CN1515690A - Mycobacterium tuberculosis diagnosis and drug resistance analysis chip, its preparation process and application method - Google Patents
Mycobacterium tuberculosis diagnosis and drug resistance analysis chip, its preparation process and application method Download PDFInfo
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- 241000187479 Mycobacterium tuberculosis Species 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 27
- 238000004458 analytical method Methods 0.000 title claims abstract description 25
- 238000003745 diagnosis Methods 0.000 title claims abstract description 20
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 8
- 239000000523 sample Substances 0.000 claims abstract description 85
- 201000008827 tuberculosis Diseases 0.000 claims abstract description 23
- 238000009396 hybridization Methods 0.000 claims abstract description 18
- 238000002493 microarray Methods 0.000 claims abstract description 8
- 239000011521 glass Substances 0.000 claims abstract description 7
- 238000003908 quality control method Methods 0.000 claims abstract description 6
- AEUTYOVWOVBAKS-UWVGGRQHSA-N ethambutol Chemical compound CC[C@@H](CO)NCCN[C@@H](CC)CO AEUTYOVWOVBAKS-UWVGGRQHSA-N 0.000 claims description 21
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 18
- MYSWGUAQZAJSOK-UHFFFAOYSA-N ciprofloxacin Chemical compound C12=CC(N3CCNCC3)=C(F)C=C2C(=O)C(C(=O)O)=CN1C1CC1 MYSWGUAQZAJSOK-UHFFFAOYSA-N 0.000 claims description 18
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 108020005187 Oligonucleotide Probes Proteins 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 238000005516 engineering process Methods 0.000 claims description 12
- 239000002751 oligonucleotide probe Substances 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 10
- IPEHBUMCGVEMRF-UHFFFAOYSA-N pyrazinecarboxamide Chemical compound NC(=O)C1=CN=CC=N1 IPEHBUMCGVEMRF-UHFFFAOYSA-N 0.000 claims description 10
- 229960003405 ciprofloxacin Drugs 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 9
- 125000003729 nucleotide group Chemical group 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 230000003321 amplification Effects 0.000 claims description 7
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 7
- 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 7
- 229960001225 rifampicin Drugs 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 6
- 238000013016 damping Methods 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 6
- 229960000285 ethambutol Drugs 0.000 claims description 6
- 239000012530 fluid Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 claims description 6
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 5
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 4
- 230000004087 circulation Effects 0.000 claims description 4
- 238000007405 data analysis Methods 0.000 claims description 4
- 239000008367 deionised water Substances 0.000 claims description 4
- 229910021641 deionized water Inorganic materials 0.000 claims description 4
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- 238000012986 modification Methods 0.000 claims description 4
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 4
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- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 claims description 3
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Abstract
The present invention relates to a genechip-mycobacterium tuberculosis diagnosis and drug-resistance analysis chip, its preparation process and application method. On a glass base plate several microarrays with different zones are distributed, in every microarray zone are respectively fixed several specific probes with mycobacterium tuberculosis type and subtype, besides, the quality control probe and several drug-resistance analysis probes are set. The correspondent probe can be used for making hybridization with amplified myobacterium DNA in which the fluorescence label is added, and then said chip is scanned by scanner, and the hybridization singnal can be processed and analyzed so as to can obtain the related information.
Description
One, technical field: the present invention relates to the medical science vitro diagnostic techniques, is a kind of gene chip and preparation technology thereof and using method concretely.
Two, background technology: the tuberculosis that is caused by Mycobacterium tuberculosis (TB) is one of the most general in the world now human infectious disease, though worldwide carry out vaccine prevention, remote districts and the low area of standard of living, even some city can also find its trace, and the control of tuberculosis is still a global health problem.China has tuberculosis patient about more than 600 ten thousand now, also have at least every year 1130000 newborn tuberculosis cases to take place, annual because of the tuberculosis death toll up to 250,000, and 75% tuberculosis case occurs in the middle of the person between twenty and fifty in 15-50 year, had a strong impact on the development of Chinese national economy and population quality.Treatment tuberculosis needs long-term prescription, and serious day by day tubercule bacillus resistance is brought very big difficulty to clinical treatment, and from the beginning of the nineties, the report of the many persisters of tubercule bacillus gradually has increase, and the waste of manpower and financial resources is inevitable.Have data to show, the Mycobacterium tuberculosis specific gene makes a variation has very big dependency in resistance.Therefore, viral hepatitis being diagnosed fast and effectively is the prerequisite of prevention and treatment hepatitis.
