CN1456685A - Multiple morbifical agent specimen detecting method - Google Patents

Abstract

A method for detecting multiple pathogen specimens by combination of gene chip with PCR technique features that based on the targeting and automatic recognition characteristics of gene bar code and the features of PCR amplification to modify terminal 5' of amplified sequence, a great number of specimens can be analyzed by chip hybridizing once, and the report probes and quenching probes are used in chip hybridization for quenching the excessive report probes, so increasing efficiency.

Description

The novel method that one kind of multiple pathogenic agent samples detect

Technical field the present invention relates to a kind of application of biological technology products, specifically utilizes genes encoding identification general-purpose chip in conjunction with round pcr, realizes analyzing simultaneously by the once hybridization of chip the purpose of many parts of pathogenic agent samples.

Background technology is along with human genome (order-checking) plan finishes and the fast development of molecular biology related discipline, on a large scale, high-throughout analytical technology more and more comes into one's own, 26S Proteasome Structure and Function genome research, sequencing, medical diagnosis on disease, drug screening etc. all be unable to do without the high throughput analysis of samples technology.Gene chip (claiming the DNA chip again) technology is exactly to comply with the product of this scientific development requirement.It melts microtronics, biology, physics, chemistry, computer science is the new technology that the height of one intersects, have simple to operate, characteristics such as level of automation is high, sequence quantity is big, detection efficiency is high, applied range, satisfied to a great extent at present needs extensive, the high throughput analysis technology.

Genes encoding identification general-purpose chip technology is that (it is used to carry out high-throughout single base point mutation at first and detects for J.MOL.BIO.1999,292:251-262) invention by F.Barany etc.It is by realizing that in conjunction with PCR, ligase enzyme detection technique (LDR) and genes encoding hybridization technique it detects principle and sees accompanying drawing 1.At first be that the synthetic many group oligonucleotide probes of design are right, one in the every pair of probe comprises target gene distinguished sequence and addressable gene bar code sequence, and 3 ' end of this probe is mutating alkali yl, another comprises target gene distinguished sequence and examining report molecule, article two, the contiguous sequence complementation of probe and target gene, like this, when the hybridization of two probes and target-gene sequence and when adjacent to each other, ligase enzyme is that template connects two sections probe sequences with the target-gene sequence, like this when hybridizing, the gene bar code sequence that contains addressing can be fixed and the chip of addressing gene bar code sequence complementary probe is caught, and shows signal by reporter molecules again.Because have only complete and template complementation when two probes, ligase enzyme could be discerned, if there is single base mutation then can not be connected, and all have one to be connected with addressable gene bar code probe in every pair of probe, have no signal to have or not to produce the sudden change etc. which kind of type sudden change and which kind of gene produce according to the point of this addressing probe correspondence like this with regard to decidable sample to be checked.The addressable gene bar code sequence that is adopted in present method is the artificial designing probe that produces at random, its sequence is unique, therefore the chip of being made up of these sequences can be used as general-purpose chip, the sudden change that is used for any gene detects, and only needs the sequence of the target nucleic acid that conversion connected after the addressable bar code sequence just can realize once.Have characteristics such as high-throughput, specificity is good, highly sensitive, background is low with suddenly change detection, snp analysis of present method.

The method that detects at present pathogenic agent clinically mostly is serological method, molecular hybridization and PCR method etc., and is the sensitiveest with the PCR method in these methods, can reach fg level (10 ⁵Copy).But it generally is through agarose gel electrophoresis with the product after the amplification that conventional PCR detects, behind the ethidium bromide staining, under ultraviolet lamp, have or not the appearance of specific fluorescence band to judge the negative and positive result by observation, it is bigger that its result's accuracy, reliability are influenced by subjectivity and objective factor, and ethidium bromide has strong carcinogenic toxicity simultaneously.Though and based on the real time pcr of fluorescent energy transfer principle can be special, detect many parts of samples simultaneously delicately, it is all very high to fluorescent probe, PCR conditional request, has slightly and does not note, just may cause to detect and fail.The application of DNA chip technology in pathogen detection becomes the effective means of assisting clinical diagnosis, treatment disease.But present widely used chip detection generally is a sample once hybridizes, and after using many fluorescent marks technology, its sample analysis ability obtains certain raising, but because examined technology limitation (general instrument can only detect 4 kinds of fluorescence at most simultaneously) still can not realize once hybridizing the purpose that is detected as ten up to a hundred parts of samples.And conventional round pcr can realize not only that in conjunction with gene bar-code identification general-purpose chip technology many samples analyze simultaneously, and has overcome that aforesaid method detects inconvenience, specificity is relatively poor or be subject to shortcoming such as experiment condition influence.

