGCGTGATCCTTTGCACACAGC

R (SEQ ID NO: 203): ACGTTGGATGGAAATCGGTGAAAGCCCTGG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 204): AGGGTCACAGTCACAGCCGAATA (5.0 to 7.0 μ M)

69.eis-c14t_1126E

F(SEQ ID NO：205)：ACGTTGGATGCGTGATCCTTTGCCAGACAC

R (SEQ ID NO: 206): ACGTTGGATGGAAATCGGTGAAACTGGCCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 207): GATTCTGTGACTGCCCTGAT (5.0 to 7.0 μ M)

70.eis_c-2a

F(SEQ ID NO：208)：ACGTTGGATGCGTGATCCTTTGCCAGACAC

R (SEQ ID NO: 209): ACGTTGGATGGAAATCGGTGAAACTGGCCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 210): TACACAGGGTCACCACAGTA (5.0 to 7.0 μ M)

71.eis_c-12t

F(SEQ ID NO：211)：ACGTTGGATGCGTGATCCTTTGCCAGACAC

R (SEQ ID NO: 212): ACGTTGGATGGAAATCGGTGAAACTGGCCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 213): TGGCCGCGGCAGCCTATA (5.0 to 7.0 μ M)

72.eis-g37t_1126E

F(SEQ ID NO：214)：ACGTTGGATGCGTGATCCTTTGCCAGACAC

R (SEQ ID NO: 215): ACGTTGGATGGAAATCGGTGAAACTGGCCG (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 216): CCTGTCGTCGTAATCATATC (5.0-7.0 μ M);

the primer group for identifying the mycobacterium tuberculosis complex flora comprises the following gene variation sites and primers:

73.TB10

F(SEQ ID NO：217)：ACGTTGGATGAAGCAGCCAATAAGCAGAAGC

r (SEQ ID NO: 218): ACGTTGGATGAGCCCATTTGCGAGGACA (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 219): GACGAATATTCGTGCCCAGG (6.0 to 8.0 μ M)

74.TB78

F(SEQ ID NO：220)：ACGTTGGATGCGGCAACAGATTTGGCGAA

R (SEQ ID NO: 221): ACGTTGGATGCGCTCCGAACAACGCGGCTAT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 222): TTAGTGCAGCCAACGCCGCGGC (5.0 to 7.0 μ M)

75.TB81

F(SEQ ID NO：223)：ACGTTGGATGCCTGCTGCACTCCATCTAC

R (SEQ ID NO: 224): ACGTTGGATGCGTCGAGTACCCGATCATAT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 225): CCCCGACGCCGAAGTTTCAG (5.0 to 7.0 μ M)

76.mce3B

F(SEQ ID NO：226)：ACGTTGGATGTTCCTGCTGATTGTCTCCGT

R (SEQ ID NO: 227): ACGTTGGATGTCGGGGTTGATGGTCCAGGT (0.1 to 0.3 μ M each)

Extension primer (SEQ ID NO: 228): CCCAACGTGTCTCTCAAG (7.0 to 9.0 μ M)

77.TB61

F(SEQ ID NO:230)：ACGTTGGATGGCCTACCTCGGGGTTTGTC

R(SEQ ID NO:231)：ACGTTGGATGGGTCCAAGGTTCGTGCTC

Extension primer (SEQ ID NO: 232): TTGTCACCGACGCCTCACGGCGCA

Extension primer (SEQ ID NO: 233): GATCGCGTCGAGGACGCTAAGGGT are provided.

The following description will be made by taking the amino acid 516 position of rpoB gene (encoding RNA polymerase beta subunit) as an example:

first, the site numbering explains:

ATCGA

12345

it will be appreciated that an amino acid is encoded by a letter consisting of three nucleotides (bases) and that if the three letters, as in the example above, are thickened TCG, position 1 refers to the base (A) preceding the first base (T) encoding the amino acid, T itself is position 2, and so on, and position 5 is the base (A) downstream of the third base (G).

Primer sequence rpoB _ D516_ 1F: represents a forward (5 '- - > 3') extension primer designed by using the base which is one bit (namely the 1 st position) before the first nucleotide of the 516 th (the amino acid coded by the site is D in the wild case) amino acid of the RNA polymerase beta subunit as a termination site of the extension primer. In primer nomenclature, letters around the numbers representing positions such as 516 indicate amino acids in the case of upper case and nucleotides in the case of lower case.

rpoB _ D516_ 3R: represents a reverse (3 '- - - > 5') extension primer designed by using the second nucleotide 516 (i.e. 3 position) base of the beta subunit amino acid of RNA polymerase as a termination site of the extension primer.

This is also the D516 site, and possible variants thereof include the following 7:

D516Y (GAC- - > TAC, corresponding amino acid from aspartic acid to tyrosine)

D516F (GAC- - > TTC, corresponding amino acid from aspartic acid to phenylalanine)

D516V (GAC- - > GTC, corresponding amino acid from aspartic acid to valine)

D516G (GAC- - > GGC, corresponding amino acid from aspartic acid to glycine)

D516A (GAC- - > GCC, corresponding amino acid from aspartic acid to alanine)

D516A (GAC- - > GCG, corresponding amino acid from aspartic acid to alanine)

D516E (GAC- - > GAG, corresponding to the amino acid change from aspartic acid to glutamic acid).

To distinguish the 7 cases, 4 different extension primers (rpoB _ D516_1F, rpoB _ D516_2F, rpoB _ D516_3R and rpoB _ D516_5R) can be used, with respective forward and reverse amplification primers (which may be the same). In order to avoid interference between different extended primers, it may be necessary to place them in different wells of MassArray for practical use. For example, the latter 5 variants can be distinguished by using rpoB _ D516_2F and rpoB _ D516_5R and placed in the same well (W4), while the former two variants need to be distinguished by combining the results of rpoB _ D516_1F and rpoB _ D516_3R, so that the two primers need to be placed in separate wells (e.g., W2 and W3) from one another and from W4.

It should be understood that not all amino acid positions have a high variation frequency of 3 nucleotide positions, and some amino acid mutations are relatively simple, and a single nucleotide position mutation is detected.

It should be noted that the above sequences may have redundancies, e.g., the forward and reverse amplification primer pairs for gene loci No. 1-6 are identical, but the extension primers are different, general principle: it is unlikely that two sites will have three identical pairs of sequences.

The drug resistance detection primer group can realize simultaneous detection of drug resistance of 16 drugs, wherein the 16 drugs comprise: rifampin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, ethionamide, prothiocyanamide, sodium para-aminosalicylate, amikacin, kanamycin, capreomycin, cycloserine, clofazimine, bedaquiline, linezolid.

The invention utilizes Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry), adopts a method combined with PCR on a MassARRAY platform, identifies whether a Mycobacterium tuberculosis complex flora exists in a clinical sample, simultaneously detects the drug-resistant nucleic acid mutation of the Mycobacterium tuberculosis complex flora on a therapeutic drug, determines the drug-resistant condition of the Mycobacterium tuberculosis in the sample, and assists in clinically selecting an effective therapeutic scheme.