Detect at present tuberculosis and mainly depend on from patient's secretory product and separate organizing or turn out tubercule bacillus, and make diagnosis in conjunction with clinical symptom.But because the biological nature of tubercule bacillus and the restriction of detection technique, microscopy is simply quick, but needs a considerable amount of tubercule bacilluss, and will be with special staining technique; Time and technical requirements that tubercule bacillus is cultivated have all limited further developing of this method.Put the method for exempting from and also be used to detection, though rapidly, can not reach very high sensitivity tubercule bacillus.For a long time, a lot of researchists are devoted to invent a kind of efficient, highly sensitive, the tubercule bacillus detection method of high specific.
Three, summary of the invention:
1, goal of the invention: the invention provides a kind of Mycobacterium tuberculosis diagnosis and resistance analysis chip and preparation technology and using method, its purpose is to solve present detection tubercule bacillus and needs a considerable amount of tubercule bacilluss, and to and can not reach efficient, the problem that aspects such as highly sensitive exist with special staining technique.
2, technical scheme: the present invention comes to be realized by the following technical programs:
A kind of Mycobacterium tuberculosis diagnosis and resistance analysis chip and preparation technology and using method, it is characterized in that: this chip is the microarray that is distributed with a plurality of different zones on the glass substrate, in each microarray zone, be fixed with Mycobacterium tuberculosis type and veriform specific probe respectively.
In the microarray of this chip, be provided with Quality Control probe and a plurality of resistance analysis probe.
The size of probe is a 15-25 base, designs 75 probes, 9 of Quality Control probes, and designed probe sequence sees below;
Preparation technology of the present invention carries out as follows:
(1), design tuberculosis specific probe and primer; From database, obtain reliable Mycobacterium tuberculosis virus gene sequence and drug-resistance characteristics sequence, according to described probe sequence designing probe;
From database, obtain reliable Mycobacterium tuberculosis virus gene sequence and drug-resistance characteristics sequence, for accurately differentiating different genotype tuberculosis substance, with primers F 3, R385, F25, R568 the tuberculosis DNA that extracts is made conventional PCR or nest-type PRC, dissimilar tubercule bacillus 16S-18S transcribed spacer sequences increase, screen 47 oligonucleotide probes, differentiate different tubercule bacillus types; Selection is increased with primers F 418, R621 to Tibutol, vazadrine, the drug-fast dissociant characteristic sequence of Rifampin, and selection 579 and 579-1 probe are differentiated the multidrug resistance gene bacterial strain; Increase to the region of variability nucleotide sequence of quinolone, Ciprofloxacin Resistant strain with primers F 2465, R2748, use oligonucleotide probe n2553 m2567 n2568 m2574 m2576n2585 m2585t m2585c m2585a m2586g m2586c m2589c discriminating wild-type and anti-quinolone, Ciprofloxacin Types of Medicine bacterial strain; Increase to the region of variability nucleotide sequence of Tibutol Resistant strain with primers F 7769, R7921, differentiate wild-type and Tibutol drug-resistant type bacterial strain with oligonucleotide probe n7855 m7868g m7870a m7870t m7870c; Increase to the region of variability nucleotide sequence of pyrazinoic acid amide Resistant strain with primers F 34, R308, differentiate wild-type and pyrazinoic acid amide Types of Medicine bacterial strain with oligonucleotide probe n169, m169;
(2), synthesising probing needle;
(3), preparation chip;
With the dissolving of synthetic probe, concentration is 200mmol/l, carries out point sample on glass substrate with deionized water; The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down; With probe damping fluid and distilled water flushing slide, dry up; With confining liquid sealing slide, wash afterwards 3 times, dry up standby.