The combined probe technology be based on fluorescent energy transmission technology (FRET) and the design a kind of new technology, be mainly used in carry out quantitative PCR research (Wang SQ et al, Anal.BioChem.2002).Compare with other method such as TaqMan, Amplisensor, Molecular beacon and LightCycler based on FRET, this law cancellation is thorough, and is synthetic easy with mark, low cost and other advantages.It is as follows that it detects principle: combined probe is made of a long fluorescent hybridization probe and short cancellation probe, fluorescent probe 5 ' termination one fluorescent reporter molecule wherein, 3 ' termination one is extended blocker molecule phosphoric acid, cancellation probe 3 ' end connects a quencher molecule-paramethyl red, cancellation probe and fluorescent probe 5 ' end is complementary, during no template, this probe hybridization forms combined probe, no fluorescence produces, when template, fluorescent probe and template hybridization, fluorescence can not be by cancellation, the fluorescence that produces is directly proportional with the template amount, thereby can carry out accurately quantitatively initially touching plate.Biochip technology need be washed after hybridization usually, removes the not tagged molecule of hybridization, in order to avoid produce higher background; but because all factors; although in experiment, occur through repeatedly washing through regular meeting, the situation that background is still very strong, influence is to the judgement of net result.

Summary of the invention

The objective of the invention is to utilize gene bar-code identification general-purpose chip to carry out many analyzing specimens, the novel method that provides a kind of many pathogenic agent sample to detect simultaneously, in conjunction with the combined probe technology, make chip detection need not to carry out the multistep washing and can reach low background simultaneously, increase work efficiency.Its basic ideas are (seeing accompanying drawing 2): specific amplification primer and the reporter probe and the cancellation probe of the synthetic pathogen gene to be checked of design, wherein a primer extends a segment addressing bar code sequence to 5 ' end, one spacerarm is arranged between this sequence and primer, can stop PCR to extend, and this sequence and the addressable capture probe complementation that is fixed on the general-purpose chip, make the end of product connect the addressable bar code sequence of the preceding paragraph by pcr amplification, then all pcr amplification products are mixed, with reporter probe, the cancellation probe joins the hybridization system together, hybridize with general-purpose chip, complementary addressing capture probe hybridization on addressable bar code sequence that connects on these PCR products and the chip like this, thereby amplified production directionally is anchored on the chip correspondence position, the PCR product that is anchored is hybridized with the special reporter probe of reporter molecules marks such as fluorescence or vitamin H again, can detect the result of every duplicate samples by analyses such as signal scannings.Because one addressing bar code sequence can corresponding a sample, there are many addressing bar code sequences just can corresponding many minutes samples, just can detect many parts of samples simultaneously by hybridization once like this, thereby realize the purpose of many samples detections.Simultaneously, in the chip hybridization system, add reporter probe and cancellation probe respectively, the cancellation probe can be hybridized with excessive reporter probe, make fluorescence by cancellation, even do not wash after the hybridization like this, the background of chip also can be very low, thereby saved the step of multistep washing behind the chip hybridization, improved working efficiency.