The MassARRAY system is a non-fluorescent detection platform that uses mass spectrometry to accurately measure PCR amplification products. The combination of mass spectrometry and end-point PCR allows for highly multiplexed reactions under universal cycling conditions to provide accurate, rapid, and cost-effective analysis. MassARRAY is a detection method based on MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectrum. This system uses PCR to amplify a gene fragment containing each SNP, in which an extension primer anneals near polymorphic bases, and the last base is complementary to the SNPs to be detected, so that the extension reaction of this fragment is terminated when the reaction is amplified to this base. Since the last base has different mass, the mass difference between different gene fragments can be detected by mass spectrometry, and the SNPs existing in the fragment can be calculated.

The pulsed ultraviolet laser ionizes the product, and the high voltage electrostatic field accelerates the ionized DNA molecules from the bottom to the top of the vacuum tube. Lighter DNA molecules may impinge on the detector at the top of the detector tube earlier because lighter ions move faster in the electric field. After each laser pulse emission, the detector records the relative time of flight of each analyte, so that the mass of the DNA fragment can be calculated and the specific type of SNPs present at the polymorphic site on the detected fragment determined. Since the entire emission of laser light to signal detection takes only a few milliseconds, up to 384 samples can be analyzed in less than 50 minutes. The MassARRAY system provides a unique solution for targeted gene detection with limited sample additions. Compared with the whole genome association analysis (GWAS), the MassARRAY system has stronger self-defining function and can accurately analyze limited SNPs with important significance, thereby improving the accuracy and the timeliness.

The invention overcomes the defects of low sensitivity, long period, high cost and narrow detection range of the current common detection method, and is purposefully developed and optimized in the aspects. The invention can be used for clinical treatment reference and epidemiological research.

The important application of the invention is that clinical samples (sputum, blood, urine, lavage fluid, cerebrospinal fluid and the like) or cultures (Loewenstein-Jensen culture medium or MGIT liquid culture medium) from human bodies can be adopted for patients with tuberculosis or other tuberculosis, and the following applications can be realized: a) determining the presence of a Mycobacterium tuberculosis complex in the lung or other tissue of the patient; b) the drug resistance of the mycobacterium tuberculosis is determined simultaneously, and comprises 16 kinds of first-line and second-line drugs: rifampin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones (ofloxacin, levofloxacin), streptomycin, ethionamide, prothiocyanamide, sodium para-aminosalicylate, amikacin, kanamycin, capreomycin, cycloserine, clofazimine, bedaquiline, linezolid.

The detection utilizes a MALDI-TOF MS combined PCR method to detect the tubercle bacillus gene mutation corresponding to different drugs, thereby identifying the drug resistance of the antituberculous drugs of specific patients. For example, rifampicin resistance is associated with site mutation of RNA polymerase beta subunit gene (rpoB), isoniazid resistance is associated with mutation of Catalase encoding gene (Catalase peroxidase, katG) and enoyl-acyl carrier protein reductase gene promoter gene (inhA-15). Except for the first-line drugs (4 drugs such as rifampicin, isoniazid, ethambutol, pyrazinamide and the like), the drug resistance of the tubercle bacillus to the second-line drugs can also be detected, and the detection can be used for detecting the drug resistance of 16 (types of) antitubercular drugs, so that the rapid, simultaneous and accurate drug resistance detection is realized.

In summary, according to the primer set and the kit for identifying the mycobacterium tuberculosis complex flora and detecting the drug resistance by using the time-of-flight mass spectrometry, provided by the invention, clinical samples (sputum, blood, urine, lavage fluid, cerebrospinal fluid and the like) or cultures (Loewenstein-Jensen culture medium or MGIT liquid culture medium) from human bodies are adopted, so that the high-resolution differential detection can be carried out on 16 common drug resistance gene mutations of first-line and second-line anti-tuberculosis drugs of the mycobacterium tuberculosis complex flora while identifying the mycobacterium tuberculosis complex flora, the rapid, simultaneous and accurate drug resistance detection is realized, and powerful assistance is further provided for clinical medication decision.

Detailed Description

The present invention will be further described with reference to the following specific examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

1.1 principle of examination

The detection method adopted by the invention is nucleic acid flight mass spectrometry. Firstly, a target sequence is amplified through PCR, then a SNP sequence specific extension primer is added, and 1 base is extended on the SNP site. The prepared sample analyte and a chip matrix are co-crystallized and then are excited by strong laser in a vacuum tube of a mass spectrometer, nucleic acid molecules are desorbed into singly charged ions, the ion flight time in an electric field is inversely proportional to the ion mass, and the accurate molecular weight of the sample analyte is obtained by detecting the flight time of the nucleic acid molecules in the vacuum tube, so that the SNP site information is detected.

1.2 major ingredients

10×PCR buffer；MgCl₂A solution; a dNTP solution; HS Taq; 3-Pt Calibrant; iPLex Buffer Plus-10 ×; iPLex Termination Mix; iPLex Enzyme; SAP Buffer; SAP Enzyme; a primer; ddH₂O; a positive control; drying the resin; and a chip board.

1.3 matching instruments

A general PCR instrument; massarray Nanodispenser model RS1000(Agena Bioscience); mass spectrometer Massarray Analyser Fourier, model Massarray Analyser Fourier System 96/24genotyping (agena bioscience).

1.4 sample requirement

The product is suitable for extracting Mycobacterium tuberculosis genome DNA from clinical samples or cultures such as sputum, blood, urine, lavage fluid, cerebrospinal fluid, etc., and the required ratio of DNA A260/A280 should be between 1.8 and 2.0. Frozen DNA samples should be stored below-20 ℃ and repeated freezing and thawing is avoided.

1.5 test methods

1.5.1PCR reaction

1) In PCR I area, each reagent (kit) was taken out from a-20 ℃ freezer, thawed on ice (4 ℃), and the amplification primers were taken out from a 4 ℃ freezer, vortexed, shaken for 10s, and centrifuged briefly for use.

2) Adding related reagent components in sequence according to the table 1 to prepare a PCR reaction mixed solution, marking, and subpackaging into a 96-well plate with 3 mu L/well; after packaging, the DNA is transferred from PCR I to PCR II through the transfer window. The composition of the PCR mixture is shown in Table 1.

Table 1: PCR mixture

3) In PCR II region, DNA template was removed from refrigerator at-20 deg.C, thawed on ice (4 deg.C), vortexed for 10s, centrifuged briefly, and a certain amount of DNA was aspirated and diluted to 5 ng/. mu.l for use.

4) Adding 2 muL of 5 ng/muL DNA template into each well of a 96-well plate, covering a tube cover, performing vortex oscillation for 10s, centrifuging briefly, transferring from a PCR II area to a PCR III area through a transfer window, and transferring from the PCR III area to a PCR IV area through the transfer window, wherein a blank control (2 muL ddH) must be set in each experiment₂O), negative control (2 μ LDNA extraction eluate) and positive control.

5) The 96-well plate was placed in the amplification apparatus, and the program was run: pcr, the specific procedure is as follows:

1.5.2 SAP reaction

After the PCR reaction was completed, an SAP mixture was prepared in a 1.5mL EP tube according to Table 2. The numbers in table 2 are calculated as a 96 well plate plus a 38% excess. This configuration was performed in PCR I.

Table 2: SAP reaction mixture

1) The prepared SAP mixture was transferred from PCR zone i to zone iv, and 2 μ L of SAP mixture was added to each well (total volume after addition of mixture: 7 μ L).

2) The plates were sealed with a membrane (Life's or other company quality membrane), vortexed and centrifuged (4000rpm for 5 seconds).

