CN1311435A - Drug tolerance test chip for Mycobacterium tuberculosis and its preparing method and utilization method - Google Patents

Drug tolerance test chip for Mycobacterium tuberculosis and its preparing method and utilization method Download PDF

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CN1311435A
CN1311435A CN 01105981 CN01105981A CN1311435A CN 1311435 A CN1311435 A CN 1311435A CN 01105981 CN01105981 CN 01105981 CN 01105981 A CN01105981 A CN 01105981A CN 1311435 A CN1311435 A CN 1311435A
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CN1153969C (en
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赵建龙
景奉香
胡忠义
孙斌
孙悦
徐元森
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Shanghai Institute of Optics and Fine Mechanics of CAS
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Shanghai Institute of Metallurgy of CAS
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Abstract

Chip for drug tolerance test for mycobacteria tuberculosis is made by designing the velevant oligonucleotides probe of the drug tolerance according to the known genic mutation information in drug tolerance to refampin and isonicid and then to fix the synthesized probe on the modified slide of form the chip for drug tolerance. To use the relevant designed priment to augment the DNA sample of mycobacteria and to add fluorescent mark treatment and then to hybridize it with chip, to use chip signal analying system to scan the chip and to analyse the hybridizing signals is the method to obtain the drug tolerance information of the mycobacteria.

Description

Mycobacterium tuberculosis drug resistance detecting chip and preparation method thereof and methods for using them
The present invention relates to method, particularly a kind of relevant Mycobacterium tuberculosis drug resistance detecting chip and preparation method thereof and methods for using them to Mycobacterium tuberculosis drug resistance fast detecting.
The DNA 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, microelectronics, precision optical machinery and life science cross-synthesis.What the DNA chip was integrated is not electronic devices and components, but utilizes or by the intensification and parallel handling principle of microelectronic chip technology, be solidificated in the dna fragmentation that has the particular sequence of biological significance in a large number on the substrate in an orderly manner; Perhaps utilize micro-electronic mechanical system technique (MEMS) and integrated optics etc. with many digital processes related in life science or the medical diagnostic procedures, processes such as for example specimen preparation, sample preparation, amplified reaction, analyzing and testing organically integrate.Utilize the DNA chip can obtain or handle a large amount of life-information (comprise that the setting of gene, detection in Gene Mutation, gene expression profile detect and to the identification of foreign molecules etc.) fast and efficiently, it has revolutionary impetus to life science, medical diagnosis, new medicament screen and judicial expertise etc.
The tuberculosis that is caused by Mycobacterium tuberculosis (TB) is one of the most general in the world now human infectious disease.The existing tuberculosis patient in the whole world is about 2,000 ten thousand, annual New Development patient 800-1000 ten thousand, and annual dead 3,000,000 people in the whole world reach a record high, and tuberculosis becomes the No.1 dead killer of infectious disease.The tuberculosis of China is popular very serious always, and epidemic situation descends slowly for many years, also gos up to some extent in some area.The existing tuberculosis patient of China is about more than 600 ten thousand, also has 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, therefore, had a strong impact on the development of Chinese national economy and population quality.In addition, because popular being on the rise of human immunodeficiency virus (HIV), have 1/3rd to infect tuberculosis simultaneously among HIV the infected, if tuberculosis can not be effectively controlled, the double infection of tuberculosis and HIV will be to bringing on a disaster property of the tuberculosis control influence of China.
Anti-multiple medicines tuberculosis (MDR-TB) is the most serious a kind of in the tuberculosis, because it is to multiple approach mycobacterium medicine resistance, treatment is difficulty very.In recent years, the U.S. has recurred the eruption and prevalence of 12 MDR-TB, has caused the common concern in the world.Estimate that according to the World Health Organization (WHO) existing 5,000 ten thousand people in the whole world are subjected to the infection of the Mycobacterium tuberculosis of anti-the multiple medicines, the tuberculosis patient more than 2/ 3 has the danger that MDR-TB takes place.This disease is propagated fast, and PD is rapid, and from diagnosing the average 4-6 of death week, mortality ratio is up to 37%.In merging HIV the infected, mortality ratio has the danger that becomes incurable disease again greatly more up to 72%-89%.
An existing treatment line medicine lungy is rifampin (RFP) and isoniazid (INH), is characterized in good effect, instant effect, and side effect is little, clinically as long as anti-rifampin of while and isoniazid promptly are diagnosed as anti-multiple medicines tuberculosis.
The resistance mechanism of RFP is fairly simple, clear and definite, only relevant with the variation of rpoB gene (encoding gene of bacteria RNA polymerase beta subunit) (Lincoln P.M., etal.Antimicrob.Agents.Chemother., 1994:38 (4): 805-811).Existing statistics shows that rpoB gene mutation and Mycobacterium tuberculosis resistance correlativity can reach more than 96%, do not find geographic difference.These sudden changes mainly occur in the nucleus-rifampin resistance determining area (RRDR) of 81 bases of rpoB gene, comprise point mutation, short insertion or deletion mutation etc. kind more than totally 40 (Telenti A., etal., Lancet, 1993:341:647-670).These sudden changes cause rifampin not combine with RNA polymerase β subunit, thereby make the tolerance of bacterium generation to rifampin.Wherein the missense mutation of 513-Ser has taken place in 43% bacterial strain, and the missense mutation of 526-His has taken place 36% bacterial strain, and the missense mutation of 516-Asp has taken place 7.3% bacterial strain.That is to say that as long as theoretically all missense mutation in these three sites of this gene are produced on the chip, we just can detect 86.3% RFP endurance strain.Other comparatively common sudden change comprises 513,514,521,532,533 codons.LiPA (linear probe test method(s), Robert C.C., etal., J.Clin.Microbiol., 1997; 35 (5): 1281-83) be a kind of business-like kit, 1993 clinical with regard to being applied to, and its principle is similar to the DNA chip, also be to utilize the method for Southern hybridization to carry out the chemical sproof detection of RFP, yet probe only comprises His-526Tyr, Ser-513Leu, Leu-533Pro.