In the preprocessing process to testing sample, pcr amplification primer sequence sees below;
Probe 5 ' end adds amino the modification.
Point sample from tens to thousands of points, the size of point is about the 50-100 micron.
The probe damping fluid is 1xSSC, 0.1%SDS; Confining liquid is 1%BSA, PH=7 0.01mol/l PB.
Using method is carried out as follows:
(1). handle sample;
It is a small amount of to get patient's cerebrospinal fluid, extracts DNA respectively, standby; Place for a long time as need, frozen down at-20 ℃;
(2) .PCR amplification
In reaction tubes, add amplification reaction mixture---Taq enzyme, sample of handling well accordingly and primer, the dUTP of the dNTP of capacity and fluorescein Cy3 mark; Amplification condition is as follows:
94℃ 5min
94℃ 30sec
56℃ 30sec
72℃ 1min
72℃ 5min
Above step is 30 circulations;
(3). hybridization;
Probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.
(4). detect;
Chip with after the scanner scanning hybridization obtains image and exports the result;
(5). data analysis
The chip analysis result is exported.
3, advantage and effect:
Gene chip is one of great science and technology progress of the tool characteristics of the times that occurred in high-tech area in recent years, and it is the high-tech of physics, chemistry, microtronics, precision optical machinery and life science cross-synthesis.What gene chip was integrated is not electronic devices and components, adopt combinatorial chemistry on the throne, microelectronic chip photoetching technique, perhaps utilize other method to be fixed on the dna probe of a large amount of particular sequences on the substrate in an orderly manner, with testing sample DNA effect after, a large amount of life-informations be can detect, gene recognition, transgenation and genetic expression etc. comprised.Utilize gene chip can obtain or handle a large amount of life-informations fast and efficiently, it has revolutionary pushing effect to life science, medical diagnosis, new medicament screen and judicial expertise etc.
The invention provides a kind of Mycobacterium tuberculosis diagnosis and resistance analysis chip, synthetic specific probe matrix is fixed in substrate surface, by chip and sample to be tested DNA hybridization, can obtain a large amount of biological informations relevant with Mycobacterium tuberculosis.Utilize this chip, just can finish the detection of Mycobacterium tuberculosis type and hypotype thereof simultaneously, and common drug is carried out the resistant characterization analysis by single job.This gene chip has the advantages that diagnosis is accurate, specificity is high, contain much information.If this chip is successfully applied to clinical, will bring great economic benefit and social benefit.
Four, embodiment: concrete steps of the invention process are:
1. design tuberculosis specific probe and primer
Obtain reliable Mycobacterium tuberculosis gene order and drug-resistance characteristics sequence etc. from databases such as NCBI, utilize information biology software design primer and specific probe.
With the strict screening of information biology related software primer, probe sequence.In order accurately to differentiate different genotype tuberculosis substance, with primers F 3, R385, F25, R568 the tuberculosis DNA that extracts is made conventional PCR or nest-type PRC, the dissimilar tubercule bacillus 16S-18S transcribed spacer sequences that increase are screened 47 oligonucleotide probes, differentiate different tubercule bacillus types.Selection is increased with primers F 418, R621 to Tibutol, vazadrine, the drug-fast dissociant characteristic sequence of Rifampin, and selection 579 and 579-1 probe are differentiated the multidrug resistance gene bacterial strain.Increase to the region of variability nucleotide sequence of quinolone, Ciprofloxacin Resistant strain with primers F 2465, R2748, use oligonucleotide probe n2553 m2567 n2568 m2574 m2576 n2585 m2585tm2585c m2585a m2586g m2586c m2589c discriminating wild-type and anti-quinolone, Ciprofloxacin Types of Medicine bacterial strain.Increase to the region of variability nucleotide sequence of Tibutol Resistant strain with primers F 7769, R7921, differentiate wild-type and Tibutol drug-resistant type bacterial strain with oligonucleotide probe n7855 m7868gm7870a m7870t m7870c.Increase to the region of variability nucleotide sequence of pyrazinoic acid amide Resistant strain with primers F 34, R308, differentiate wild-type and pyrazinoic acid amide Types of Medicine bacterial strain with oligonucleotide probe n169, m169.