3) Place the plate on a PCR instrument for the following procedures:

the temperature of the mixture is controlled to be 37 ℃ for 40 minutes,

the temperature of the mixture is 85 ℃ for 5 minutes,

keeping the temperature at 4 ℃.

1.5.3 extension reaction

1) The SAP reaction plate was removed and centrifuged at 2000rpm for 1 min.

2) An iPLEX extension reaction was prepared in a 1.5mL tube according to Table 3. The numbers in table 3 are calculated as one 96-well plate plus a 38% excess. Please adjust the number according to the actual number of responses. This configuration was performed in PCR I.

Table 3: iPLEX extension reaction liquid

3) The iPLEX extension mix was transferred from PCR zone I to zone IV and 2. mu.L of iPLEX extension mix was added to each well and mixed (total volume after addition of mix: 9 μ L).

4) The plates were sealed with a membrane, vortexed and centrifuged (4000rpm for 5 seconds).

5) The 96-well plate was placed on a PCR instrument for the following thermal cycling:

1.5.4 Conditioning (sample desalting)

The following procedure was set for one 96-well plate, please adjust the procedure based on the actual number of wells. Wear gloves and goggles.

1) Clean Resin (Resin) was spread flat on 96/15mg of sample plate and air dried for a minimum of 10 minutes.

Note that: resin is firstly paved on a plate by a spoon, then the resin is scraped from left to right or from right to left by a scraper, so that 96 holes are filled with the resin, and after the 96 holes are filled, the resin is lightly scraped by the scraper and the residual resin on the surface is scraped off, so that the next step of film pasting is prevented from being interfered. When the resin changed from dark yellow to light yellow, it was shown that the resin had dried almost completely.

2) To each well of the sample plate, 41. mu.L of water was added, and the membrane was sealed (using a common membrane), followed by centrifugation.

3) Add 15mg of clean Resin (Resin): the sample plate was gently inverted in the air and placed on the resin-loaded sample plate with a hole! The sample plate is then inverted (the plates cannot move horizontally in the process) to allow the resin to fall into the wells.

4) The plates were sealed with a membrane (using a common membrane) and placed on a rotator and shaken upside down for 15 minutes.

5) Plates were centrifuged for 5 minutes at 3200g (4000rpm of standard plate centrifuge).

1.5.5 spotting

The PCR product was transferred to the chip plate according to the Massarray Nanodispenser protocol.

1.5.6 Mass Spectrometry

1) The chip plate was removed from the spotting instrument.

2) The chip plate was transferred to a Massarray Analyser fountain chip tray with the side of the chip printed with text facing outward using tweezers, and the chip was squeezed in the lower left direction using tweezers.

3) Flight mass spectrometry detection and result readout were performed according to Massarray Analyser Four protocol.

1.6 interpretation of test results

1) And (3) judging the effectiveness of the kit: the standard substance can be used for detecting corresponding genotype and blank reference substance (ddH)₂O) no signal is detected, when the weak positive control can detect the corresponding positive signal, the detection result is valid, otherwise, the detection result is invalid.

1.7 limitations of the test method

1) The method may be affected by the quality of the detected sample DNA, and if the quality of the detected sample DNA is poor, a false negative result may occur.

2) The detection result is only used for clinical medication reference, is used for guiding individualized medication, and cannot be used as the only basis of clinical medication.

3) When the genotype of the corresponding site detected by the product is sensitive, the product cannot exclude the mutation of other sites of the gene.

Case one

1. The characteristics of the cases are as follows:

1.1 Shen somewhere, male, 69 years old, farmer, found tuberculosis infection one week admission, with a history of "type II diabetes".

1.2 admission examination results: clear mind, slight fatigue, stable respiration, no cyanosis of lips, no superficial lymph node swelling, centered trachea, normal heart auscultation, no deformity of thorax, no increase and decrease of intercostal space, thick respiratory sounds of two lungs, no dry and wet gong sounds heard, soft abdomen, no tenderness and no back pain. The lower limbs of the liver and spleen are not in the reach, turbid yin is moved, the lower limbs are not swollen, physiological reflex exists, and pathological reflex is not led out.

1.3 auxiliary detection: drug resistance and rapid drug resistance tests for isonicotinic acid, rifampicin, streptomycin and ethambutol.

2. The basis of diagnosis is as follows: patients, elderly men, hospitalized for "finding multidrug resistant tuberculosis infection", physical examination: superficial lymph nodes are not swollen and the respiratory sounds of both lungs are coarse and the sounds are not heard. Mycobacterium tuberculosis drug resistance and rapid drug resistance tests for isoniazid, rifampin, streptomycin and ethambutol.

3. Differential diagnosis: the diagnosis of the patient's multidrug resistance is definite.

4. And (3) diagnosis: the lung tuberculosis of two lung groups is primarily treated by daubing yang to resist multidrug, II type diabetes.

5. The diagnosis and treatment scheme is as follows:

5.1 to perfect the clear diagnosis and illness state of the relevant necessary examination (bronchofiberscope, hearing, visual field, etc.);

5.2 treatment with a drug-resistant regimen was given after exclusion of contraindications.

6. Results of Gene detection

6.1 results of detection of Mycobacterium

6.2 first-line drug resistance Gene mutation test results (TB)

6.3 second line drug resistance Gene mutation test results (TB)

Case two

1. The characteristics of the cases are as follows: old a certain one, male, age 67, farmer.

2. Admission diagnosis: secondary tuberculosis of two lungs, primary treatment, positive smear detection, arrhythmia, complete right bundle branch block, primary hyperthyroidism Graves disease, thyroid nodule, leukopenia and thrombocytopenia.

3. At present, diagnosis is as follows: secondary tuberculosis of two lungs, primary treatment, positive smear detection, isoniazid resistance, diabetes, arrhythmia, complete right bundle branch block, left front branch block, primary hyperthyroidism Graves disease, thyroid nodule, leukopenia and thrombocytopenia.

4. The current disease condition is as follows: patients complain of cough and expectoration unobvious, slight abdominal discomfort, feeling of lower limb pain, no nausea and vomiting, no intolerance of cold and fever, no chest distress and shortness of breath, physical examination: shenqing, steady respiration, no cyanosis of lips, no irritability of jugular vein, no protrusion of precordial region, no excessive tremor, and large heart boundary under percussion.

Arrhythmia, unhealthy smell and marked noise. The lung is clear in breathing sound, and the wet gong sound is not heard. The abdomen is flat and soft, no tenderness exists, and edema does not exist on the lower limbs.

Possible accidents and risks: 1. the condition of the patient is aggravated, and complications such as hemoptysis, pneumothorax, pleural effusion and the like occur, even hemoptysis appears, a blood suspected block blocks a respiratory tract, and death is caused by asphyxia. 2. Exacerbation of the disease, exacerbation of pulmonary infections, and even respiratory failure, leading to multiple organ failure and even death. 3. For example, tuberculosis infection, tuberculosis spread, and other organ tissue tuberculosis, such as tuberculous meningitis, digestive tract tuberculosis, urinary system tuberculosis, etc. 4. Drug reaction and infusion reaction occur in the treatment process, and liver and kidney function damage, fever, rash and the like caused by antituberculosis drugs, even anaphylactic shock threatens life. 5. Patients are older, and unexpected accidents such as pulmonary embolism, cardiovascular and cerebrovascular accidents, cerebral hemorrhage, cerebral infarction and the like occur in the treatment process, so that the life is threatened. 6. There may be drug resistance of other drugs, which is a multi-drug resistant tuberculosis infection. 7. The patients have more basic diseases and poor tuberculosis prognosis.