Compare with rifampin, the isoniazid mechanism of drug resistance is wanted the many of complexity, becomes the focus of Mycobacterium tuberculosis drug resistance research gradually.Discover recently; isoniazid drug resistance of tubercle bacillus and katG (hydrogen peroxide-peroxidase) gene; the promoter region of inhA (acyl group transport protein reductase) gene and ahpC (alkyl hydrogen hydrogen-peroxide reduction enzyme) gene, and kasA (β-keto-aldehyde transport protein synzyme) gene changes relevant.Acyl group transport protein reductase (inhA) and β-keto-aldehyde transport protein synzyme (kasA) participates in Mycobacterium tuberculosis mycolic acid route of synthesis; the isoniazid is transformed into activity conformation under hydrogen peroxide-peroxidase (katG) catalytic action; this conformation acts on inhA or kasA; so that blocked the synthetic of tubercle bacillus mycolic acid, thereby killing bacteria.The physiological action of alkyl hydroperoxide reductase (ahpC) is similar to katG, the ahpC promoter region is undergone mutation, the high expressed that causes ahpC is a Mycobacterium tuberculosis from katG gene delection, or a kind of compensatory physiological reaction during the complete or most of loss of activity of katG.Different with rifampin, the chemical sproof geographic difference in isoniazid is bigger, and in China, existing report is thought, the correlativity of katG and isoniazid resistance can reach about 50%-70%, collaborative can explain isoniazid drug resistance more than 90% to the detection of inhA and ahpC.
The method of traditional detection of drugs susceptibility is absolute concentration method or the rule of three based on cultivation, and method is more reliable, need not specific apparatus, but greatest weakness is time-consuming, and generally going to a doctor to from patient obtains.Drug sensitivity tests needs 2-3 month.During this period, the doctor can only the universal law medication, and these medicines may be invalid to patient, so that delay treatment.Be applied to clinical Bactec method (Huanghai Sea honor etc., Chinese tuberculosis and breathing magazine, 2000, Vol23 (11)) in recent years and Drug Resistance Detection can be advanceed to 2-4 week, but still can not meet clinical needs, and relate to inconveniences such as isotopic application and processing; Dna sequence analysis method general terminal cessation method of two deoxidations that adopt, its visual result has been got rid of the unreliability of the experimental result that human factor causes, and have the objective advantage of result, and can obtain the result in one day, but prices are rather stiff, clinical can't acceptance; Molecular beacon method (Piatek A.S., etal., Nat.Biotech., 1998; 16:359-363) simple, easy operating can in time obtain the result, and required sample size is less, can directly detect clinical samples such as phlegm, hydrothorax, exudate, very meaningful to instructing clinical application, but reaction is to carry out in liquid environment, and the probe amount that primary first-order equation can detect is limited; RNA/RNA base incorrect posting method is 100% to the recall rate of ropB gene mutation, but can't determine the definite type and the position that suddenly change, can not operate simultaneously a plurality of genes, and RNA is carried out operation easier than higher, is difficult to carry out clinical practice; LiPA is a kind of business-like kit, be used to measure the situation of the anti-rifampin of tubercle bacillus, it is the blank of biochip, because probe stationary is on diaphragm, relate to limited mutational site, can only carry out scalping to the rpoB gene, powerless to the detection in low mutation rate site, therefore also be difficult to promote clinically.
Design of the present invention is in order to overcome the deficiency of above-mentioned prior art, utilize the method for DNA chip, with synthetic oligonucleotide probe dot matrix and be fixed in slide surface, DNA or RNA and chip hybridization by sample to be tested, can obtain a large amount of gene mutation information relevant, thereby determine its drug resistance with the Mycobacterium tuberculosis drug resistance.
The object of the invention provides a kind of Mycobacterium tuberculosis drug resistance detecting chip and preparation method thereof and methods for using them, to realize the Mycobacterium tuberculosis drug resistance particularly to chemical sproof low costs such as Rimactazid, highly sensitive, special, detection fast.
The object of the present invention is achieved like this: a kind of tuberculosis molecule bacillus drug resistance detecting chip, on a modified microslide, be fixed with a series of oligonucleotides dot matrix probes, and it is characterized in that
(1) said probe designs at the sudden change of rpoB gene, KatG gene, inhA gene, KasA gene, ahpC gene etc.;
(2) each probe length is at least 10 bases, and each probe length is identical;
(3) catastrophe point is positioned in the middle of the probe as far as possible;
Said probe is primarily aimed at 496 of rpoB gene, 505,507,509,510,511,512,513,514,515,516,518,520,521,522,523,524,526,528,531,533,541,553,569,579, the sudden change of 583 codons such as grade, 1 of katG gene, 20,63,99,108,122-125,138,140,142,160,172,180,262,275,295,300,302,304,315,328,335,336,350,463,477,501,515,567,587,593,629, the sudden change of 710 codons such as grade, 16 of inhA gene, 21,47,78,94,95, the sudden change of 194 codons such as grade, 66 of kasA gene, 121,269,312,387,2 of the sudden change of 413 codons such as grade and ahpC gene, 3,73,191,-46,-44,-39,-34,-32,-12,-10,-6,-4,4, the sudden change of 33 codons such as grade;
Said probe mainly is the sudden change at codons such as 533,532,531,526,516,514,513,511 of rpoB gene;
At the sudden change of codons such as 533,532,531,526,516,514,513,511 of rpoB gene and designed probe is as shown in the table:
Probe number Sequence Genotype (G-C)％