2. synthesising probing needle
Synthetic by Shanghai biotechnology company limited.Probe 5 ' end adds amino the modification.
3. preparation chip
Set the point sample program as required, matrix distribution requires according to probe hybridization kinetics and whether point sample makes things convenient for and arrange.With the dissolving of synthetic probe, concentration is 200mmol/l with deionized water.The glass substrate that uses CEL company to produce carries out point sample with the point sample instrument of BioRobotics company by pre-set program.Require from tens according to difference that the size of point is about the 50-100 micron to thousands of points, dot spacing is decided according to the quantity of point.The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down.(1xSSC 0.1%SDS) and the distilled water flushing slide, dries up with the probe damping fluid.With confining liquid (1%BSA, PH=7 0.01mol/l PB) sealing slide, wash afterwards 3 times.Dry up standby.
The application method of above-mentioned Mycobacterium tuberculosis diagnosis and resistance analysis chip may further comprise the steps:
1. processing sample
It is a small amount of to get patient's sputum, extracts DNA with tubercule bacillus PCR hybridization diagnostic kit, standby.Place for a long time as need, frozen down at-20 ℃.
2.PCR amplification
In reaction tubes, add amplification reaction mixture, Taq enzyme, sample of handling well and primer, the dUTP of the dNTP of capacity and fluorescein (Cy3) mark.Amplification condition is as follows:
96℃ 5min
96℃ 30sec
56℃ 30sec
72℃ 1min
72℃ 5min
Above step is 30 circulations;
3. hybridization
Probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.
4. detect
Chip with after the scanner scanning hybridization of Genomic Solutions company obtains image and exports the result.
5. data analysis
The supporting analysis software of the scanner of Genomic Solutions company is exported the chip analysis result.
Be further described below in conjunction with the technical scheme of specific embodiment invention:
Embodiment:
Design Mycobacterium tuberculosis primer and specific probe 75 (9 of Quality Control probes) are individual.The size of probe is a 15-25 base.Synthetic by Shanghai biotechnology company limited.Probe 5 ' end adds amino the modification.
The preparation gene chip:
With the glass substrate that CEL company produces, use the point sample instrument of BioRobotics company to carry out point sample by pre-set program.With the dissolving of synthetic probe, concentration is 200mmol/l with deionized water.The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down.(1xSSC 0.1%SDS) and the distilled water flushing slide, dries up with the probe damping fluid.With confining liquid (1%BSA, PH=7 0.01mol/l PB) sealing slide, wash afterwards 3 times.Dry up standby.
Handle sample: it is a small amount of for tubercular's sputum to get Clinical Laboratory, extracts DNA with tubercule bacillus PCR hybridization diagnostic kit, standby.
Pcr amplification: the primer sequence of employing is: 5`-tgggacgaag tcgtaacaag g, 5`-tgccaaggca tccaccat
The reaction cumulative volume is 20 μ l, adds 10X buffer 2 μ l in reaction tubes, Taq enzyme 0.2 μ l, and each 2 μ l of sample 0.8 μ l that handles well and upstream and downstream primer, the dUTP1.2 μ l of dNTP0.8 μ l and Cy3 mark, remaining uses H
2O.Amplification condition is as follows:
96℃ 5min
96℃ 30sec
56℃ 40sec
72℃ 1min
72℃ 5min
Above step is 30 circulations
Hybridization: the probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.
Detect: the chip with after the scanner scanning hybridization of Genomic Solutions company obtains image and exports the result.Data analysis: the supporting analysis software of the scanner of Genomic Solutions company is exported chip results.Compare coincidence rate 100% with the Clinical Laboratory result.