5. The precautionary measures are as follows: and perfecting the clear test of the medicine. 2. Take care of rest, strengthen nutrition, avoid smoking and drinking. 3. During the hospitalization period, the examination and treatment are matched with medical staff. 4. Leaving the hospital during hospitalization must not be done without permission. 5. The progress and deterioration of the disease may occur during the hospitalization period, and the patient may be considered to be transferred to the higher-level hospital for continuous treatment.

6. Results of Gene detection

6.1 results of detection of Mycobacterium

6.2 first-line drug resistance Gene mutation test results (TB)

6.3 second line drug resistance Gene mutation test results (TB)

The above embodiments are merely preferred embodiments of the present invention, which are not intended to limit the scope of the present invention, and various changes may be made in the above embodiments of the present invention. All simple and equivalent changes and modifications made according to the claims and the content of the specification of the present application fall within the scope of the claims of the present patent application. The invention has not been described in detail in order to avoid obscuring the invention.

SEQUENCE LISTING

<110> Shanghai Kangli diagnostic technology Co., Ltd

<120> related gene, method, primer group and kit for identification and drug resistance detection of mycobacterium tuberculosis complex flora

<160> 233

<170> PatentIn version 3.5

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acgttggatg tcacaccgcc taccatcat 29

<210> 11

<211> 29

<212> DNA

<213> Artificial sequence

<400> 11

acgttggatg attctcagct gacaggtcg 29

<210> 12

<211> 18

<212> DNA

<213> Artificial sequence

<400> 12

gggtccatga attgcctg 18

<210> 13

<211> 29

<212> DNA

<213> Artificial sequence

<400> 13

acgttggatg tcacaccgcc taccatcat 29

<210> 14

<211> 29

<212> DNA

<213> Artificial sequence

<400> 14

acgttggatg attctcagct gacaggtcg 29

<210> 15

<211> 24

<212> DNA

<213> Artificial sequence

<400> 15

ttattggtcg cggcgatcaa gcag 24

<210> 16

<211> 29

<212> DNA

<213> Artificial sequence

<400> 16

acgttggatg tcacaccgcc taccatcat 29

<210> 17

<211> 29

<212> DNA

<213> Artificial sequence

<400> 17

acgttggatg attctcagct gacaggtcg 29

<210> 18

<211> 23

<212> DNA

<213> Artificial sequence

<400> 18

tttttacacc agccagcaga cgc 23

<210> 19

<211> 30

<212> DNA

<213> Artificial sequence

<400> 19

acgttggatg gatcaaggag ttctctgcga 30

<210> 20

<211> 29

<212> DNA

<213> Artificial sequence

<400> 20

acgttggatg acgctcacgt gcaagcacg 29

<210> 21

<211> 15

<212> DNA

<213> Artificial sequence

<400> 21

accagccagc agtgc 15

<210> 22

<211> 30

<212> DNA

<213> Artificial sequence

<400> 22

acgttggatg gatcaaggag ttctctgcga 30

<210> 23

<211> 29

<212> DNA

<213> Artificial sequence

<400> 23

acgttggatg acgctcacgt gcaagcacg 29

<210> 24

<211> 17

<212> DNA

<213> Artificial sequence

<400> 24

ttgttctggt ccgataa 17

<210> 25

<211> 30

<212> DNA

<213> Artificial sequence

<400> 25

acgttggatg gatcaaggag ttctctgcga 30

<210> 26

<211> 29

<212> DNA

<213> Artificial sequence

<400> 26

acgttggatg acgctcacgt gcaagcacg 29

<210> 27

<211> 15

<212> DNA

<213> Artificial sequence

<400> 27

agcgggttgt gtctg 15

<210> 28

<211> 30

<212> DNA

<213> Artificial sequence

<400> 28

acgttggatg gatcaaggag ttctctgcga 30

<210> 29

<211> 29

<212> DNA

<213> Artificial sequence

<400> 29

acgttggatg acgctcacgt gcaagcacg 29

<210> 30

<211> 16

<212> DNA

<213> Artificial sequence

<400> 30

acttgtgggt cccaac 16

<210> 31

<211> 30

<212> DNA

<213> Artificial sequence

<400> 31

acgttggatg gatcaaggag ttctctgcga 30

<210> 32

<211> 29

<212> DNA

<213> Artificial sequence

<400> 32

acgttggatg acgctcacgt gcaagcacg 29

<210> 33

<211> 16

<212> DNA

<213> Artificial sequence

<400> 33

gctgagccaa tattcg 16

<210> 34

<211> 30

<212> DNA

<213> Artificial sequence

<400> 34

acgttggatg gatcaaggag ttctctgcga 30

<210> 35

<211> 29

<212> DNA

<213> Artificial sequence

<400> 35

acgttggatg acgctcacgt gcaagcacg 29

<210> 36

<211> 22

<212> DNA

<213> Artificial sequence

<400> 36

gttgacccac aagcgctcag cg 22

<210> 37

<211> 30

<212> DNA

<213> Artificial sequence

<400> 37

acgttggatg gatcaaggag ttctctgcga 30

<210> 38

<211> 29

<212> DNA

<213> Artificial sequence

<400> 38

acgttggatg acgctcacgt gcaagcacg 29

<210> 39

<211> 16

<212> DNA

<213> Artificial sequence

<400> 39

cagaccgccg cccggc 16

<210> 40

<211> 30

<212> DNA

<213> Artificial sequence

<400> 40

acgttggatg gatcaaggag ttctctgcga 30

<210> 41

<211> 29

<212> DNA

<213> Artificial sequence

<400> 41

acgttggatg acgctcacgt gcaagcacg 29

<210> 42

<211> 17

<212> DNA

<213> Artificial sequence

<400> 42

accagaacaa cctgccg 17

<210> 43

<211> 30

<212> DNA

<213> Artificial sequence

<400> 43

acgttggatg gatcaaggag ttctctgcga 30

<210> 44

<211> 29

<212> DNA

<213> Artificial sequence

<400> 44

acgttggatg acgctcacgt gcaagcacg 29

<210> 45

<211> 18

<212> DNA

<213> Artificial sequence

<400> 45

aactacagtc ggcttcgg 18

<210> 46

<211> 30

<212> DNA

<213> Artificial sequence

<400> 46

acgttggatg gatcaaggag ttctctgcga 30

<210> 47

<211> 29

<212> DNA

<213> Artificial sequence

<400> 47

acgttggatg acgctcacgt gcaagcacg 29

<210> 48

<211> 17

<212> DNA

<213> Artificial sequence

<400> 48

aacagcgggt tcgtttg 17

<210> 49

<211> 30

<212> DNA

<213> Artificial sequence

<400> 49

acgttggatg gatcaaggag ttctctgcga 30

<210> 50

<211> 29

<212> DNA

<213> Artificial sequence

<400> 50

acgttggatg acgctcacgt gcaagcacg 29

<210> 51

<211> 21

<212> DNA