????1a ????1b ????2a ????2b ????3a ????3b ????3c ????3d ????3e ????4a ????4b ????4c ????4d ????4e ????4f ????4g ????5a ????5b ????5c ????6a ????6b ????6c ????7a ????7b ????7c ????8a ????8b ????8c 5＇CTG?TCG?GCG?CTG?GGG?CCC?GG3＇ 5＇CTG?TCG?GCG?CcG?GGG?CCC?GG3＇ 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ 5＇CGA?CTG?TCG?GtG?CTG?GGG?CC3＇ 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ 5＇GC?CGA?CTG?TtG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?TgG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Ttc?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Tac?GCG?CTG?GGG3＇ 5＇GGG?TTG?ACC?CAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?tAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CtC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CgC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gcC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?aAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gAC?AAG?CGC?CG3＇ 5＇AA?TTC?ATG?GAC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?GtC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?tAC?CAG?AAC?AAC3＇ 5＇CTG?AGC?CAA?TTC?ATG?GAC?CA3＇ 5＇TG?AGC?CAA?ttc?TCC?ATG?GAC3＇ 5′CTG?AGC?CAA?gTC?ATG?GAC?CA3＇ 5＇CAG?CTG?AGC?CAA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?CcA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?aAA?TTC?ATG?GA3＇ 5＇CC?AGC?CAG?CTG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CcG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CgG?AGC?CAA?TTC3＇ ?Wt ?Mt ?Wt ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt 85％ 90％ 80％ 75％ 80％ 75％ 80％ 75％ 75％ 70％ 65％ 70％ 75％ 75％ 65％ 70％ 40％ 40％ 35％ 50％ 50％ 55％ 50％ 55％ 45％ 60％ 65％ 65％
Probe length is all 20mer mutually, and wherein Mt, Wt represent saltant and wild type respectively;
The method for making of above-mentioned Mycobacterium tuberculosis drug resistance detecting chip comprises the steps:
(1) carries out the synthetic of probe according to designed oligonucleotide probe;
(2) with deionized water with synthetic probe dilution, and mix with spotting solution (spoting solution) equal-volume, making final concentration is 75pmol/ μ l;
(3) slide surface is with aldehyde group modified;
(4) the micro-array chip manufacturing system of utilizing Cartesian company with the probe dot matrix with aldehyde group modified slide surface;
(5) place relative humidity 70%, fixed through 48-72 hour under the room temperature condition;
(6) under the room temperature slide immersed among the 0.2%SDS vibration several minutes, immersed in the pure water vibration several minutes, immerse again among the 0.2%SDS three times, each 1 minute, immersed again in 100 ℃ of pure water 2 seconds, volatilization is done then, and is standby;
5 ＇ ends of said oligonucleotide probe need add-linking arm (PolyT) of measured length (8-20 is poly-), and 5 ＇ ends of this linking arm need add amino the modification;
The application process of the noisy property of medicine detection chip of above-mentioned Mycobacterium tuberculosis may further comprise the steps:
(1) sample preparation, it is a small amount of to get patient's sputum, extracts DNA with tubercle bacillus PCR hybridization diagnostic kit;
(2) fluorescence labeling:
Obtain after the DNA of testing sample, need carry out fluorescence labeling and handle:
(1) getting the DNA sample 5 μ l that prepare mixes with 20 μ lPCR amplification systems, (amplification system comprises 2.5 μ l PCR reaction buffers, 5nmols dATP, 5nmols dGTP, 5nmols dCTP, 2nmols dTTP, 1nmol Cy5-dUTP, the upstream of each 15pmols and downstream primer, 1.25U pfuDNA polymerase) prepare fluorescently-labeled DNA sample purpose segment by following thermal cycle process: at first 94 ℃, 5mins, with 94 ℃, 40s, 58 ℃, 1min, 72 ℃, 1min circulation 30 times, last, 72 ℃ are extended 5mins;
(2) get fluorescently-labeled target DNA sample 10 μ l, use Dnase I (nuclease I) digestion to be the dna fragmentation about 200bp;
(3) chip hybridization
(4) detection and signal analysis, the chip signal analytic system Scanarray 3000 with General Scanning company scans and analysis results at last;
Said chip hybridization is exactly:
The target DNA segment 5 μ l that get mark and interrupt, 99 ℃ of sex change 5mins are then with 20 μ l EasyHyb hybridization solution (Roche Holding Ag) mixings, get 10 μ l and drip in chip surface, covered places 60 ℃ of wet boxes to hybridize 30mins, put then to swing in 45 ℃ of washing lotions and wash 10mins, airing.
Said upstream and downstream primer is as follows:
Gene Primer sequence Tm Product length (bp)
????RpoB ?5’-AGG?ACG?TGG?AGG?CGA?TCA?CA-3’ 63.3 ???350
?5’-GGG?TTG?ACC?CGC?GCG?TA-3’ 63.1
????KatG ?5’-CC?GGC?TTC?CTG?TTG?GAC?GAC-3’ 64.9 ??2447
?5’-CGG?CGC?GAT?TTG?TCA?GAC?C-3’ 64.5
?Inh ?A Promoter ?5’-CT?CGC?TGC?CCA?GAA?AGG?GA-3’ 63.3 ???248
?5’-CCC?CGG?TTT?CCT?CCG?GT-3’ 62.4
Structure ?5’-TT?CAT?GAC?AGG?ACT?GCT?GGA?CG-3’ 63.2
?5’-CT?AGA?GCA?ATT?GGG?TGT?GCG?C-3’ 63.1
????KasA ?5’-ATT?GAG?TCG?GAC?AAC?CCC?GA-3’ 62.3 ??1389
?5’-CCT?TCC?ATA?TCG?GTC?CGA?CTC-3’ 61.9
?Ah ?pC Promoter ?5’-CCG?ATG?AGA?GCG?GTG?AGC?TG-3’ 63.8 ???236
?5’-C?ACT?GCT?TTG?CCG?CCA?CC-3’ 62.9
Structure ?5’-CTT?GCG?GCA?CTG?CTG?AAC?C-3’ 62.3
?5’-GCT?GCT?GCG?GGT?GAT?TGA?G-3’ 62.2
Advantage of the present invention and effect:
A. simple, the easy row of probe design method that Mycobacterium tuberculosis drug resistance detecting chip of the present invention is used;
B. Mycobacterium tuberculosis drug resistance detecting chip of the present invention has the detection sensitivity height, can differentiate the difference of single base;
C. Mycobacterium tuberculosis drug resistance detecting chip of the present invention can detect the gene mutation information in the resistance site relevant with Rimactazid etc., coverage rate height;
The invention will be further described below in conjunction with embodiment and accompanying drawing.
Fig. 1 is the result that the Mycobacterium tuberculosis drug resistance detecting chip that the present invention includes 32 kinds of probes is used to detect actual sample.
Embodiment one is according to the newly-designed chip probe of Mycobacterium tuberculosis genomic dna sequence, and is as shown in table 1:
The probe that table 1 is designed according to the Mycobacterium tuberculosis genomic dna sequence
Gene Codon Probe sequence Genotype
????rpoB ????496 ????tc?aac?atc?cgg?ccg?gtg?gtc ????wt
????tc?aac?atc?cga?ccg?gtg?gtc ????mt
????505 ????tc?aag?gag?ttc?ttc?ggc?acc ????wt
????tc?aag?gag?ttg?ttc?ggc?acc ????mt
????507 ????ag?ttc?ttc?ggc?acc?agc?cag ????wt
????ag?ttc?ttc?gac?acc?agc?cag ????mt
????509 ????tc?ggc?acc?agc?cag?ctg?agc ????wt
????tc?ggc?acc?ttc?cag?ctg?agc ????mt
????510 ????gc?acc?agc?cag?ctg?agc?caa ????wt
????gc?acc?agc?cat?ctg?agc?caa ????mt
????511 ????cc?agc?cag?ctg?agc?caa?ttc ????wt
????cc?agc?cag?ccg?agc?caa?ttc ????mt
????512 ????agc?cag?ctg?agc?caa?ttc?at ????wt
????agc?cag?ctg?cgc?caa?ttc?at ????mt
????agc?cag?ctg?acc?caa?ttc?at ????mt
????cc?agc?cag?cgg?agc?caa?ttc ????mt
????513 ????cag?ctg?agc?caa?ttc?atg?ga ????wt
????cag?ctg?agc?cca?ttc?atg?ga ????mt
????cag?ctg?agc?aaa?ttc?atg?ga ????mt
????cag?ctg?agc?gaa?ttc?atg?ga ????mt
????cag?ctg?agc?cta?ttc?atg?ga ????mt
????cag?ctg?agc?cag?ttc?atg?ga ????mt
????514 ????ctg?agc?caa?ttc?atg?gac?ca ????wt
????ctg?agc?caa?gtc?atg?gac?ca ????mt
????tg?agc?caa?ttc?ttc?atg?gac ????mt
????c?cag?ctg?agc?atg?gac?cag?a ????mt
????c?caa?ttc?atg?ttc?atg?gac?c ????mt
????515 ????gc?caa?ttc?atg?gac?cag?aac ????wt
????gc?caa?ttc?gtg?gac?cag?aac ????mt
????gc?caa?ttc?ata?gac?cag?aac ????mt
????516 ????aat?tca?tgg?acc?aga?aca?ac ????wt
????aat?tca?tgg?tcc?aga?aca?ac ????mt
????aat?tca?tgt?acc?aga?aca?ac ????mt
????aat?tca?tgg?gag?aga?aca?ac ????mt
????aat?tca?tgg?ggc?aga?aca?ac ????mt
????518 ????tg?gac?cag?aac?aac?ccg?ctg ????wt
????tg?gac?cag?acc?aac?ccg?ctg ????mt
????tg?gac?cag?cac?aac?ccg?ctg ????mt
????c?atg?gac?cag?aac?cag?ctg?t ????mt
????520 ????c?cag?aac?aac?ccg?ctg?tcg?g ????wt
????521 ????aac?aac?ccg?ctg?tcg?ggg?tt ????wt
????aac?aac?ccg?atg?tcg?ggg?tt ????mt
????522 ????ac?ccg?ctg?tcg?ggg?ttg?ac ????wt
????ac?ccg?ctg?ttg?ggg?ttg?ac ????mt
????523 ????ccg?ctg?tcg?ggg?ttg?acc?ca ????wt
????ccg?ctg?tcg?tgg?ttg?acc?ca ????mt
????524 ????tg?tcg?ggg?ttg?acc?cac?aag ????wt
????tg?tcg?ggg?tgg?acc?cac?aag ????mt
????526 ????ggg?ttg?acc?cac?aag?cgc?cg ????wt
????ggg?ttg?acc?tac?aag?cgc?cg ????mt
????ggg?ttg?acc?ccc?aag?cgc?cg ????mt
????ggg?ttg?acc?gcc?aag?cgc?cg ????mt
????ggg?ttg?acc?ctc?aag?cgc?cg ????mt
????ggg?ttg?acc?cgc?aag?cgc?cg ????mt
????ggg?ttg?acc?cag?aag?cgc?cg ????mt
????ggg?ttg?acc?aac?aag?cgc?cg ????mt
????ggg?ttg?acc?tgc?aag?cgc?cg ????mt
????ggg?ttg?acc?caa?aag?cgc?cg ????mt
????ggg?ttg?acc?gac?aag?cgc?cg ????mt
????528 ????cc?cac?aag?cgc?cga?ctg?tcg ????wt
????cc?cac?aag?cgt?cga?ctg?tcg ????mt
????531 ????cga?ctg?tcg?gcg?ctg?ggg?cc ????wt
????gc?cga?ctg?ttg?gcg?ctg?ggg ????mt
????gc?cga?ctg?tac?gcg?ctg?ggg ????mt
????gc?cga?ctg?tgg?gcg?ctg?ggg ????mt
????gc?cga?ctg?ttc?gcg?ctg?ggg ????mt
????gc?cga?ctg?cag?gcg?ctg?ggg ????mt
????gc?cga?ctg?tgt?gcg?ctg?ggg ????mt
????533 ????ctg?tcg?gcg?ctg?ggg?ccc?gg ????wt
????ctg?tcg?gcg?ccg?ggg?ccc?gg ????mt
????ctg?tcg?gcg?cct?ggg?ccc?gg ????mt
????541 ????tg?tca?cgt?gag?cgt?gcc?ggg ????wt
????tg?tca?cgt?gat?cgt?gcc?ggg ????mt
????553 ????tg?cac?ccg?tcg?cac?tac?ggc ????wt
????tg?cac?ccg?gcg?cac?tac?ggc ????mt
????569 ????ggg?ccc?aac?atc?ggt?ctg?at ????wt
????ggg?ccc?aac?gtc?ggt?ctg?at ????mt
????579 ????cg?gtg?tac?gcg?cgg?gtc?aac ????wt
????cg?gtg?tac?ggg?cgg?gtc?aac ????mt
????583 ????gg?gtc?aac?ccg?ttc?ggg?ttc ????wt
????gg?gtc?aac?cgt?ttc?ggg?ttc ????mt
????cgg?gtc?aac?cg?ttc?ggg?ttc ????mt
????katG ????1 ????agg?aat?gct?gtg?ccc?gag?ca ????wt
????agg?aat?gct?gcg?ccc?gag?ca ????mt
????20 ????gc?aac?ggc?tgt?ccc?gtc?gtg ????wt
????gc?aac?ggc?tcc?ccc?gtc?gtg ????mt
????63 ????gcg?gcg?ttc?gac?tat?gcc?gc ????wt
????gcg?gcg?ttc?cac?tat?gcc?gc ????mt
????99 ????ggc?cac?tac?ggg?ccg?ctg?tt ????wt
????ggc?cac?tac?gag?ccg?ctg?tt ????mt
????108 ????tg?gcg?tgg?cac?gct?gcc?ggc ????wt
????tg?gcg?tgg?caa?gct?gcc?ggc ????mt
????tg?gcg?tgg?cag?gct?gcc?ggc ????mt
????122- ????125 ????c?ggc?gcc?ggg?ggc?ggc?atg?c ????wt
????c?cgc?ggc?ggc?atg?cag?cgg?t ????mt
????138 ????tgg?ccc?gac?aac?gcc?agc?tt ????wt
????tgg?ccc?gac?agc?gcc?agc?tt ????mt
????tgg?ccc?gac?cac?gcc?agc?tt ????mt
????140 ????gac?aac?gcc?agc?ttg?gac?aa ????wt
????gac?aac?gcc?aac?ttg?gac?aa ????mt
????142 ????gcc?agc?ttg?gac?aag?gcg?cg ????wt
????gcc?agc?ttg?gcc?aag?gcg?cg ????mt
????160 ????aag?aag?ctc?tca?tgg?gcg?ga ????wt
????aag?aag?ctc?gcg?tgg?gcg?ga ????mt
????172 ????ggc?aac?tgc?gcg?ctg?gaa?tc ????wt
????ggc?aac?tgc?acg?ctg?gaa?tc ????mt
????180 ????ggc?ttc?aag?acg?ttc?ggg?tt ????wt
????ggc?ttc?aag?aag?ttc?ggg?tt ????mt
????262 ????gac?gtc?gaa?aca?gcg?gcg?ct ????wt
????gac?gtc?gaa?aga?gcg?gcg?ct ????mt
????275 ????ttc?ggt?aag?acc?cat?ggc?gcc ????wt
????ttc?ggt?aag?ccc?cat?ggc?gcc ????mt
????295 ????ccg?ctg?gag?cag?atg?ggc?tt ????wt
????ccg?ctg?gag?tag?atg?ggc?tt ????mt
????300 ????ggc?ttg?ggc?tgg?aag?agc?tc ????wt
????ggc?ttg?ggc?tag?aag?agc?tc ????mt
????ggc?ttg?ggc?tga?aag?agc?tc ????mt
????ggc?ttg?ggc?ggg?aag?agc?tc ????mt
????302 ????ggc?tgg?aag?agc?tcg?tat?gg ????wt
????ggc?tgg?aag?cgc?tcg?tat?gg ????mt
????304 ????aag?agc?tcg?tat?ggc?acc?gg ????wt
????aag?agc?tcg?tgt?ggc?acc?gg ????mt
????315 ????cg?atc?acc?agc?ggt?atc?gag ????wt
????cg?atc?acc?aac?ggt?atc?gag ????mt
????cg?atc?acc?aca?ggt?atc?gag ????mt
????cg?atc?acc?aac?ggt?atc?gag ????mt
????328 ????ccg?acg?aaa?tgg?ggc?aac?ag ????wt
????ccg?acg?aaa?ggg?ggc?aac?ag ????mt
????335 ????ttc?ctc?gag?atc?ctg?tac?gg ????wt
????ttc?ctc?gag?acc?ctg?tac?gg ????mt
????336 ????ctc?gag?atc?ctg?tac?ggc?ta ????wt
????ctc?gag?atc?cgg?tac?ggc?ta ????mt
????350 ????cct?gct?ggc?gct?tgg?caa?ta ????wt
????cct?gct?ggc?tct?tgg?caa?ta ????mt
????463 ????agc?cag?atc?cgg?gca?tcg?gg ????wt
????agc?cag?atc?ctg?gca?tcg?gg ????mt
????477 ????cg?acc?gca?tgg?gcg?gcg?gcg ????wt
????cg?acc?gca?tga?gcg?gcg?gcg ????mt
????501 ????cga?ctgcag?cca?caa?gtc?gg ????wt
????cga?ctgcag?gca?caa?gtc?gg ????mt
????515 ????ggg?gat?ctg?cgc?aag?gtc?at ????wt
????ggg?gat?ctg?tgc?aag?gtc?at ????mt
????567 ????acg?gtg?ccc?ttc?acc?ccg?gg ????wt
????acg?gtg?ccc?tcc?acc?ccg?gg ????mt
????587 ????ttt?gcc?gtg?ctg?gag?ccc?aa ????wt
????ttt?gcc?gtg?ccg?gag?ccc?aa ????mt
????593 ????aag?gca?gat?ggc?ttc?cga?aa ????wt
????aag?gca?gat?gac?ttc?cga?aa ????mt
????629 ????gtg?ctg?gta?ggt?ggc?ctg?cg ????wt
????gtg?ctg?gta?agt?ggc?ctg?cg ????mt
????710 ????ctt?gtc?gag?gtc?tat?ggc?gcc ????wt
????ctt?gtc?gag?gcc?tat?ggc?gcc ????mt
?inhA ????16 ????agc?gga?atc?atc?acc?gac?tc ????wt
????agc?gga?atc?acc?acc?gac?tc ????mt
????21 ????gac?tcg?tcg?atc?gcg?ttt?ca ????wt
????gac?tcg?tcg?gtc?gcg?ttt?ca ????mt
????47 ????ctg?cgg?ctg?att?cag?cgc?at ????wt
????ctg?cgg?ctg?act?cag?cgc?at ????mt
????78 ????gcc?ggc?cgg?gtg?acc?gag?gc ????wt
????gcc?ggc?cgg?gcg?acc?gag?gc ????mt
????94 ????gtg?gtg?cat?tcg?att?ggg?tt ????wt
????gtg?gtg?cat?gcg?att?ggg?tt ????mt
????95 ????gtg?cat?tcg?att?ggg?ttc?at ????wt
????gtg?cat?tcg?cct?ggg?ttc?at ????mt
????194 ????gca?ggc?cct?atc?cgg?acg?ct ????wt
????gca?ggc?cct?acc?cgg?acg?ct ????mt
?kasA ????66 ????cac?ctc?aag?gat?ccg?gtc?ga ????wt
????cac?ctc?aag?aat?ccg?gtc?ga ????mt
????121 ????gga?gcc?gag?agg?att?gtc?ga ????wt
????gga?gcc?gag?aag?att?gtc?ga ????mt
????269 ????ctg?ggt?gcc?ggt?atc?acc?tc ????wt
????ctg?ggt?gcc?agt?atc?acc?tc ????mt
????312 ????aac?gcg?cac?ggc?acg?gcg?ac ????wt
????aac?gcg?cac?agc?acg?gcg?ac ????mt
????387 ????gtc?gtc?gcc?ggc?gaa?ccg?cg ????wt
????gtc?gtc?gcc?gac?gaa?ccg?cg ????mt
????413 ????cg?ctt?gcc?ttc?ggg?cgt?tac ????wt
????cg?ctt?gcc?tta?ggg?cgt?tac ????mt
?ahpC ????2 ????gc?gtc?atg?cca?ctg?cta?acc ????wt
????gc?gtc?atg?cct?ctg?cta?acc ????mt
????3 ????gtc?atg?cca?ctg?cta?acc?at ????wt
????gtc?atg?cca?aag?cta?acc?at ????mt
????73 ????aag?ctc?aat?gac?gag?ttc?ga ????wt
????aag?ctc?aat?cac?gag?ttc?ga ????mt
????191 ????ggc?gaa?ctc?ctc?aag?gct?tc ????wt
????ggc?gaa?ctc?cgc?aag?gct?tc ????mt
????-46 ????aat?atg?gtg?tga?tat?atc?ac ????wt
????aat?atg?gtg?taa?tat?atc?ac ????mt
????t?atg?gtg?tga?tat?ata?tca?c ????mt
????-44 ????atg?gtg?tga?tat?atc?acc?tt ????wt
????atg?gtg?tga?aat?atc?acc?tt ????mt
????-39 ????tg?tga?tat?atc?acc?ttt?gcc ????wt
????tg?tga?tat?att?acc?ttt?gcc ????mt
????-34 ????tat?atc?acc?ttt?gcc?tga?ca ????wt
????tat?atc?acc?tct?gcc?tga?ca ????mt
????-32 ????atc?acc?ttt?gcc?tga?cag?cg ????wt
????atc?acc?ttt?acc?tga?cag?cg ????mt
????-12 ????ga?ctt?cac?ggc?acg?atg?gaa ????wt
????ga?ctt?cac?ggt?acg?atg?gaa ????mt
????-10 ????tt?cac?ggc?acg?atg?gaa?tgt ????wt
????tt?cac?ggc?aag?atg?gaa?tgt ????mt
????tt?cac?ggc?aca?atg?gaa?tgt ????mt
????-6 ????ac?ggc?acg?atg?gaa?tgt?cgc ????wt
????ac?ggc?acg?ata?gaa?tgt?cgc ????mt
????-4 ????gc?acg?atg?gaa?tgt?cgc?aac ????wt
????gc?acg?atg?gga?tgt?cgc?aac ????mt
????4 ????tg?gaa?tgt?cgc?aac?caa?atg ????wt
????tg?gaa?tgt?cgt?aac?caa?atg ????mt
????33 ????tt?tga?tga?tga?gga?gag?tca ????wt
????tt?tga?tga?taa?gga?gag?tca ????mt
Annotate: wt represents wild type; Mt represents saltant
Embodiment two probe design and chip manufacturing
According to the situation of known target DNA fragment sequence (tubercle bacillus gene group) and anti-rifampin mutational site (533,532,531,526,516,514,513,511 point mutation) thereof, the probe of design length identical (20mer).Show as table 2: wherein: Mt, Wt represent saltant and wild type respectively.
Table 2
Probe number Sequence Genotype (G-C)％
????1a ????1b 5＇CTG?TCG?GCG?CTG?GGG?CCC?GG3＇ 5＇CTG?TCG?GCG?CcG?GGG?CCC?GG3＇ ?Wt ?Mt 85％ 90％
?2a ?2b ?3a ?3b ?3c ?3d ?3e ?4a ?4b ?4c ?4d ?4e ?4f ?4g ?5a ?5b ?5c ?6a ?6b ?6c ?7a ?7b ?7c ?8a ?8b ?8c 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ 5＇CGA?CTG?TCG?GtG?CTG?GGG?CC3＇ 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ 5＇GC?CGA?CTG?TtG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?TgG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Ttc?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Tac?GCG?CTG?GGG3＇ 5＇GGG?TTG?ACC?CAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?tAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CtC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CgC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gcC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?aAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gAC?AAG?CGC?CG3＇ 5＇AA?TTC?ATG?GAC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?GtC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?tAC?CAG?AAC?AAC3＇ 5＇CTG?AGC?CAA?TTC?ATG?GAC?CA3＇ 5＇TG?AGC?CAAttc?TCC?ATG?GAC3＇ 5＇CTG?AGC?CAA?gTC?ATG?GAC?CA3＇ 5＇CAG?CTG?AGC?CAA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?CcA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?aAA?TTC?ATG?GA3＇ 5＇CC?AGC?CAG?CTG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CcG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CgG?AGC?CAA?TTC3＇ ?Wt ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt 80％ 75％ 80％ 75％ 80％ 75％ 75％ 70％ 65％ 70％ 75％ 75％ 65％ 70％ 40％ 40％ 35％ 50％ 50％ 55％ 50％ 55％ 45％ 60％ 65％ 65％
Synthetic oligonucleotides is soluble in water, mix with the Spoting Solution of equal volume then, making final concentration is 75pmol/ μ l, with the micro-array chip manufacturing system dot matrix of Cartesian company in aldehyde group modified slide surface, place under the room temperature, 70% relative humidity is preserved and was fixed in 48-72 hour, then, under the room temperature slide immersed among the 0.2%SDS vibration several minutes, immerse in the pure water vibration several minutes again, immerse 0.2% SDS again three times, each 1 minute, immersed in 100 ℃ of pure water 2 seconds, volatilization is done standby then again.
The fluorescence labeling of embodiment three target DNA fragments
Design a pair of primer, produce fluorescently-labeled target DNA fragment with pcr amplification, wherein template is tubercle bacillus gene group DNA, and primer sequence is 5 ＇ AGGACGTGGAGGCGATCACA3 ＇ and 5 ＇ AACGGGTTGACCCGCGCGTA3 ＇.Amplification system is as follows: 50mM KCl, 10mMTris-HCl (PH9.0at25 ℃), 0.1%Tritonx-100,1.5mM MgCl 2, 500 μ M dGTP, dCTP, dATP, the upstream of 200 μ MdTTP, 100 μ M Cy5-dUTP, 0.4mM and downstream primer, 0.1ng/ μ l template.This system 94 ℃ the insulation 5min, then with 94 ℃ 40 seconds, 58 ℃ of 1min, 72 ℃ 40 seconds the circulation 35 times, last 72 ℃ the insulation 5min, amplified production length 307bp.
The purpose fragment of embodiment four marks and the hybridization of chip
To mix with the hybridization solution that contains various concentration TMACL that 20 μ l prepare after 99 ℃ of sex change in 5 minutes of fluorescently-labeled purpose fragment 5 μ l, drip, 60 ℃ of hybridization 30 minutes in chip.Then in 45 ℃ TMACL washing lotion swing wash 30min after airing.Get 5 μ l PCR products simultaneously and mix, drip in another chip block surface with 20 μ l hybridization solutions.In 25 ℃ of hybridization 30min in contrast, swing with 2 * SSC (containing 0.2%SDS) under the room temperature and wash 10min, and wash airing with 0.1 * SSC solution.
Embodiment five input and analysis
Chip after the hybridization carries out scanning analysis with the chip signal analytic system Scanarray3000 of General Scanning company, and according to the results of hybridization information of being suddenlyd change accordingly, the sequencing result with respective sample compares (shown in the table 3) then.(the sample order-checking entrusts Bo Ya company to finish)
3 H37Rv S Wt wt 1.1 R 526:CAC→GAC 526:CAC→GAC 1.2 R 531:TCG→TAC 531:TCG→TAC 2.1 R * 583:CCG→CG 513:CAA→AAA 2.2 R 513:CAA→AAA 569:ATC→GTC 2.3 R 531:TCG→TTG 531:TCG→TTG 583:CCG→CG 2.5 R 526:CAC→CGC 526:CAC→CGC 2.6 R 531:TCG→TTG 531:TCG→TTG 2.7 R 514:TTC insert 514:TTC insert 2.8 R 531:TCG→TTC 531:TCG→TTC 2.9 R * 583:CCG→CG 4.2 R 526:CAC→AAC 526:CAC→AAC 4.4 R 526:CAC→GCC 526:CAC→GCC 4.5 R 531:TCG→TTG 531:TCG→TTG 4.6 R 531:TCG→TTG 531:TCG→TTG 4.7 R--4.9 R 526:CAC→GAC 526:CAC→GAC4.10 R 531:TCG→TTG 531:TCG→TTG
Annotate: H37Rv: type strain; S: drug sensitivity; R: drug resistance wt: wild type; "-": do not see sudden change;
*:, therefore do not detect owing to do not design corresponding probe on the chip.
By table 3 as seen, the coincidence rate of chip detection result and sequencing result is 100%.This shows that detect the drug resistance susceptibility that is caused by the rpoB sudden change with the Mycobacterium tuberculosis drug resistance detecting chip and can reach more than 90%, specificity is 100%.
Use the Mycobacterium tuberculosis drug resistance detecting chip, from the specimen preparation to the pcr amplification,, only need 4 hours time, shortened patient's Diagnostic Time greatly to obtaining testing result.In addition, because the required sample size of this method is few, and the quantity of information that chip comprised is greatly, can obtain the drug resistance situation of a bacterial strain to all line antituberculosis drugs things with few sample.If synthetic probe comprises all mutation types, can detect 100% sudden change by single test.

Claims (9)

1. Mycobacterium tuberculosis drug resistance detecting chip on a modified microslide, is fixed with a series of oligonucleotides dot matrix probes, it is characterized in that:
(1) said probe designs at the sudden change of rpoB gene, katG gene, inhA gene, kasA gene, ahpC gene;
(2) each probe length is at least 10 bases, and each probe length is identical;
(3) catastrophe point is positioned in the middle of the probe as far as possible.
2. Mycobacterium tuberculosis drug resistance detecting chip according to claim 1, it is characterized in that said probe is primarily aimed at 496 of rpoB gene, 505,507,509,510,511,512,513,514,515,516,518,520,521,522,523,524,526,528,531,533,541,553,569,579, the sudden change of 583 codons, 1 of katG gene, 20,63,99,108,122-125,138,140,142,160,172,180,262,275,295,300,302,304,315,328,335,336,350,463,477,501,515,567,587,593,629, the sudden change of 710 codons, 16 of inhA gene, 21,47,78,94,95, the sudden change of 194 codons, 66 of kasA gene, 121,269,312,387,2 of the sudden change of 413 codons and ahpC gene, 3,73,191,-46,-44,-39,-34,-32,-12,-10,-6,-4,4, the sudden change of 33 codons.
3. Mycobacterium tuberculosis drug resistance detecting chip according to claim 2 is characterized in that said probe mainly is the sudden change at 533,532,531,526,516,514,513,511 codons of rpoB gene.
4. Mycobacterium tuberculosis drug resistance detecting chip according to claim 3 is characterized in that at the sudden change of 533,532,531,526,516,514,513,511 codons of rpoB gene and designed probe is as shown in the table: Probe number Sequence Genotype (G-C)％ ????1a ????1b ????2a 5＇CTG?TCG?GCG?CTG?GGG?CCC?GG3＇ 5＇CTG?TCG?GCG?CcG?GGG?CCC?GG3＇ 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ ?Wt ?Mt ?Wt 85％ 90％ 80％
?2b ?3a ?3b ?3c ?3d ?3e ?4a ?4b ?4c ?4d ?4e ?4f ?4g ?5a ?5b ?5c ?6a ?6b ?6c ?7a ?7b ?7c ?8a ?8b ?8c 5＇CGA?CTG?TCG?GtG?CTG?GGG?CC3＇ 5＇CGA?CTG?TCG?GCG?CTG?GGG?CC3＇ 5＇GC?CGA?CTG?TtG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?TgG?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Ttc?GCG?CTG?GGG3＇ 5＇GC?CGA?CTG?Tac?GCG?CTG?GGG3＇ 5＇GGG?TTG?ACC?CAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?tAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CtC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?CgC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gcC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?aAC?AAG?CGC?CG3＇ 5＇GGG?TTG?ACC?gAC?AAG?CGC?CG3＇ 5＇AA?TTC?ATG?GAC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?GtC?CAG?AAC?AAC3＇ 5＇AA?TTC?ATG?tAC?CAG?AAC?AAC3＇ 5＇CTG?AGC?CAA?TTC?ATG?GAC?CA3＇ 5′TG?AGC?CAA?ttc?TCC?ATG?GAC3＇ 5′CTG?AGC?CAA?gTC?ATG?GAC?CA3＇ 5＇CAG?CTG?AGC?CAA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?CcA?TTC?ATG?GA3＇ 5＇CAG?CTG?AGC?aAA?TTC?ATG?GA3＇ 5＇CC?AGC?CAG?CTG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CcG?AGC?CAA?TTC3＇ 5＇CC?AGC?CAG?CgG?AGC?CAA?TTC3＇ ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Mt ?Mt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt ?Wt ?Mt ?Mt 75％ 80％ 75％ 80％ 75％ 75％ 70％ 65％ 70％ 75％ 75％ 65％ 70％ 40％ 40％ 35％ 50％ 50％ 55％ 50％ 55％ 45％ 60％ 65％ 65％
Probe length is all 20mer mutually, and wherein Mt, Wt represent saltant and wild type respectively.
5. according to claim 1 or 2 or the method for making of 3 or 4 described Mycobacterium tuberculosis drug resistance detecting chips, it is characterized in that this method comprises the steps:
(1) carries out the synthetic of probe according to designed oligonucleotide probe;
(2) with deionized water with synthetic probe dilution, and mix with spotting solution (spoting solution) equal-volume, making final concentration is 75pmol/ μ l;
(3) slide surface is with aldehyde group modified;
(4) the micro-array chip manufacturing system of utilizing Cartesian company with the probe dot matrix with aldehyde group modified slide surface;
(5) place relative humidity 70%, fixed through 48-72 hour under the room temperature condition;
(6) under the room temperature slide immersed among the 0.2%SDS vibration several minutes, immersed in the pure water vibration several minutes, immerse again among the 0.2%SDS three times, each 1 minute, immersed again in 100 ℃ of pure water 2 seconds, volatilization is done then, and is standby.
6. the method for making of Mycobacterium tuberculosis drug resistance detecting chip according to claim 5, it is characterized in that 5 ＇ ends of said oligonucleotide probe need add the linking arm (Poly T) of certain-length (8-20 is poly-), 5 ＇ ends of this linking arm need add amino the modification.
7. the application process of Mycobacterium tuberculosis drug resistance detecting chip according to claim 1 is characterized in that this application process may further comprise the steps:
(1) sample preparation, it is a small amount of to get patient's sputum, extracts DNA with tubercle bacillus PCR hybridization diagnostic kit;
(2) fluorescence labeling:
Obtain after the DNA of testing sample, need carry out fluorescence labeling handles: 1. get the DNA sample 5 μ l that prepare and mix with 20 μ l pcr amplification systems, (amplification system comprises 2.5 μ l PCR reaction buffers, 5nmols dATP, 5nmols dGTP, 5nmols dCTP, 2nmols dTTP, 1nmol Cy5-dUTP, the upstream of each 15pmols and downstream primer, 1.25U the pfu archaeal dna polymerase) prepare fluorescently-labeled DNA sample purpose segment: at first 94 ℃ by following thermal cycle process, 5mins, with 94 ℃, 40s, 58 ℃, 1min, 72 ℃, 1min circulation 30 times, at last, 72 ℃ are extended 5mins.2. get fluorescently-labeled target DNA sample 10 μ l, use Dnase I (nuclease I) digestion to be the dna fragmentation about 200bp.
(3) chip hybridization
(4) detection and signal analysis, the chip signal analytic system Scanarray 3000 with General Scanning company scans and analysis results at last.
8. the application process of Mycobacterium tuberculosis drug resistance detecting chip according to claim 7 is characterized in that said chip hybridization is exactly:
The target DNA segment 5 μ l that get mark and interrupt, 99 ℃ of sex change 5mins are then with 20 μ l EasyHyb hybridization solution (Roche Holding Ag) mixings, get 10 μ l and drip in chip surface, covered places 60 ℃ of wet boxes to hybridize 30mins, put then to swing in 45 ℃ of washing lotions and wash 10mins, airing.
9. the application process of Mycobacterium tuberculosis drug resistance detecting chip according to claim 7 is characterized in that said upstream and downstream primer is as follows: Gene Primer sequence Tm Product length (bp) ????RpoB ?5’-AGG?ACG?TGG?AGG?CGA?TCA?CA-3’ 63.3 ???350 ?5’-GGG?TTG?ACC?CGC?GCG?TA-3’ 63.1 ????KatG ?5’-CC?GGC?TTC?CTG?TTG?GAC?GAC-3’ 64.9 ??2447 ?5’-CGG?CGC?GAT?TTG?TCA?GAC?C-3’ 64.5 ?Inh ?A Promoter ?5’-CT?CGC?TGC?CCA?GAA?AGG?GA-3’ 63.3 ???248 ?5’-CCC?CGG?TTT?CCT?CCG?GT-3’ 62.4 Structure ?5’-TT?CAT?GAC?AGG?ACT?GCT?GGA?CG-3’ 63.2 ?5’-CT?AGA?GCA?ATT?GGG?TGT?GCG?C-3’ 63.1 ????KasA ?5’-ATT?GAG?TCG?GAC?AAC?CCC?GA-3’ 62.3 ??1389 ?5’-CCT?TCC?ATA?TCG?GTC?CGA?CTC-3’ 61.9 ?Ah ?pC Promoter ?5’-CCG?ATG?AGA?GCG?GTG?AGC?TG-3’ 63.8 ???236 ?5’-C?ACT?GCT?TTG?CCG?CCA?CC-3’ 62.9 Structure ?5’-CTT?GCG?GCA?CTG?CTG?AAC?C-3’ 62.3 ?5’-GCT?GCT?GCG?GGT?GAT?TGA?G-3’ 62.2
CNB011059818A 2001-04-13 2001-04-13 Drug tolerance test chip for Mycobacterium tuberculosis and its preparing method and utilization method Expired - Lifetime CN1153969C (en)

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Cited By (10)

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CN102618625A (en) * 2011-01-27 2012-08-01 博奥生物有限公司 Method and kit for detecting drug resistance of mycobacterium tuberculosis
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CN100412202C (en) * 2003-04-14 2008-08-20 赵雨杰 Mycobacterium tuberculosis diagnosis and drug resistance analysis chip, its preparation process and application method
CN102010907B (en) * 2010-11-09 2013-03-20 广州益善生物技术有限公司 Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection
CN102010907A (en) * 2010-11-09 2011-04-13 广州益善生物技术有限公司 Specific primer and liquid-phase chip for katG, inhA and ndh genetic mutation detection
CN102618625B (en) * 2011-01-27 2013-09-11 博奥生物有限公司 Method and kit for detecting drug resistance of mycobacterium tuberculosis
CN102618625A (en) * 2011-01-27 2012-08-01 博奥生物有限公司 Method and kit for detecting drug resistance of mycobacterium tuberculosis
CN102304574B (en) * 2011-08-25 2013-03-20 广东省结核病控制中心 Diagnostic method for rifampicin resistant mycobacterium tuberculosis
CN102304574A (en) * 2011-08-25 2012-01-04 广东省结核病控制中心 Diagnostic method for rifampicin resistant 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
CN103045716A (en) * 2011-10-11 2013-04-17 上海市肺科医院 Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology
CN103045716B (en) * 2011-10-11 2015-04-08 上海市肺科医院 Method for detecting isoniazid drug resistance of mycobacterium tuberculosis by using pyrosequencing technology
US11180816B2 (en) 2014-10-10 2021-11-23 Rutgers, The State University Of New Jersey Polymerase chain reaction primers and probes for Mycobacterium tuberculosis
JP2018500024A (en) * 2014-12-18 2018-01-11 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ Compositions and methods for detection of drug-resistant Mycobacterium tuberculosis
JP7036595B2 (en) 2014-12-18 2022-03-15 エフ．ホフマン－ラ ロシュ アーゲー Compositions and Methods for Detection of Drug-Resistant M. Tuberculosis
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CN105316349A (en) * 2015-11-19 2016-02-10 昆明理工大学 Mycobacterium tuberculosis KatG mutant gene and application thereof

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