The principle schematic accompanying drawing 2 genes encodings identification general-purpose chip of principle schematic (b) the general-purpose chip check point sudden change that appended drawings 1 ligase enzyme detection technique detects in conjunction with principle schematic (a) ligase enzyme of genes encoding identification general-purpose chip check point sudden change detects the schematic appearance of synoptic diagram accompanying drawing 3 general-purpose chips of many samples and dot matrix layout accompanying drawing 4 reporter probe consumptions influences influence (a) scanning image of accompanying drawing 5 hybridization time to hybridization signal to hybridization signal: according to from left to right, putting in order from top to bottom is followed successively by 2,5,10,20,30, and scanning image (b) data analysis result accompanying drawing 6 hybridization temperatures of 60 minutes correspondences are to the application of the accompanying drawing 7 combined probe technology that influence in hybridization detects of hybridization signal

From left to right be followed successively by first group (no cancellation probe does not wash after the hybridization), second group (no cancellation probe, the multistep washing of hybridization back), the 3rd group (add the cancellation probe, do not wash after the hybridization), the 4th group (adding the cancellation probe, the multistep washing of hybridization back).The application of accompanying drawing 8 genes encodings identification general-purpose chip in clinical samples detects

Introduce principle of the present invention, main contents and specific operation process in detail below in conjunction with embodiment.

The specific embodiment present embodiment with HBV DNA as detected object.1. the design of addressing bar code capture probe is with synthetic

According to (J.MOL.BIO.1999 such as F.Barany, 292:251-262) the addressing bar code probe design principle of Jie Shaoing, design 16 (number of probes can increase and decrease on demand) long 24mer, Tm value close (by the OLIGO6.0 computed in software), and the probe that does not have homology each other, its sequence and corresponding numbering see Table 1.Synthesize on 391 dna synthesizers (Applied Biosystems) and finish, 3 of probe ' end carries out amido modified.Utilization is based on the cyanoethyl-N ' of four condensed ethandiols, and the inferior phosphinylidyne ammonia reagent of N '-di-isopropyl is introduced spacerarm between amino reagent and oligonucleotide probe.Synthetic 55 ℃ of deprotections of the back strong aqua/cutting 15hr that finishes, through the reversed-phase column purifying, the quantitative final vacuum of ultraviolet is concentrated into dried, and-20 ℃ of preservations are standby.

The sequence and the numbering of table 1 addressing bar code capture probe

Numbered sequence (5 ' → 3 ') Tm (℃)

Bar1 GTCT?TCTG?GCAA?TCGT?CTCA?CGTT-spacer-NH2 71.1

Bar2 GCAA?TCTG?ACCT?TCGT?CCAT?CGTT-spacer-NH2 72.9

Bar3 ACCT?TCTG?AGGA?TCGT?TGTC?CGTT-spacer-NH2 71.7

Bar4 AGGA?TCTG?GTCT?TCGT?CTTG?CGTT-spacer-NH2 72.0

Bar5 TCTG?TGTC?TCGT?GCAA?CGTT?CTCA-spacer-NH2 71.4

Bar6 TCGT?TGTC?CGTT?GCAA?CTGT?CTCA-spacer-NH2 71.4

Bar7 CGTT?TGTC?CTGT?GCAA?TCTG?CTCA-spacer-NH2 73.6

Bar8 CTGT?TGTC?TCTG?GCAA?TCGT?CTCA-spacer-NH2 70.7

Bar9 CGTT?GGTA?CTCA?TGTC?CCAT?CTGT-spacer-NH2 69.4

Bar10?CTCA?GGTA?CTTG?CGTT?TGTC?CCAT-spacer-NH2 71.0

Bar11?CTGT?GGTA?CCAT?TGTC?CGTT?GAGT-spacer-NH2 71.4

Bar12?CTTG?CCAT?CTCA?GGTA?TGTC?GGTT-spacer-NH2 70.9

Bar13?TCTG?CGAA?TGTC?CTGT?ACCT?TCGT-spacer-NH2 71.5

Bar14?TGTC?CAGA?AGTG?CTTG?CTGT?TCGT-spacer-NH2 72.5

Bar15?TCGT?AGTG?CTTG?ACCT?TCTG?CTGT-spacer-NH2 69.7

The preparation of Bar16 CTTG GCAA AGTG CTGT TCGT ACTC-spacer-NH2 69.72. general-purpose chip