<213> Artificial sequence

<400> 51

tcttcggcac cagccatggc a 21

<210> 52

<211> 30

<212> DNA

<213> Artificial sequence

<400> 52

acgttggatg tagcgacgag atcgtcatgc 30

<210> 53

<211> 32

<212> DNA

<213> Artificial sequence

<400> 53

acgttggatg catgtagtcc acctcagaga cg 32

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence

<400> 54

aattcgccga tcgcgtagga 20

<210> 55

<211> 30

<212> DNA

<213> Artificial sequence

<400> 55

acgttggatg gatcaaggag ttcttgccga 30

<210> 56

<211> 29

<212> DNA

<213> Artificial sequence

<400> 56

acgttggatg acgctcacgt gacaccagg 29

<210> 57

<211> 15

<212> DNA

<213> Artificial sequence

<400> 57

tgagccaatt tgcag 15

<210> 58

<211> 30

<212> DNA

<213> Artificial sequence

<400> 58

acgttggatg ctgttgtccc attctgtcgg 30

<210> 59

<211> 30

<212> DNA

<213> Artificial sequence

<400> 59

acgttggatg tggaagagct cgtgtgacac 30

<210> 60

<211> 18

<212> DNA

<213> Artificial sequence

<400> 60

tccatacgac ctagtcgc 18

<210> 61

<211> 30

<212> DNA

<213> Artificial sequence

<400> 61

acgttggatg ctgttgtccc attctgtcgg 30

<210> 62

<211> 30

<212> DNA

<213> Artificial sequence

<400> 62

acgttggatg tggaagagct cgtgtgacac 30

<210> 63

<211> 25

<212> DNA

<213> Artificial sequence

<400> 63

tgttcgtcca tacgacctta ggccc 25

<210> 64

<211> 30

<212> DNA

<213> Artificial sequence

<400> 64

acgttggatg ctgttgtccc attctgtcgg 30

<210> 65

<211> 30

<212> DNA

<213> Artificial sequence

<400> 65

acgttggatg tggaagagct cgtgtgacac 30

<210> 66

<211> 15

<212> DNA

<213> Artificial sequence

<400> 66

aaggacgcgt aaccc 15

<210> 67

<211> 30

<212> DNA

<213> Artificial sequence

<400> 67

acgttggatg actgaacggg ataagctagg 30

<210> 68

<211> 30

<212> DNA

<213> Artificial sequence

<400> 68

acgttggatg ctcgtggaca tactgctgtc 30

<210> 69

<211> 18

<212> DNA

<213> Artificial sequence

<400> 69

ttggcagtca ccacgcca 18

<210> 70

<211> 30

<212> DNA

<213> Artificial sequence

<400> 70

acgttggatg cctcgctgcc cagagagaga 30

<210> 71

<211> 30

<212> DNA

<213> Artificial sequence

<400> 71

acgttggatg gaaatcggta tgtcgaccag 30

<210> 72

<211> 21

<212> DNA

<213> Artificial sequence

<400> 72

tcacaccgac aaacgctacg a 21

<210> 73

<211> 30

<212> DNA

<213> Artificial sequence

<400> 73

acgttggatg actgaacggg atactagagg 30

<210> 74

<211> 30

<212> DNA

<213> Artificial sequence

<400> 74

acgttggatg ctcgtggaca tactcatgtc 30

<210> 75

<211> 17

<212> DNA

<213> Artificial sequence

<400> 75

ccccgacaac ctgtact 17

<210> 76

<211> 30

<212> DNA

<213> Artificial sequence

<400> 76

acgttggatg acgaggaata gtgtcgcgtg 30

<210> 77

<211> 30

<212> DNA

<213> Artificial sequence

<400> 77

acgttggatg caaccaagga ctaatccctc 30

<210> 78

<211> 17

<212> DNA

<213> Artificial sequence

<400> 78

aggtgtgccg aaggagt 17

<210> 79

<211> 30

<212> DNA

<213> Artificial sequence

<400> 79

acgttggatg accagcggaa ataggtgatc 30

<210> 80

<211> 30

<212> DNA

<213> Artificial sequence

<400> 80

acgttggatg aattcgtcgg acgagcgtca 30

<210> 81

<211> 20

<212> DNA

<213> Artificial sequence

<400> 81

ggtgggtcgg cgacgtgcgc 20

<210> 82

<211> 30

<212> DNA

<213> Artificial sequence

<400> 82

acgttggatg ttctcggtat accatccgcg 30

<210> 83

<211> 30

<212> DNA

<213> Artificial sequence

<400> 83

acgttggatg tcatcctgac cgtgtgtgtc 30

<210> 84

<211> 20

<212> DNA

<213> Artificial sequence

<400> 84

ccaacaccgt tgacgagtgc 20

<210> 85

<211> 29

<212> DNA

<213> Artificial sequence

<400> 85

acgttggatg tcgatcagca cattgaggc 29

<210> 86

<211> 30

<212> DNA

<213> Artificial sequence

<400> 86

acgttggatg cgtggatgcc gtaccataac 30

<210> 87

<211> 20

<212> DNA

<213> Artificial sequence

<400> 87

gctagccgag cgcgtgtaag 20

<210> 88

<211> 30

<212> DNA

<213> Artificial sequence

<400> 88

acgttggatg tgatattcgg cttccgcttc 30

<210> 89

<211> 30

<212> DNA

<213> Artificial sequence

<400> 89

acgttggatg agcgccagca ggttatatga 30

<210> 90

<211> 21

<212> DNA

<213> Artificial sequence

<400> 90

ggacgaggct acatcgtgcg c 21

<210> 91

<211> 30

<212> DNA

<213> Artificial sequence

<400> 91

acgttggatg agaccatggg caaccacatc 30

<210> 92

<211> 29

<212> DNA

<213> Artificial sequence

<400> 92

acgttggatg tccaccagcg ggtcgcgaa 29

<210> 93

<211> 20

<212> DNA

<213> Artificial sequence

<400> 93

taaccacccg cagcgacgcg 20

<210> 94

<211> 30

<212> DNA

<213> Artificial sequence

<400> 94

acgttggatg agaccatggg caactcaacc 30

<210> 95

<211> 29

<212> DNA

<213> Artificial sequence

<400> 95

acgttggatg tccaccagcg ggtgagcca 29

<210> 96

<211> 15

<212> DNA

<213> Artificial sequence

<400> 96

atgcgcaccg agtgg 15

<210> 97

<211> 30

<212> DNA

<213> Artificial sequence

<400> 97

acgttggatg gcggcaagat catctatagg 30

<210> 98

<211> 30

<212> DNA

<213> Artificial sequence

<400> 98

acgttggatg cgtgatgatc gcctagtcat 30

<210> 99

<211> 22

<212> DNA

<213> Artificial sequence

<400> 99

cgcaccaggg tgtcgtacgt ag 22

<210> 100

<211> 30

<212> DNA

<213> Artificial sequence

<400> 100

acgttggatg agaccatggg caaccaatcc 30

<210> 101

<211> 29

<212> DNA

<213> Artificial sequence

<400> 101

acgttggatg tccaccagcg ggtcgcgaa 29

<210> 102

<211> 23

<212> DNA

<213> Artificial sequence

<400> 102

caggctgtcg tagatcgcac ggt 23

<210> 103

<211> 30

<212> DNA

<213> Artificial sequence

<400> 103

acgttggatg gcggcaagat catcatatgg 30

<210> 104

<211> 30

<212> DNA

<213> Artificial sequence

<400> 104

acgttggatg cgtgatgatc gcctgtacat 30

<210> 105

<211> 20

<212> DNA

<213> Artificial sequence

<400> 105

aagcaactac caccccacgg 20

<210> 106

<211> 30

<212> DNA

<213> Artificial sequence

<400> 106

acgttggatg gcggcaagat catcatgtag 30

<210> 107

<211> 30

<212> DNA