Mycobacterium tuberculosis diagnosis and resistance analysis chip primer sequence table
The tuberculosis somatotype
primer Sequence
F3 5`-tgggacgaag?tcgtaacaag?g
R385 5`-tgccaaggca?tccaccat
F25 5`-acgttc?ccgggccttg
R568 5`-tgacagctcc?ccgaggc
Resistance
primer Sequence
ethambutol F418 5`-ggctcatatc?gagaatgc
rifampin R621 5`-ctcatcatca?aagcggac
primer Sequence
quinolone F2465 5`-cgggtg?ctctatgcaa?tgttc
ciprofloxacin R2748 5`-tcctcgtcga?tttccctcag
primer Sequence
F7769 5`-tggcgcacct?tcaccct
ethambutol R7921 5`-gaaccagcgg?aaatagttgg?ac
primer Sequence
F34 5`-gacttctgcgagggtggct
pyrazinamide R308 5`-tacgctccggtgtaggcac
Mycobacterium tuberculosis diagnosis and the sequence table of resistance analysis chip probe
The tuberculosis somatotype
Probe Sequence
prob?all atagtggtt?gcgagcatc
1abscessus ATCTAAACATAGCCTCGCT
2africanum ATCAATGGATACGCTGCC
3avium ATCTAGATGAGCGCATGG
4bovis ATCAATGGATACGCTGCC
5chelonae ATCTAACAAGCCTCGCTC
6diernhoferi ATCTAGCACGCAAGAGGA
7gastri ATCAAATGGATGCGTTGC
7.1 TGTCTTGGACTCGTCCAA
8gilvum ATCGAAAGATGGTGCACA
9gordonae ATCAAAATGTATGCGTTGTC
10intracellulare ATCTAGATGAGCGCATAGT
11kansasii ATCAAATGGATGCGTTGCC
11.1 AACACTCGGGCTCTGTTC
12marinum ATCAATTGGATGCGCTGC
12.1 ATCTCTGTTGGTTTCGGG
13phlei ATCTGATACTTGATGCTCCT
14scrofulaceum ATCTAAACGGATGCGTTGC
15smegmatis ATCTAGTTCGTAAGAGTGTG
16szulgai ATCAATTGGATGCGCTGC
16.1 ACTCAGGCTTGGCCAGAG
17terrae ATCTAACAAGCAGATTTTTGG
18triviale ATCTAGCAGATGAGATCTCT
19tuberculosis ATCAATGGATACGCTGCC
20vaccae ATCTGAATGCACAGCGCT
21xenopi ATCTGGCAAAGACTGTGG
22abscessus ACCCTGCTTGGTGGTGG
23africanum AGGTGTTGTCCCACCGC
24avium CCTCCATCTTGGTGGTG
25bovis AGGTGTTGTCCCACCG
26chelonae ACCCTGCTTGGTGGTG
27diernhoferi CGGTGTCTGTTGTTGCTC
28gastri AGAGTGTTGTCCCACCAT
29gilvum GGGTCGGTGTGTTGTTG
30gordonae GGGTGCTGTCCCCCCA
31intracellulare CCTCCATCTTGGTGGTGG
32kansasii AGAGTTGTCCCACCATCT
33marinum GGGATGTTGTCCCACCAT
34phlei GGGTGCCGGTGTGTTG
35scrofulaceum TGAGTGGTGTCCCTCCA
36smegmatis GGCGTGTTGTTGCCCTG
37szulgai GAGCTGTTGTCCCACCA
38terrae GCCTCACACTTGGTGGT
39triviale TCACCGGTTTTGGTGTGG
40tuberculosis GGTGTTGTCCCACCGC
41vaccae GGGTCGGCGTGTTGTT
42xenopi TGGTGGCGGGGTGTGG
43fortuitum ATCACCTCCTTTCTAAGGAGCAC
Resistance
Probe Seq?uence
579 5`-atggaatgtc?gcaaccaaat?g
ethambutol
579-1 5`-atagaatgtc?gcaaccaaat?g
isoniazid
rifampin
primer Sequence
F2465 5`-cgggtg?ctctatgcaa?tgttc
R2748 5`-tcctcgtcga?tttccctcag
Probe Sequence
quinolone
n2553 5`-actac?accac?ccgcacggc
ciprofloxacin
m2567 5`-actaccac?ccgcactgcg
n2568 5`-gcg?acgcgtcgat?ctacg
m2574 5`-ggcg?acgtgtcgat?ctacg
m2576 5`-gcg?acgcgtcgat?cta
n2585 5`-at?ctacgacagc?ctggtgcg
m2585t 5`-at?ctactacagc?ctggtgcgc
m2585c 5`-t?ctaccacagc?ctggtgcgc
m2585a 5`-ctacaacagc?ctggtgcgc
m2586g 5`-cgat?ctacggcagc?ctgg
m2586c 5`-cgat?ctacgccagc?ctgg
m2589c 5`-tcgat?ctacgacacc?ctggtg
Probe Sequence
n7855 5`-ctacatcctg?ggcatggcc
m7868c 5`-tacatcctg?ggcctggcc
m7868g 5`-catcctg?ggcgtggcc
m7870a 5`-gggcatagcc?cgagtcg