Make square grid with thick about 50 microns waterproof paper, it closely is affixed on the slide of aldehyde radicalization, make slide be divided into 10 square sub-districts separated from each other, the length of side in each district is 9 millimeters.Then the concentration of amido modified capture probe with 50 μ mol/L is dissolved in 3 * SSC solution, put in each square sub-district on the aldehyde radical slide with Pix sys 5500 chip preparing instruments (Catesian Technologies), so just obtained comprising the chip (arrangement of probe matrix and chip synoptic diagram are seen accompanying drawing 3) of 10 just the same probe matrixes, dry after chip point system finishes, put on the Glass carrier box room temperature and place standby.3.HBV the design of DNA special primer, reporter probe and cancellation probe is with synthetic

With reference to the design of primers principle, choose the sequence that is positioned at nt2267-2288 and nt2356-2378 on the HBV dna sequence dna and detect upstream and downstream primers F 1, the R1 (sequence sees Table 2) of HBV as pcr amplification, F1 is positioned on the normal chain of dna sequence dna, and R1 is positioned at the minus strand of sequence.According to combined probe analysis principle design report probe FL and cancellation probe Q, primer and probe are all synthetic with 391A type dna synthesizer.Reporter probe and the complementation of target sequence minus strand are positioned at the nt2326-2353 of target sequence, are made up of 28 Nucleotide, and its 5 ' end is with a fluorescein molecule; The long 21mer of cancellation probe, complementary with fluorescent probe 5 ' end, its 3 ' end connects a quencher molecule-paramethyl red.

Table 2 is at the primer and the probe sequence of HBV DNA design

Numbered sequence (5 ' → 3 ') sequence location (nt)

F1 GGA?GTG?TGG?ATT?CGC?ACT?CCT?C 2267-2288

R1 TTC?TTC?TTC?TAG?GGG?ACC?TGC?CT 2378-2356

FL Cy3-ACT?TCC?GGA?AAC?TAC?TGT?TGT?TAG?ACG?A 2326-2353

Q ACA ACA GTA GTT TCC GGA AGT-Dabcyl 2346-23264. addressing gene bar code primer design

Because designed reporter probe and the complementation of target sequence minus strand, this just decision be should be minus strand by what chip was caught, just mean that also addressing bar code sequence should be positioned at minus strand, according to this point, we will be connected with the 5 ' end of HBV downstream primer R1 with addressing capture probe complementary sequence, introduce an ethylene glycol when synthetic as spacer groups between two sequences, to stop pcr amplification, each primer sequence and numbering see Table 3.

Table 3 detects the primer that contains gene bar code sequence of HBV DNA

Numbered sequence (5 ' → 3 ')

Bar1R1 AACGTGAGACGATTGCCAGAAGAC-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar2R1 AACGAGTTACGAAGGTCAGATTGC-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar3R1 AACGGACAACGATCCTCAGAAGGT-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar4R1 AACGCAAGACGAAGACCAGATCCT-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar5R1 TGAGAACGTTGCACGAGACACAGA-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar6R1 TGAGAACGTTGCACGAGACACAGA-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar7R1 TGAGCAGATTGCACAGGACAAACG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar8R1 TGAGACGATTGCCAGAGACAACAG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar9R1 ACAGATGGGACATGAGTACCAACG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar10R1?ATGGGACAAACGCAAGTACCTGAG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar11R1?ACTCAACGGACAATGGTACCACAG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar12R1?AACCGACATACCTGAGATGGCAAG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar13R1?ACGAAGGTACAGGACATTCGCAGA-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar14R1?ACGAACAGCAAGCACTTCTGGACA-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

Bar15R1?ACAGCAGAAGGTCAAGCACTACGA-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCCT

The pcr amplification of Bar16R1 GAGTACGAACAGCACTTTGCCAAG-OCH2CH2O-TTCTTCTTCTAGGGGACCTGCC T5.HBV DNA

Get patients serum 50ul, add 10 μ l Lysis Buffer, boil 15min, the centrifugal 10min of 12000rpm, get supernatant 5 μ l, add in the following reaction system: PCR Mix 20 μ l, upstream primer F1 0.5 μ M, downstream addressing gene bar code primer BarR1 1 μ M, TaqPolymerase (5u/ μ l) 0.2 μ l.94℃?5min；[94℃?30?Sec，55℃?30Sec，72℃?30Sec]40?cycles；72℃?3min。Each PCR pipe is done with addressing bar code primer and corresponding sample and is indicated accordingly, in case mix up.Establish positive control (is template with the plasmid that contains the HBV sequence) simultaneously and do not have the template negative control.With these PCR product equal-volume mixings, prepare to carry out chip hybridization and detect after the amplification.6. the oligonucleotide general-purpose chip is handled