<213> Artificial sequence

<400> 107

acgttggatg cgtgatgatc gcctgtaact 30

<210> 108

<211> 17

<212> DNA

<213> Artificial sequence

<400> 108

acaactacca ccaccgc 17

<210> 109

<211> 30

<212> DNA

<213> Artificial sequence

<400> 109

acgttggatg agaccatggg caactcaacc 30

<210> 110

<211> 29

<212> DNA

<213> Artificial sequence

<400> 110

acgttggatg tccaccagcg ggtgagcca 29

<210> 111

<211> 22

<212> DNA

<213> Artificial sequence

<400> 111

aaggccatgc gcaccgaagg tt 22

<210> 112

<211> 30

<212> DNA

<213> Artificial sequence

<400> 112

acgttggatg agaccatggg caactcaacc 30

<210> 113

<211> 29

<212> DNA

<213> Artificial sequence

<400> 113

acgttggatg tccaccagcg ggtgagcca 29

<210> 114

<211> 22

<212> DNA

<213> Artificial sequence

<400> 114

aagggcatgc gcaccgtcga gt 22

<210> 115

<211> 30

<212> DNA

<213> Artificial sequence

<400> 115

acgttggatg gcggcaagat catctatagg 30

<210> 116

<211> 30

<212> DNA

<213> Artificial sequence

<400> 116

acgttggatg cgtgatgatc gcctagtcat 30

<210> 117

<211> 15

<212> DNA

<213> Artificial sequence

<400> 117

gaccgggtga atacg 15

<210> 118

<211> 30

<212> DNA

<213> Artificial sequence

<400> 118

acgttggatg gcggcaagat catctatagg 30

<210> 119

<211> 30

<212> DNA

<213> Artificial sequence

<400> 119

acgttggatg cgtgatgatc gcctagtcat 30

<210> 120

<211> 17

<212> DNA

<213> Artificial sequence

<400> 120

tgatcgcctg acatctg 17

<210> 121

<211> 30

<212> DNA

<213> Artificial sequence

<400> 121

acgttggatg ggcaagatca tcagatgtga 30

<210> 122

<211> 30

<212> DNA

<213> Artificial sequence

<400> 122

acgttggatg atcttgtggt agcgactcgt 30

<210> 123

<211> 24

<212> DNA

<213> Artificial sequence

<400> 123

gtgatgatcg cctgaactct gtgg 24

<210> 124

<211> 30

<212> DNA

<213> Artificial sequence

<400> 124

acgttggatg ggcaagatca tcagatgtga 30

<210> 125

<211> 30

<212> DNA

<213> Artificial sequence

<400> 125

acgttggatg atcttgtggt agcgactcgt 30

<210> 126

<211> 24

<212> DNA

<213> Artificial sequence

<400> 126

gtgctaaaga acaccgaatg actg 24

<210> 127

<211> 30

<212> DNA

<213> Artificial sequence

<400> 127

acgttggatg ggcaagatca tcagatgtga 30

<210> 128

<211> 30

<212> DNA

<213> Artificial sequence

<400> 128

acgttggatg atcttgtggt agcgactcgt 30

<210> 129

<211> 19

<212> DNA

<213> Artificial sequence

<400> 129

gcccagcgcc gtgagttac 19

<210> 130

<211> 30

<212> DNA

<213> Artificial sequence

<400> 130

acgttggatg ggcaagatca tcagatgtga 30

<210> 131

<211> 30

<212> DNA

<213> Artificial sequence

<400> 131

acgttggatg atcttgtggt agcgactcgt 30

<210> 132

<211> 22

<212> DNA

<213> Artificial sequence

<400> 132

ctatcgaact cgtcgtgagc tc 22

<210> 133

<211> 30

<212> DNA

<213> Artificial sequence

<400> 133

acgttggatg cgcaagtccg aacttggatt 30

<210> 134

<211> 30

<212> DNA

<213> Artificial sequence

<400> 134

acgttggatg cgctttctcc acatgtgata 30

<210> 135

<211> 22

<212> DNA

<213> Artificial sequence

<400> 135

caagtccgaa ctgtatgctt ga 22

<210> 136

<211> 30

<212> DNA

<213> Artificial sequence

<400> 136

acgttggatg cgcaagtccg aacttggatt 30

<210> 137

<211> 30

<212> DNA

<213> Artificial sequence

<400> 137

acgttggatg cgctttctcc acatgtgata 30

<210> 138

<211> 23

<212> DNA

<213> Artificial sequence

<400> 138

gccatggaac atcgaatccg agc 23

<210> 139

<211> 30

<212> DNA

<213> Artificial sequence

<400> 139

acgttggatg cgcaagtccg aacttggatt 30

<210> 140

<211> 30

<212> DNA

<213> Artificial sequence

<400> 140

acgttggatg cgctttctcc acatgtgata 30

<210> 141

<211> 19

<212> DNA

<213> Artificial sequence

<400> 141

ttagaaccgc cggcgcgat 19

<210> 142

<211> 30

<212> DNA

<213> Artificial sequence

<400> 142

acgttggatg gatgatcttg tagccagacc 30

<210> 143

<211> 30

<212> DNA

<213> Artificial sequence

<400> 143

acgttggatg aacctgcagg agcatcacgt 30

<210> 144

<211> 20

<212> DNA

<213> Artificial sequence

<400> 144

atcgcacacc aggcatgcgc 20

<210> 145

<211> 30

<212> DNA

<213> Artificial sequence

<400> 145

acgttggatg gatgatcttg tagccagacc 30

<210> 146

<211> 30

<212> DNA

<213> Artificial sequence

<400> 146

acgttggatg aacctgcagg agcatcacgt 30

<210> 147

<211> 22

<212> DNA

<213> Artificial sequence

<400> 147

gtatgcaccc gcgtgtcacc aa 22

<210> 148

<211> 30

<212> DNA

<213> Artificial sequence

<400> 148

acgttggatg gatgatcttg tagccagacc 30

<210> 149

<211> 30

<212> DNA

<213> Artificial sequence

<400> 149

acgttggatg aacctgcagg agcatcacgt 30

<210> 150

<211> 25

<212> DNA

<213> Artificial sequence

<400> 150

tttccgaagc gccgagttct tcggc 25

<210> 151

<211> 30

<212> DNA

<213> Artificial sequence

<400> 151

acgttggatg tgagcatcta ctccaagacc 30

<210> 152

<211> 29

<212> DNA

<213> Artificial sequence

<400> 152

acgttggatg tccatcaacg cgatgctcc 29

<210> 153

<211> 17

<212> DNA

<213> Artificial sequence

<400> 153

gggtcaggct catcggg 17

<210> 154

<211> 30

<212> DNA

<213> Artificial sequence

<400> 154

acgttggatg ttgctcaccc acaggtagtc 30

<210> 155

<211> 30

<212> DNA

<213> Artificial sequence

<400> 155

acgttggatg ttgtcgtaga tgtagtggcc 30

<210> 156

<211> 20

<212> DNA

<213> Artificial sequence

<400> 156

cggtcggcga gttctgcatg 20

<210> 157

<211> 30

<212> DNA

<213> Artificial sequence

<400> 157

acgttggatg agtttgaggt gggggagaat 30

<210> 158

<211> 28

<212> DNA

<213> Artificial sequence

<400> 158

acgttggatg aatcccttgg caagacct 28

<210> 159

<211> 16

<212> DNA

<213> Artificial sequence

<400> 159

acaggaagtc gaactc 16

<210> 160

<211> 30

<212> DNA

<213> Artificial sequence

<400> 160

acgttggatg ccggtacggc tacttttgca 30

<210> 161

<211> 30

<212> DNA

<213> Artificial sequence

<400> 161

acgttggatg aaagtcggta acacacagcg 30

<210> 162

<211> 23

