CN1273364A - Detection method of special DNA chip for diagnosis of pathogenic bacteria and disease-related gene mutation - Google Patents

Abstract

The utility model discloses a new detection method of a DNA chip special for pathogenic bacteria and disease related gene mutation diagnosis. It utilizes semiconductor microelectronic processing technology to form medium-density micro-gold electrode array on the glass slide, and fixes different DNA probes on the surface of the array electrode. The specific gene of various pathogenic bacteria and the mutation of the gene related to diseases are detected by a specific DNA probe hybridization method on the surface of an electrode array. Detecting hybridization signals by an electrode electrochemical method or a laser-excited fluorescence scanning method. The invention can simultaneously detect a plurality of pathogenic bacteria and disease-related gene mutation and has the characteristics of parallel analysis and multiple analysis.

Description

The detection method of pathogen and disease-correlative gene mutation DNA chip dedicated for diagnosing

The present invention relates to the detection method of a kind of pathogen and disease-correlative gene mutation DNA chip dedicated for diagnosing.

In the prior art, the detection of pathogen mainly is to utilize micro-biological process, serological method and PCR method.Existing detection method as enterohemorrhagic Escherichia coli O 157: H7 is: sample thief 25g, the mEC nutrient culture media 225ml of first day interpolation ovobiocin (increasing bacterium cultivates) (42 ℃, 18～24 hours); Added SIB agar medium (isolation medium) (35 ℃, 24 hours), and select 20 above bacterium colonies in second day; Added CLIG agar slant culture-medium (confirming to cultivate) (35 ℃, 18～24 hours) on the 3rd day and carry out O157 aggregation (seroreaction) to positive; Carried out toxin on the 4th day and produce experiment, the H type is judged.

The detection of disease-correlative gene mutation mainly contains (1) PCR-RFLP (restriction fragmentlength polymorphism), this method is utilized multiple restriction enzyme that gene amplification product is carried out enzyme and is cut, different gene orders will produce different electrophoresis pattern (Mercier, B.et al, Eur.J.lmmunogenetics, 21,105,1994).This method can only be applied to when sudden change has changed a certain restriction enzyme site.The improvement of this method is to introduce restriction enzyme site method, i.e. PCR-AIRS (Mut.Res, 285,125,1993) in the mutational site.(2) PCR-SSO (sequence specificoligonucleotide) or PCR-ASO (allele specific oligonucleotide) (Cotton, R.G.H.Mut.Res, 285,1251993; Saiki, R K et al, Nature, 324,163,1986), testing gene is behind pcr amplification, respectively with the wild type and the saltant probe hybridization of 15-20bp mark.This method need be carried out marks such as isotope labeling or digoxin, biotin, peroxidase, and the analytic process complexity is not suitable for express-analysis.(3) PCR-SSP (sequence specific primers) or ASPCR (allelespecific PCR), its principle is that the character according to the known mutations site designs a base mismatch in primer, makes it only can increase saltant or wild type gene.This method is comparatively fast and convenient.(Wu，D.Y，etal，Proc，Natl.Acad.Sci.USA，86，2757，1989；Rust，S，etal，Nucleic?AcidsResearch，21，3623，1993；Newton，R?et?al，Nucleic?Acids?Research，17，2503，1989)。(4) PCR-SSCP (single-stranded conformation polymorphism), SSCP be meant single strand dna in neutral PAGE electrophoretic mobility with the character of its conformational change, therefore can be used as the detection method of genetic mutation, be used for oncogene point mutation and people's gene group polymorphism research (Orita by Orita the earliest, M, et al, Proc Natl Acad Sci USA, 86,2766,1989), this method only can detect the existence of genetic mutation, and can not determine the position and the content of sudden change.(5) dna sequencing method, this is the most directly perceived, method the most accurately.But technical sophistication costs an arm and a leg, can not be as conventional method.

In sum, in the prior art of relevant pathogen and disease-correlative gene mutation diagnosis, have trivial operations, required time is long, and cost is higher, is difficult to problems such as robotization and great amount of samples parallel analysis.

Purpose of the present invention is exactly at the deficiencies in the prior art, proposes the detection method of a kind of pathogen and disease-correlative gene mutation DNA chip dedicated for diagnosing.

The present invention takes following measures in order to achieve the above object:

Utilize the semiconductor microelectronic processing technique on slide, form in density (＞2000/microscopeslide) little gold electrode array is fixed in array electrode surface with the self assembly technique for fixing with different dna probes with array point sample instrument (Arrayer) in the different gold electrode surfaces of electrod-array.Detect specific gene and the disease-correlative gene mutation of various pathogens with the specificity DNA probing needle hybridizing method of electrode array surface.Crossover process can freely be controlled by different electrode in the electrod-array with the hybridization zone.(electrogenerated chemiluminescence, ECL), electrode electro Chemical method or LASER Excited Fluorescence scan method detect hybridization signal with electrochemiluminescence.

Advantage of the present invention:

In a microscope slide surface, set up 10 or more regional microelectrode array (10 * 10) with the semiconductor microelectronic processing technique, therefore can detect various pathogens and disease-correlative gene mutation simultaneously.Have the multiple analysis characteristics, and the hybridization of each area array and testing process can independently be controlled and detect by electrochemical method.A kind of area array is used for the multiple relevant sudden change of several related genes of parallel repeated detection or a kind of gene, has the characteristics of parallel analysis and multiple analysis simultaneously.Stationary probe crossover process on sample DNA and the chip is accelerated in the low current pulse, and oppositely the low current pulse can be distinguished and join hybridization and the hybridization of single base mispairing entirely.Electrochemical luminous detection method does not need expensive confocal fluorescent microscope or fluorescent scanning instrument.The detection of pathogenic bacteria and disease-correlative gene mutation can foreshorten in 5 hours and finish.

Elaborate below in conjunction with drawings and Examples:

Fig. 1 is area array electrode and terminal pin synoptic diagram;

Fig. 2 is the gold-plated making synoptic diagram of area electrodes;

Fig. 3 is that the DNA special chip is made synoptic diagram;

Fig. 4 is a DNA chip hybridization control action synoptic diagram.

The manufacturing process of little gold electrode array of the present invention is: pattern Figure 11 of first design specialized chip, on slide, plate thechromium 15 of one deck 20nm with certain pattern, plate thegold 16 of one deck 200nm again at the chromium laminar surface, the mask mode that utilization designs, asinsulation course 17, make upgold electrode array 13 with 2 μ m silicon nitrides in different gold thin film zones.Each area electrodes array utilizes same lead-inwire 14 and electrode change-over switch 18, can carry out galvanochemistry control 19.

Being fixed as of dna probe: with sulfydryl modification dna probe (SH-(CH₂)₆-DNA oligo), utilizes sulfhydryl compound to form self assembled monolayer character, dna probe is fixed in electrode surface in active gold surface.Or with ll-Mercaptoundecanoic acid (HS-(CH₂)₁₀-COOH) form self assembled monolayer in gold surface, make gold electrode surfaces have pendant carboxylic group, under EDC and NHS effect, amido modified dna probe can be fixed in the array electrode surface.

DNA chip surface crossover process is: the DNA chip of structure inserts DNA chip dedicated hybridization control enclosure, use the low electric conductivity hybridization solution, utilization to electrode and the pulse of area array electrode field current (toelectrode 12 for negative,area array electrode 13 is for just), impel the DNA sample to concentrate, accelerate stationary probe crossover process on sample DNA and the chip in hybridization array electrode zone; Reverse current pulses stimulates, and removes the DNA and the base mispairing hybrid dna of non-specific adsorption.With the low current pulse action hybridization was finished in 10 minutes; Oppositely the low current pulse can be distinguished and join hybridization and the hybridization of single base mispairing entirely.

Hybridization signal detects: the present invention detects hybridization signal with two kinds of methods, uses the fluorescence labeling sample DNA for first kind, and DNA chip hybridization signal is detected with confocal fluorescent microscope or fluorescent scanning instrument in the hybridization back; Second kind is after the hybridization of non-marked sample DNA, adds specific bond double-stranded DNA reagent, as Ru (Phen)₃²⁺, electrochemiluminescence detects in the presence of tri-n-propylamine.

The embodiment design is as the chip mode of Fig. 1, on glass sheet, plate thechromium 15 of one deck 20nm with this pattern,, utilize the mask mode of 10 * 10 arrays again at thegold 16 of chromium laminar surface plating one deck 200nm, asinsulation course 17, make upgold electrode array 13 with 2 μ m silicon nitrides in different gold thin film zones.Make then and detect pathogenic entero becteria: toxin originality Escherichia coli, intestinal bleeding Escherichia coli O 157: H7, Salmonella, staphylococcus aureus Staphylococcus aursus, the DNA chip of the resistance to the action of a drug somatotype of vibrios Vibrio cholerae and tubercle bacillus Mycobacterium tuberculosis.

As previously mentioned, the existing method of pathogenic entero becteria detection is microbe culture and serological method.The shortcoming that life period is long, general 3～5 days.Required time for the resistance to the action of a drug somatotype of tubercle bacillus is longer, because growth of bacillus tubercle is very slow, detects and resistance to the action of a drug analysis generally needed for 6～8 weeks.Whole world mycobacterium tuberculosis infection number accounts for 1/3 of population, annual dead about 3,000,000 people.

1. several pathogenic entero becterias and tubercle bacillus resistance to the action of a drug somatotype specific DNA probe design toxin originality Escherichia coli: with heat-sensitive toxin I (LTI) and heat-sensitive toxin II (LTII) is the gene of target; Designed probe is

The probe of LTI: LTI-1, GGTCTCGGTCATATATGTGATTC and LTI-2, TAGAGAGGATAGTAACGCCGTAA is fixed in I, the III electrode position ofarray region 1 respectively with 5 * 5 arrays.

The probe of LTII: LTII-1, CAATAAAATCATCTTCGCTCATG and LTII-2, TTTGCTGAAACAGAAAATGATAT is respectively with II, the IV electrode position of 5 * 5 array regions 1.Intestinal bleeding Escherichia coli O 157: H7: with vero toxin stx2 gene is the gene of target, and the designed probe sequence is that GCCGTATTAACGAACCCGGG is fixed inarea array 2 electrode positions.Salmonella: belonging to specificity is target sequence with 16SrDNA, and probe sequence is TGCTGCCGTTATTAACCACCACA, is fixed in I, the II electrode position ofarea array 6; For STyphimurirm, be the gene of target with its enterotoxin STN gene, the designed probe sequence is GATATTATTACTCACTCCCTG and GTTGCCAGTGATAGCGAGACA, is individually fixed in III, the IV electrode position of area array 6.Staphylococcus aureus: with its enterotoxin genes is the gene of target, and the designed probe sequence is TTCAGTAATGCCACCATAGGC and GTATTTTGTTTACCGTCTAGC, is individually fixed in I, the II electrode position of area array 4.Vibrios Vibrio cholerae: with the CT toxin gene is the gene of target, and designed probe is TTAAACATCCAAAGCAAGCT and TTTGGCTAAGGGCGATTGAA, is individually fixed in III, the IV electrode position of area array 4.The resistance to the action of a drug somatotype of tubercle bacillus Mycobaclerium tuberculosis: different cigarette callosity resistance to the action of a drug somatotype---area array 5

The sudden change of KatG (catalase-peroxidase) gene-correlation; The S315T detection that suddenlys change;

The sudden change of ImhA (NADH-dependent2-trans enoyl-acyl carrier protein reductase) gene-correlation

The relevant sudden change of zone line of ahpC (alkylhydroperoxide reductase) gene-correlation sudden change oxyR gene and ahpC gene: 641-GACTCTCCTCATCATCAAATGAT ATATCACACCATATTTATCGGG-745 (Genbank, Accession No.U16243), G651A, C680T, A687G, G689A, G692A, C693A/T, C695T, G715A, T717C, C722T, T727A, G729A.With primer GCTTGATGTCCGAGAGCATCG and GGTCGCGTAGGCAGTGCCCC amplification zone line segment.Rifampin resistance to the action of a drug somatotype----area array 3

The sudden change of rpoB (β subunit of RNA polymerase) gene-correlation, there is sudden change in 96% rifampicin resistance tubercle bacillus at the 81bp of rpoB gene zone line (508AAA-532AA), as His526Tyr, Ser531Leu etc.Design point sudden change distinguished sequence is fixed in area array 7.Sample increases with primer GGCCGGTGGTCGCCGCG and ACGTGACAGACCGCCGGGC.The Met306 sudden change of ethambutol (ethambutol) resistance to the action of a drug somatotype----area array 7embB gene:(Genbank, Accession No.U68480) G AC T

C

Design detects the point mutation probe of A7868 → G/C and G7870 → A/T/C.Streptomycin resistance division----area array 8

The relevant sudden change of rrs (16s rRNA) gene: C491T, C512T, A513T, C798T, G877A, A904G and A906C.With primer GAGAGTTTGATCCTGGCTCAG and TGCACACAGGCCACAAGGGGA amplification rrs gene segment.

The relevant sudden change of RpsL (ribosomal protein S12) gene; K43R (AAG → AGG) and K88Q (AAG → CAG).With primer GGCCGACAAACAGAACGT and GTTCACCAACTGGGTGAC amplification rpsL gene segment.Pyrazinamide resistance to the action of a drug somatotype----area array 9

The sudden change of pnaA (pyranzinamidase) gene-correlation: relevant sudden change mainly concentrates on A13-C36, C205-G255 and G394-G426 section Genbank, Access ion No.U59967), the relevant mutant probe of design is fixed in area array 9.Fluoroquinolones resistance to the action of a drug somatotype----area array 10

The sudden change of gyrA (A subunit of DNA gryase) gene-correlation; Relevant sudden change mainly concentrates on 2566-GGCGACGCGTCGATCTACGACAG-2590 section (Genbank, Accession No.L27512), as G2567T, and C2574T, T2576C, G2585T/C/A, A2586G/C, G2589C.The relevant mutant probe of design is fixed in area array 10.With primer CAGCTACATCGACTATGCGA and GGGCTTCGGTGTACCTCAT amplification gyrA gene segment.

2. sample preparation:

The gene or the sudden change zone of the target that detects with the PCR primer amplification of design as detecting with fluorescent method, add the dNTP of Cy5 mark during pcr amplification.

3. chip surface hybridization and detection:

Characteristics per sample, (to electrode is negative to the current impulse between electrode and the area array electrode in utilization, the area array electrode is for just), impel the DNA sample to concentrate at the specific array electrode zone, accelerate stationary probe crossover process on sample DNA and the chip: reverse current pulses stimulates, and removes the DNA and the base mispairing hybrid dna of non-specific adsorption.Use the confocal fluorescent microscopic examination.

Like this, the DNA chip that makes up can the fast detecting pathogenic entero becteria: toxin originality Escherichia coli, intestinal bleeding Escherichia coli O 157: H7, Salmonella, staphylococcus aureus Staphylococcus aureus, the resistance to the action of a drug somatotype of vibrios Vibrio cholerae and tubercle bacillus.

Claims (4)

1. The detection new method of pathogen and disease-correlative gene mutation DNA chip dedicated for diagnosing it is characterized in that utilizing the semiconductor microelectronic processing technique on slide, form in little gold electrode array of density, different gold electrode surfaces at electrod-array are fixed in the array electrode surface with the array point sample instrument with different dna probes, detect specific gene and the disease-correlative gene mutation of various pathogens with the specificity DNA probing needle hybridizing method of electrode array surface, crossover process is freely controlled by different electrode in the electrod-array with the hybridization zone, use electrochemiluminescence, electrode electro Chemical method or LASER Excited Fluorescence scan method etc. detect hybridization signal.
2. 2. the detection new method of pathogen according to claim 1 and disease-correlative gene mutation DNA chip dedicated for diagnosing is characterized in that the manufacturing process of said little gold electrode array is: the mode chart of first design specialized chip [11], on slide, plate one deck chromium [15] with certain pattern, again at chromium laminar surface plating one deck gold [16], utilize the mask mode, with silicon nitride as insulation course [17], make up gold electrode array [13] in different gold thin film zones, each area electrodes array utilizes same lead-in wire [14] and electrode change-over switch [18] to carry out galvanochemistry control [19].
3. 3. what it is characterized in that said dna probe according to the detection new method of claim 1 or 2 pathogens and disease-correlative gene mutation DNA chip dedicated for diagnosing fixedly is to use the sulfydryl modification dna probe, utilize sulfhydryl compound to form self assembled monolayer character in active gold surface, the self assembled monolayer that dna probe is fixed in electrod-array or forms in gold surface, make gold electrode surfaces have pendant carboxylic group, amido modified dna probe is fixed in the array electrode surface.
4. 4. the detection new method according to claim 1 or 2 pathogens and disease-correlative gene mutation DNA chip dedicated for diagnosing is characterized in that said DNA chip surface crossover process is: the DNA chip of structure inserts DNA chip dedicated hybridization control enclosure, use the low electric conductivity hybridization solution, utilization is to electrode and the pulse of area array electrode field current, impel the DNA sample to concentrate, accelerate stationary probe crossover process on sample DNA and the chip in hybridization array electrode zone; Reverse current pulses stimulates, and removes the DNA and the base mispairing hybrid dna of non-specific adsorption, with the low current pulse action hybridization is finished in 10 minutes; Oppositely the low current pulse separation is joined hybridization and the hybridization of single base mispairing entirely.

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