Oligochip cleans 2 times with 0.2%SDS earlier, then cleans 2 times the air airing with water.Use NaBH ₄[100ml contains and adds 1.3g NaBH in the PBS solution of 20%7 alcohol solution ₄] effect 10min, the aldehyde radical of sealing surface of glass slide.0.2%SDS cleans 1 time, and water cleans 1 time, is used for hybridization after drying.7. hybridization and optimization of hybridization conditions thereof: with PCR mixture and hybridization solution in proportion behind the mixing, get 10 μ l and be added on each square sub-district of chip, and place hybridizing box, hybridize certain hour at a certain temperature, usefulness washing lotion A after chip hybridization is finished (1 * SSC, 0.2%SDS); Washing lotion B (0.2 * SSC) and washing lotion C (0.1 * SSC) respectively cleaned 5 minutes, with the Axon4000 scanner it was scanned then, and write down the result, according to strength of signal the result was judged.

(1) composition of hybridization system: 5 * SSC, 0.2%SDS, 10% formyl ammonia, PCR mixture, reporter probe.

(2) consumption of reporter probe: in containing 10 μ l hybridization systems of same amount PCR mixture, the reporter probe that adds different amounts, make the final concentration of reporter probe in each system be respectively 0.01 μ M, 0.02 μ M, 0.04 μ M, 0.08 μ M, 0.1 μ M. is added in these systems on each sub-district of chip then, and in 45 ℃ of hybridization 1 hour.Scanning result analysis (seeing accompanying drawing 4) is shown the consumption of reporter probe is advisable about with 0.02 μ M, cross that I haven't seen you for ages influence strength of signal, cross that the background meeting is higher at most.

(3) determining of hybridization time: same hybridization system is divided into many parts, be added in respectively on many chips in 45 ℃ and hybridize, in hybridization 2,5,10,20,30, and took out a chip respectively in 60 minutes and scan (seeing accompanying drawing 5), analyze and the comparative result demonstration, with the growth of hybridization time, signal strengthens gradually, and signal is saturated substantially after 30 minutes.

(4) determining of hybridization temperature: same hybridization system is divided into many parts, be added in respectively on many chips, in 37 ℃, 42 ℃, 45 ℃, 50 ℃, 55 ℃ of hybridization were taken out chip and are scanned after 30 minutes, analyze and comparative result demonstration (seeing accompanying drawing 6), 42 ℃ of-50 ℃ of hybridization signal intensity no significant differences hybridize that then specificity is relatively poor for 37 ℃, and 55 ℃ of hybridization signals weaken.8. general-purpose chip specificity analyses

Gene bar code primer Bar1R1, Bar3R1, Bar6R1, Bar8R1, Bar9R1, Bar11R1, Bar14R1, Bar16R1 are used to the positive template that increases, and the negative controls that are used to increase such as Bar2R1, Bar4R1, Bar5R1, hybridize detection after then these PCR products being mixed, the result shows that this chip can be distinguished the yin and yang attribute sample well.When chip prepares, also put simultaneously and made blank sampling liquid point and be positioned at HBV probe outside the amplification region as negative control, with the specificity of chip monitoring.9. chip detection sensitivity analysis

To contain 10 times of serial dilutions of plasmid of HBV dna sequence dna, making concentration is 10 ⁸-10 ²The series mask of copy/ul with increase the respectively plasmid of same concentration known of Bar1R1-Bar16R1, mixes the PCR product back hybridization then, and the results of hybridization analysis is shown that the detectability of each point can both reach 10 on the chip ⁴Copy.10. combined probe The Application of Technology