<212> DNA

<213> Artificial sequence

<400> 162

aaggtgggat cggcgatgtg agc 23

<210> 163

<211> 31

<212> DNA

<213> Artificial sequence

<400> 163

acgttggatg tcgggttgta aaccttctct a 31

<210> 164

<211> 30

<212> DNA

<213> Artificial sequence

<400> 164

acgttggatg cgagctcttt acgagtccca 30

<210> 165

<211> 25

<212> DNA

<213> Artificial sequence

<400> 165

aagcaccggc caactacgca gtgcc 25

<210> 166

<211> 31

<212> DNA

<213> Artificial sequence

<400> 166

acgttggatg tcgggttgta aaccttctct a 31

<210> 167

<211> 30

<212> DNA

<213> Artificial sequence

<400> 167

acgttggatg cgagctcttt acgagtccca 30

<210> 168

<211> 22

<212> DNA

<213> Artificial sequence

<400> 168

ctcgcaccct acgtaccgtt ac 22

<210> 169

<211> 30

<212> DNA

<213> Artificial sequence

<400> 169

acgttggatg ttcgggtgtt accttcgatc 30

<210> 170

<211> 30

<212> DNA

<213> Artificial sequence

<400> 170

acgttggatg taatcgcaga tcaacggcac 30

<210> 171

<211> 20

<212> DNA

<213> Artificial sequence

<400> 171

tgttaccgac tttcaactgg 20

<210> 172

<211> 31

<212> DNA

<213> Artificial sequence

<400> 172

acgttggatg tcgggttgta aactttcctc a 31

<210> 173

<211> 30

<212> DNA

<213> Artificial sequence

<400> 173

acgttggatg cgagctcttt acgagtccca 30

<210> 174

<211> 24

<212> DNA

<213> Artificial sequence

<400> 174

aagcaccggc caactacgtc agcg 24

<210> 175

<211> 31

<212> DNA

<213> Artificial sequence

<400> 175

acgttggatg tcgggttgta aactttcctc a 31

<210> 176

<211> 30

<212> DNA

<213> Artificial sequence

<400> 176

acgttggatg cgagctcttt acgagtccca 30

<210> 177

<211> 19

<212> DNA

<213> Artificial sequence

<400> 177

aacgacgaag gtggtccgt 19

<210> 178

<211> 30

<212> DNA

<213> Artificial sequence

<400> 178

acgttggatg aagctatagt gaacccggtg 30

<210> 179

<211> 30

<212> DNA

<213> Artificial sequence

<400> 179

acgttggatg actcggcgaa attctagcac 30

<210> 180

<211> 22

<212> DNA

<213> Artificial sequence

<400> 180

tatattgaag gtcccgtcgg gt 22

<210> 181

<211> 30

<212> DNA

<213> Artificial sequence

<400> 181

acgttggatg aagctatagt gaccctggag 30

<210> 182

<211> 30

<212> DNA

<213> Artificial sequence

<400> 182

acgttggatg actcggcgaa attctagcac 30

<210> 183

<211> 19

<212> DNA

<213> Artificial sequence

<400> 183

gtgaaggtcc cgtctgggt 19

<210> 184

<211> 30

<212> DNA

<213> Artificial sequence

<400> 184

acgttggatg acctgacgtt cgaggttccg 30

<210> 185

<211> 30

<212> DNA

<213> Artificial sequence

<400> 185

acgttggatg agcacatttc acgctactgg 30

<210> 186

<211> 20

<212> DNA

<213> Artificial sequence

<400> 186

atcgcggtgt atgtgagcta 20

<210> 187

<211> 30

<212> DNA

<213> Artificial sequence

<400> 187

acgttggatg accggcatct actctggaac 30

<210> 188

<211> 28

<212> DNA

<213> Artificial sequence

<400> 188

acgttggatg acgcccagat ccactgtc 28

<210> 189

<211> 24

<212> DNA

<213> Artificial sequence

<400> 189

tgccgaccag gtagttctag tcca 24

<210> 190

<211> 30

<212> DNA

<213> Artificial sequence

<400> 190

acgttggatg tcagtcgtcc tctgttccgc 30

<210> 191

<211> 30

<212> DNA

<213> Artificial sequence

<400> 191

acgttggatg ggatctgttg gcaatatgtg 30

<210> 192

<211> 19

<212> DNA

<213> Artificial sequence

<400> 192

ccccttcgct ggtatctgc 19

<210> 193

<211> 30

<212> DNA

<213> Artificial sequence

<400> 193

acgttggatg ccaaattgga tcatccgcag 30

<210> 194

<211> 30

<212> DNA

<213> Artificial sequence

<400> 194

acgttggatg tgctggtgtg tgacgatccg 30

<210> 195

<211> 15

<212> DNA

<213> Artificial sequence

<400> 195

tggtgctccc gatcc 15

<210> 196

<211> 30

<212> DNA

<213> Artificial sequence

<400> 196

acgttggatg gtcatcagtg agctacggac 30

<210> 197

<211> 30

<212> DNA

<213> Artificial sequence

<400> 197

acgttggatg cgatatcttt ggatgggcag 30

<210> 198

<211> 19

<212> DNA

<213> Artificial sequence

<400> 198

agctgcggat acccccagc 19

<210> 199

<211> 29

<212> DNA

<213> Artificial sequence

<400> 199

acgttggatg tttcgccggc acgaacatg 29

<210> 200

<211> 29

<212> DNA

<213> Artificial sequence

<400> 200

acgttggatg atccgggtgc ccaacttga 29

<210> 201

<211> 22

<212> DNA

<213> Artificial sequence

<400> 201

ttatttgggc tccatgcggc ga 22

<210> 202

<211> 30

<212> DNA

<213> Artificial sequence

<400> 202

acgttggatg cgtgatcctt tgcacacagc 30

<210> 203

<211> 30

<212> DNA

<213> Artificial sequence

<400> 203

acgttggatg gaaatcggtg aaagccctgg 30

<210> 204

<211> 23

<212> DNA

<213> Artificial sequence

<400> 204

agggtcacag tcacagccga ata 23

<210> 205

<211> 30

<212> DNA

<213> Artificial sequence

<400> 205

acgttggatg cgtgatcctt tgccagacac 30

<210> 206

<211> 30

<212> DNA

<213> Artificial sequence

<400> 206

acgttggatg gaaatcggtg aaactggccg 30

<210> 207

<211> 20

<212> DNA

<213> Artificial sequence

<400> 207

gattctgtga ctgccctgat 20

<210> 208

<211> 30

<212> DNA

<213> Artificial sequence

<400> 208

acgttggatg cgtgatcctt tgccagacac 30

<210> 209

<211> 30

<212> DNA

<213> Artificial sequence

<400> 209

acgttggatg gaaatcggtg aaactggccg 30

<210> 210

<211> 20

<212> DNA

<213> Artificial sequence

<400> 210

tacacagggt caccacagta 20

<210> 211

<211> 30

<212> DNA

<213> Artificial sequence

<400> 211

acgttggatg cgtgatcctt tgccagacac 30

<210> 212

<211> 30

<212> DNA

<213> Artificial sequence

<400> 212

acgttggatg gaaatcggtg aaactggccg 30

<210> 213

<211> 18

<212> DNA

<213> Artificial sequence

<400> 213

tggccgcggc agcctata 18

<210> 214

<211> 30

<212> DNA

<213> Artificial sequence

<400> 214

acgttggatg cgtgatcctt tgccagacac 30

<210> 215

<211> 30

<212> DNA

<213> Artificial sequence

<400> 215

acgttggatg gaaatcggtg aaactggccg 30

<210> 216

<211> 20

<212> DNA

<213> Artificial sequence

<400> 216

cctgtcgtcg taatcatatc 20