m7870t 5`-gggcattgcc?cgagtcg
ethambuto m7870c 5`-gggcatcgcc?cgagtc
Probe Sequence
n169 5`-acc?cgggtgacga?cttctc
pyrazinamide m169 5`-acc?cgggtgacca?cttctc
Claims (9)
1. a Mycobacterium tuberculosis is diagnosed and resistance analysis chip and preparation technology and using method, it is characterized in that: this chip is the microarray that is distributed with a plurality of different zones on the glass substrate, in each microarray zone, be fixed with Mycobacterium tuberculosis type and veriform specific probe respectively.
2. Mycobacterium tuberculosis diagnosis according to claim 1 and resistance analysis chip and preparation technology and using method is characterized in that: in the microarray of this chip, be provided with Quality Control probe and a plurality of resistance analysis probe.
3. Mycobacterium tuberculosis diagnosis according to claim 1 and resistance analysis chip and preparation technology and using method, it is characterized in that: the size of probe is a 15-25 base, designs 75 probes, 9 of Quality Control probes, designed probe sequence is as follows:
The tuberculosis somatotype
Probe Sequence
prob?all atagtggtt?gcgagcatc
1abscessus ATCTAAACATAGCCTCGCT
2africanum ATCAATGGATACGCTGCC
3avium ATCTAGATGAGCGCATGG
4bovis ATCAATGGATACGCTGCC
5chelonae ATCTAACAAGCCTCGCTC
6diemhoferi ATCTAGCACGCAAGAGGA
7gastri ATCAAATGGATGCGTTGC
7.1 TGTCTTGGACTCGTCCAA
8gilvum ATCGAAAGATGGTGCACA
9gordonae ATCAAAATGTATGCGTTGTC
10?intracellulare ATCTAGATGAGCGCATAGT
11kansasii ATCAAATGGATGCGTTGCC
11.1 AACACTCGGGCTCTGTTC
12marinum ATCAATTGGATGCGCTGC
12.1 ATCTCTGTTGGTTTCGGG
13phlei ATCTGATACTTGATGCTCCT
14scrofulaceum ATCTAAACGGATGCGTTGC
15smegmatis ATCTAGTTCGTAAGAGTGTG
16szulgai ATCAATTGGATGCGCTGC
16.1 ACTCAGGCTTGGCCAGAG
17terrae ATCTAACAAGCAGATTTTTGG
18triviale ATCTAGCAGATGAGATCTCT
19tuberculosis ATCAATGGATACGCTGCC
20vaccae ATCTGAATGCACAGCGCT
21xenopi ATCTGGCAAAGACTGTGG
22abscessus ACCCTGCTTGGTGGTGG
23africanum AGGTGTTGTCCCACCGC
24avium CCTCCATCTTGGTGGTG
25bovis AGGTGTTGTCCCACCG
26chelonae ACCCTGCTTGGTGGTG
27diernhoferi CGGTGTCTGTTGTTGCTC
28gastri AGAGTGTTGTCCCACCAT
29gilvum GGGTCGGTGTGTTGTTG
30gordonae GGGTGCTGTCCCCCCA
31intracellulare CCTCCATCTTGGTGGTGG
32kansasii AGAGTTGTCCCACCATCT
33marinum GGGATGTTGTCCCACCAT
34phlei GGGTGCCGGTGTGTTG
35scrofulaceum TGAGTGGTGTCCCTCCA
36smegmatis GGCGTGTTGTTGCCCTG
37szulgai GAGCTGTTGTCCCACCA
38terrae GCCTCACACTTGGTGGT
39triviale TCACCGGTTTTGGTGTGG
40tuberculosis GGTGTTGTCCCACCGC
41vaccae GGGTCGGCGTGTTGTT
42xenopi TGGTGGCGGGGTGTGG
43fortuitum ATCACCTCCTTTCTAAGGAGCAC
Resistance
Probe Sequence
ethambutol 579 5`-atggaatgtc?gcaaccaaat?g
isoniazid 579-1 5`-atagaatgtc?gcaaccaaat?g
rifampin
primer Sequence
F2465 5`-cgggtg?ctctatgcaa?tgttc
R2748 5`-tcctcgtcga?tttccctcag
Probe Sequence
n2553 5`-actaccac?ccgcacggc
m2567 5`-actaccac?ccgcactgcg
n2568 5`-gcg?acgcgtcgat?ctacg
m2574 5`-ggcg?acgtgtcgat?ctacg
m2576 5`-gcg?acgcgtcgat?cta
n2585 5`-at?ctacgacagc?ctggtgcg
m2585t 5`-at?ctactacagc?ctggtgcgc
m2585c 5`-t?ctaccacagc?ctggtgcgc
m2585a 5`-ctacaacagc?ctggtgcgc
m2586g 5`-cgat?ctacggcagc?ctgg
m2586c 5`-cgat?ctacgccagc?ctgg
qumolone m2589c 5`-tcgat?ctacgacacc?ctggtg
ciprofloxacin
Probe Sequence
ethambuto n7855 5`-ctacatcctg?ggcatggcc
m7868c 5`-tacatcctg?ggcctggcc
m7868g 5`-catcctg?ggcgtggcc
m7870a 5`-gggcatagcc?cgagtcg
m7870t 5`-gggcattgcc?cgagtcg
m7870c 5`-gggcatcgcc?cgagtc
Probe Sequence
n169 5`-acc?cgggtgacga?cttctc
pyrazinamide m169 5`-acc?cgggt9acca?cttctc
4. Mycobacterium tuberculosis diagnosis according to claim 1 and resistance analysis chip and preparation technology and using method, it is characterized in that: preparation technology of the present invention carries out as follows:
(1), design tuberculosis specific probe and primer;
From database, obtain reliable Mycobacterium tuberculosis virus gene sequence and drug-resistance characteristics sequence, according to described probe sequence designing probe;
From database, obtain reliable Mycobacterium tuberculosis virus gene sequence and drug-resistance characteristics sequence, for accurately differentiating different genotype tuberculosis substance, with primers F 3, R385, F25, R568 the tuberculosis DNA that extracts is made conventional PCR or nest-type PRC, dissimilar tubercule bacillus 16S-18S transcribed spacer sequences increase, screen 47 oligonucleotide probes, differentiate different tubercule bacillus types; Selection is increased with primers F 418, R621 to Tibutol, vazadrine, the drug-fast dissociant characteristic sequence of Rifampin, and selection 579 and 579-1 probe are differentiated the multidrug resistance gene bacterial strain; Increase to the region of variability nucleotide sequence of husky star Resistant strain in quinolone, the ring with primers F 2465, R2748, use oligonucleotide probe n2553 m2567 n2568 m2574 m2576n2585 m2585t m2585c m2585a m2586g m2586c m2589c discriminating wild-type and anti-quinolone, Ciprofloxacin Types of Medicine bacterial strain; Increase to the region of variability nucleotide sequence of Tibutol Resistant strain with primers F 7769, R7921, differentiate wild-type and Tibutol drug-resistant type bacterial strain with oligonucleotide probe n7855 m7868g m7870a m7870t m7870c; Increase to the region of variability nucleotide sequence of pyrazinoic acid amide Resistant strain with primers F 34, R308, differentiate wild-type and pyrazinoic acid amide Types of Medicine bacterial strain with oligonucleotide probe n169, m169;
(2), synthesising probing needle;
(3), preparation chip;
With the dissolving of synthetic probe, concentration is 200mmol/l, carries out point sample on glass substrate with deionized water; The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down; With probe damping fluid and distilled water flushing slide, dry up; With confining liquid sealing slide, wash afterwards 3 times, dry up standby.
5. the using method of Mycobacterium tuberculosis diagnosis according to claim 4 and resistance analysis chip, it is characterized in that: in the preprocessing process to testing sample, pcr amplification primer sequence is as follows:
The tuberculosis somatotype
primer Sequence
F3 5`-tgggacgaag?tcgtaacaag?g
R385 5`-tgccaaggca?tccaccat
F25 5`-acgttc?ccgggccttg
R568 5`-tgacagctcc?ccgaggc
Resistance
primer Sequence
ethambutol F418 5`-ggctcatatc?gagaatgc
rifampin R621 5`-ctcatcatca?aagcggac
primer Sequence
quinolone F2465 5`-cgggtg?ctctatgcaa?tgttc
ciprofloxacin R2748 5`-tcctcgtcga?tttccctcag
primer Sequence
F7769 5`-tggcgcacct?tcaccct
ethambutol R7921 5`-gaaccagcgg?aaatagttgg?ac
primer Sequence
F34 5`-gacttctgcgagggtggct
pyrazinamide R308 5`-tacgctccggtgtaggcac
6. the using method of Mycobacterium tuberculosis diagnosis according to claim 4 and resistance analysis chip is characterized in that: probe 5 ' end adds amino the modification.
7. the using method of Mycobacterium tuberculosis according to claim 4 diagnosis and resistance analysis chip is characterized in that: point sample from tens to thousands of points, the size of point is about the 50-100 micron.
8. the using method of Mycobacterium tuberculosis diagnosis according to claim 4 and resistance analysis chip, it is characterized in that: the probe damping fluid is 1xSSC, 0.1%SDS; Confining liquid is 1%BSA, PH=7 0.01mol/l PB.
9. the using method of Mycobacterium tuberculosis diagnosis according to claim 1 and resistance analysis chip, it is characterized in that: using method is carried out as follows:
(1). handle sample;
It is a small amount of to get patient's cerebrospinal fluid, extracts DNA respectively, standby; Place for a long time as need, frozen down at-20 ℃;
(2) .PCR amplification
In reaction tubes, add amplification reaction mixture---Taq enzyme, sample of handling well accordingly and primer, the dUTP of the dNTP of capacity and fluorescein Cy3 mark; Amplification condition is as follows:
94℃ 5min
94℃ 30sec
56℃ 30sec
72℃ 1min
72℃ 5min
Above step is 30 circulations;
(3). hybridization;
Probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.
(4). detect;
Chip with after the scanner scanning hybridization obtains image and exports the result;
(5). data analysis
The chip analysis result is exported.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
| CN101580879B (en) * | 2009-04-30 | 2012-04-11 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN107267633A (en) * | 2017-07-20 | 2017-10-20 | 杭州更蓝生物科技有限公司 | A kind of method of utilization genechip detection mycobacterium tuberculosis |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1153969C (en) * | 2001-04-13 | 2004-06-16 | 中国科学院上海冶金研究所 | Drug tolerance test chip for Mycobacterium tuberculosis and its preparing method and utilization method |
-
2003
- 2003-04-14 CN CNB031114539A patent/CN100412202C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101580879B (en) * | 2009-04-30 | 2012-04-11 | 广西医科大学 | Drug-resistance gene film chip for detecting mycobacterium tuberculosis |
| CN102312001A (en) * | 2011-09-14 | 2012-01-11 | 四川大学 | Method for detecting drug resistance of Mycobacterium tuberculosis with multiple PCR-reverse dot blot technique |
| CN102312001B (en) * | 2011-09-14 | 2016-04-13 | 四川大学 | Multiplex PCR-reverse dot blot hybridization technique detects the method for Drug Resistance of Mycobacterium Tuberculosis |
| CN107267633A (en) * | 2017-07-20 | 2017-10-20 | 杭州更蓝生物科技有限公司 | A kind of method of utilization genechip detection mycobacterium tuberculosis |
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| CN100412202C (en) | 2008-08-20 |
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