In order to determine whether adding cancellation probe can influence results of hybridization and whether wash in hybridization scanning result is had or not influence, experiment divides four groups, only add reporter probe during first group of hybridization, do not wash after the hybridization, only add reporter probe during second group of hybridization, but carry out the multistep washing after the hybridization, add reporter probe and cancellation probe during the 3rd group of hybridization, do not wash after the hybridization, add reporter probe and cancellation probe during the 4th group of hybridization, but carry out the multistep washing after the hybridization, then this four core assemblies sheet is scanned, interpretation of result relatively shows (seeing accompanying drawing 7), the three groups of signal intensity differences that detect in back are little, and background is low and even, and first group of background is very inhomogeneous, and influence is to result's judgement.Therefore, when hybridization, add cancellation probe, the effectively excessive reporter probe of cancellation, and can not have influence on hybridization signal, and need not washing and just can reach better background, can save the plenty of time, this point more can embody when using many chips to detect at the same time.11. gene bar-code identification general-purpose chip is in conjunction with combined probe technical Analysis clinical samples

To be combined into the performing PCR amplification with BarR1-Bar15R1 and F1 respectively from 15 parts of clear and definite serum specimens of the yin and yang attribute of hospital, the Bar16R1 positive control that increases, respectively getting the 2uLPCR product mixes, add 0.02 μ M reporter probe, 0.02 μ M cancellation probe, 5 * SSC, 0.2%SDS, 10% formyl ammonia, making final volume is 50 μ L, takes out 10 μ L and is added on the sub-district of general-purpose chip, put into hybridizing box, place 45 ℃ of hybridization to take out after 30 minutes, get rid of the hybridization solution on surface, use the Axon4000 scanner scanning, its scanning result is seen Fig. 8, show that by analysis institute's measurement result and expected results match, show and utilize this method can realize accurate mensuration many samples.

Claims (7)

1. the novel method that detects of pathogenic agent sample more than a kind.It is characterized in that, specific amplification primer and the reporter probe and the cancellation probe of the synthetic pathogen gene to be checked of design, wherein a primer extends a segment addressing bar code sequence to 5 ' end, one spacerarm is arranged between this sequence and primer, can stop PCR to extend, and this sequence and the addressable capture probe complementation that is fixed on the general-purpose chip, make the end of product connect the addressable bar code sequence of the preceding paragraph by pcr amplification, then all pcr amplification products are mixed, with reporter probe, the cancellation probe joins the hybridization system together, hybridize with general-purpose chip, complementary addressing capture probe hybridization on addressable bar code sequence that connects on these PCR products and the chip like this, thereby amplified production directionally is anchored on the chip correspondence position, the PCR product that is anchored again with the special reporter probe hybridization of reporter molecules mark such as fluorescence, can detect the result of every duplicate samples by analyses such as signal scannings.

2. according to claim 1, addressing bar code sequence can corresponding a sample, have many addressing bar code sequences just can corresponding many increments this, like this by hybridization once just can detect simultaneously many increments this, thereby realize the purpose of multiple sample detection.

3. according to claim 2, it can be the same pathogenic agent that detects Different Individual, the different pathogens of same individuality that multiple sample detects, and also can be the different pathogens of Different Individual, can decide according to particular case.

4. the pathogenic agent in the claim 1 can be the pathogenic agent that virus, chlamydozoan, mycoplasma, bacterium, fungi, spirochete, rickettsia etc. can cause the human infectious disease.

5. the special reporter probe of the target in the claim 1, long 15-30mer is positioned in the middle of the amplified fragments, reporter molecules such as 5 ' end mark fluorescein or vitamin H, and with the chain complementation that primer extended that is connected with addressable bar code sequence.The cancellation probe is than few 5-10 the base of reporter probe, and 3 ' end connects a quencher molecule-paramethyl red, and its sequence and fluorescent probe 5 ' end is complementary.When two probe hybridizations form dipolymer, the fluorescent energy transmission can take place, make reporter probe can not send fluorescence.

Spacerarm in the claim 1 can be-OCH2CH2O-,-O-or-groups such as NH2-.

7. according to claim 1 and 5, in the hybridization system excessive reporter probe can with the cancellation probe hybridization, make its fluorescence by cancellation, even do not wash after the hybridization like this, the background of chip also can be very low, thereby saved the step of multistep washing behind the chip hybridization, improves detection efficiency.

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