<210> 217

<211> 31

<212> DNA

<213> Artificial sequence

<400> 217

acgttggatg aagcagccaa taagcagaag c 31

<210> 218

<211> 28

<212> DNA

<213> Artificial sequence

<400> 218

acgttggatg agcccatttg cgaggaca 28

<210> 219

<211> 20

<212> DNA

<213> Artificial sequence

<400> 219

gacgaatatt cgtgcccagg 20

<210> 220

<211> 29

<212> DNA

<213> Artificial sequence

<400> 220

acgttggatg cggcaacaga tttggcgaa 29

<210> 221

<211> 31

<212> DNA

<213> Artificial sequence

<400> 221

acgttggatg cgctccgaac aacgcggcta t 31

<210> 222

<211> 22

<212> DNA

<213> Artificial sequence

<400> 222

ttagtgcagc caacgccgcg gc 22

<210> 223

<211> 29

<212> DNA

<213> Artificial sequence

<400> 223

acgttggatg cctgctgcac tccatctac 29

<210> 224

<211> 30

<212> DNA

<213> Artificial sequence

<400> 224

acgttggatg cgtcgagtac ccgatcatat 30

<210> 225

<211> 20

<212> DNA

<213> Artificial sequence

<400> 225

ccccgacgcc gaagtttcag 20

<210> 226

<211> 30

<212> DNA

<213> Artificial sequence

<400> 226

acgttggatg ttcctgctga ttgtctccgt 30

<210> 227

<211> 30

<212> DNA

<213> Artificial sequence

<400> 227

acgttggatg tcggggttga tggtccaggt 30

<210> 228

<211> 18

<212> DNA

<213> Artificial sequence

<400> 228

cccaacgtgt ctctcaag 18

<210> 229

<211> 15

<212> DNA

<213> Artificial sequence

<400> 229

atgggcaccg agcgg 15

<210> 230

<211> 29

<212> DNA

<213> Artificial sequence

<400> 230

acgttggatg gcctacctcg gggtttgtc 29

<210> 231

<211> 28

<212> DNA

<213> Artificial sequence

<400> 231

acgttggatg ggtccaaggt tcgtgctc 28

<210> 232

<211> 24

<212> DNA

<213> Artificial sequence

<400> 232

ttgtcaccga cgcctcacgg cgca 24

<210> 233

<211> 24

<212> DNA

<213> Artificial sequence

<400> 233

gatcgcgtcg aggacgctaa gggt 24

Claims

1. The related gene for identifying the mycobacterium tuberculosis complex flora is characterized by comprising the following gene sites and characteristic bases:

gene locus Characteristic base TB81 T mce3B C TB10 G TB78 G TB61 G

。

2. The related gene for detecting the drug resistance of the mycobacterium tuberculosis is characterized by comprising the following drugs, detection genes and detection sites or mutations:

1) rifampicin

2) Isoniazid

3) Pyrazinamides

Serial number Detection of genes Detecting sites or mutations 1 pncA H57D

4) Ethambutol

5) Fluoroquinolones

6) Streptomycin

7) Ethionamide

8) Propylthioisonicotinamine

9) Para-aminosalicylic acid sodium salt

10) Amikacin

11) Kanamycin

12) Capreomycin

13) Cyclic serine

14) Clofazimine

15) Bedaquinoline

16) Linezolid

Serial number Detection of genes Detecting sites or mutations 1 rplC T460C

。

3. A method for the identification of complex populations of mycobacterium tuberculosis for non-disease diagnostic purposes, comprising detecting the identity of a characteristic base in the genetic locus associated with the identification of complex populations of mycobacterium tuberculosis as defined in claim 1, and if so determining the presence of a corresponding complex population of mycobacterium tuberculosis:

4. a method for detecting drug resistance in mycobacterium tuberculosis for non-disease diagnostic purposes, the method further comprising detecting the presence of the following sensitive, drug-resistant nucleic acid mutations in the mycobacterium tuberculosis drug resistance-associated genetic locus as defined in claim 2:

1) rifampicin

2) Isoniazid

3) Pyrazinamides

4) Ethambutol

5) Fluoroquinolones

6) Streptomycin

7) Ethionamide

8) Propylthioisonicotinamine

9) Para-aminosalicylic acid sodium salt

10) Amikacin

11) Kanamycin

12) Capreomycin

13) Cyclic serine

14) Clofazimine

15) Bedaquinoline

16) Linezolid

5. A method for identification of mycobacterium tuberculosis complex and detection of drug resistance for non-disease diagnostic purposes, comprising the method for identification of mycobacterium tuberculosis complex according to claim 3 and the method for detection of drug resistance of mycobacterium tuberculosis according to claim 4.

6. The method of claim 5, wherein the identification of Mycobacterium tuberculosis complex flora and the detection of drug resistance are performed simultaneously by time-of-flight mass spectrometry.

7. A primer group for identification and drug resistance detection of Mycobacterium tuberculosis complex flora is characterized by comprising a drug resistance detection primer group and a Mycobacterium tuberculosis complex flora identification primer group, wherein the drug resistance detection primer group comprises the following gene variation sites and primers:

the primer group for identifying the mycobacterium tuberculosis complex flora comprises the following gene sites and primers:

gene locus Amplification primers Extension primer TB10 SIQ ID NO:217～218 SIQ ID NO:219 TB78 SIQ ID NO:220～221 SIQ ID NO:222 TB81 SIQ ID NO:223～224 SIQ ID NO:225 mce3B SIQ ID NO:226～227 SIQ ID NO:228 TB61 SEQ ID NO:230～231 SEQ ID NO:232～233

。

8. A kit for identification of mycobacterium tuberculosis complex and detection of drug resistance, comprising the primer set for identification of mycobacterium tuberculosis complex and detection of drug resistance according to claim 7.

9. The kit according to claim 8, wherein the molar concentrations of the amplification primer and the extension primer in the reaction system are as shown in the following table:

10. the kit according to claim 8, wherein the reaction system of PCR amplification of the kit is as follows:

the SAP reaction system of the kit is as follows:

SAP buffer 0.17. mu.L

SAP enzyme 0.5U

Replenishing water to 2 mu L;

the iPLEX extension reaction system of the kit is as follows: